Expression pattern of <Emphasis Type="Italic">serine protease inhibitor kazal type 3 (Spink3)</Emphasis> during mouse embryonic development

Recent evidence shows that the serine protease inhibitor Kazal type 3 (Spink3) has more diverse functions than expected. To gain insight into its function, we analyzed the spatiotemporal expression profile of Spink3, using in situ hybridization (ISH) and a Spink3 ( +/lacZ ) knock-in mouse, in which lacZ was inserted into the Spink3 locus. Spink3 ( lacZ ) expression was first observed in the foregut, midgut, hindgut and the forebrain/midbrain junction region at 9.5 days post coitus (dpc). In the pancreas, Spink3 mRNA was detected at 11.5 dpc, before formation of the typical shape of the exocrine structure of the pancreas. Acinar cell expression was clearly identified by 13.5 dpc. After differentiation of the intestinal tract, Spink3 ( lacZ ) expression was observed in the large intestine at 11.5 dpc, followed by expression in the small intestine at 13.5 dpc, before appearance of intestinal digestive enzymes. Spink3 mRNA and Spink3 ( lacZ ) activity were also detected in other tissues, including the mesonephric tubules and the urogenital ridge at 11.5 dpc, the genital swelling at 13.5 dpc, the ductus epididymis at 17.5 dpc, and the seminal vesicle at 8 weeks. These data suggest that Spink3 may play important roles in proliferation and/or differentiation of various cell types during development.     

Mutations in CSTA, encoding Cystatin A, underlie exfoliative ichthyosis and reveal a role for this protease inhibitor in cell-cell adhesion

Autosomal-recessive exfoliative ichthyosis presents shortly after birth as dry, scaly skin over most of the body with coarse peeling of nonerythematous skin on the palms and soles, which is exacerbated by excessive moisture and minor trauma. Using whole-genome homozygosity mapping, candidate-gene analysis and deep sequencing, we have identified loss-of-function mutations in the gene for protease inhibitor cystatin A (CSTA) as the underlying genetic cause of exfoliative ichthyosis. We found two homozygous mutations, a splice-site and a nonsense mutation, in two consanguineous families of Bedouin and Turkish origin. Electron microscopy of skin biopsies from affected individuals revealed that the level of detachment occurs in the basal and lower suprabasal layers. In addition, in vitro modeling suggests that in the absence of cystatin A protein, there is a cell-cell adhesion defect in human keratinocytes that is particularly prominent when cells are subject to mechanical stress. We show here evidence of a key role for a protease inhibitor in epidermal adhesion within the lower layers of the human epidermis.     

Pilot trial of interleukin-2 and zoledronic acid to augment γδ T cells as treatment for patients with refractory renal cell carcinoma

Prior to the advent of VEGF-targeted therapies, renal cell carcinoma (RCC) was among the few solid tumors shown to respond to cytokine-based therapies such as interleukin-2 (IL-2) and interferon alpha. Previous work has shown that aminobisphosphonates, including zoledronic acid (ZA), are capable of activating human Vγ9 Vδ2 T cells in vitro, and these cells can be further expanded with IL-2. Moreover, these Vγ9 Vδ2 T cells have cytolytic activity in vitro to multiple human tumor cell lines. In the current report, we have conducted a pilot trial in patients with metastatic RCC, evaluating different doses of ZA in combination with low-dose IL-2 to determine whether combining these agents can promote in vivo proliferation of Vγ9 Vδ2 T cells and elicit an antitumor response. In 12 patients evaluated, no objective clinical responses were observed by RECIST criteria; however, two patients experienced prolonged stable disease. A modest increase in Vγ9 Vδ2 T-cell frequency could be detected by Day 8 of therapy in four of the nine patients who received at least one cycle of therapy, but not to the magnitude anticipated from preclinical models. Repeated administration of IL-2 and ZA resulted in both a diminished in vivo percentage of Vγ9 Vδ2 T cells as well as impaired expansion in vitro after the first cycle of therapy. These results suggest that repeated administration of IL-2 and ZA, at the doses and schedules used in this trial, may actually inhibit the proliferative capacity of Vγ9 Vδ2 T cell in patients with metastatic RCC.     

The Anti-Botulism Triterpenoid Toosendanin Elicits Calcium Increase and Exocytosis in Rat Sensory Neurons

Toosendanin, a triterpenoid from Melia toosendan Sieb et Zucc, has been found before to be an effective anti-botulism agent, with a bi-phasic effect at both motor nerve endings and central synapse: an initial facilitation followed by prolonged depression. Initial facilitation may be due to activation of voltage-dependent calcium channels plus inhibition of potassium channels, but the depression is not fully understood. Toosendanin has no effect on intracellular calcium or secretion in the non-excitable pancreatic acinar cells, ruling out general toosendanin inhibition of exocytosis. In this study, toosendanin effects on sensory neurons isolated from rat nodose ganglia were investigated. It was found that toosendanin stimulated increases in cytosolic calcium and neuronal exocytosis dose dependently. Experiments with membrane potential indicator bis-(1,3-dibutylbarbituric acid)trimethine oxonol found that toosendanin hyperpolarized capsaicin-insensitive but depolarized capsaicin-sensitive neurons; high potassium-induced calcium increase was much smaller in hyperpolarizing neurons than in depolarizing neurons, whereas no difference was found for potassium-induced depolarization in these two types of neurons. In neurons showing spontaneous calcium oscillations, toosendanin increased the oscillatory amplitude but not frequency. Toosendanin-induced calcium increase was decreased in calcium-free buffer, by nifedipine, and by transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine. Simultaneous measurements of cytosolic and endoplasmic reticulum (ER) calcium showed an increase in cytosolic but a decrease in ER calcium, indicating that toosendanin triggered ER calcium release. These data together indicate that toosendanin modulates sensory neurons, but had opposite effects on membrane potential depending on the presence or absence of capsaicin receptor/TRPV 1 channel.     

'Alzheimer-like' pathology in a murine model of arterial hypertension

Genetic AD (Alzheimer's disease) accounts for only few AD cases and is almost exclusively associated with increased amyloid production in the brain. Instead, most patients are affected with the sporadic form of AD and typically have altered clearance mechanisms. The identification of factors that influence the onset and progression of sporadic AD is a key step towards understanding its mechanism(s) and developing successful therapies. An increasing number of epidemiological studies describe a strong association between AD and cardiovascular risk factors, particularly hypertension, that exerts detrimental effects on the cerebral circulation, favouring chronic brain hypoperfusion. However, a clear demonstration of a pathophysiological link between cardiovascular risk factors and AD aetiology is still missing. To increase our knowledge of the mechanisms involved in the brain's response to hypertension and their possible role in promoting amyloid deposition in the brain, we have performed and investigated in depth different murine models of hypertension, induced either pharmacologically or mechanically, leading in the long term to plaque formation in the brain parenchyma and around blood vessels. In the present paper, we review the major findings in this particular experimental setting that allow us to study the pathogenetic mechanisms of sporadic AD triggered by vascular risk factors.     

Egomotion estimation with optic flow and air velocity sensors

We develop a method that allows a flyer to estimate its own motion (egomotion), the wind velocity, ground slope, and flight height using only inputs from onboard optic flow and air velocity sensors. Our artificial algorithm demonstrates how it could be possible for flying insects to determine their absolute egomotion using their available sensors, namely their eyes and wind sensitive hairs and antennae. Although many behaviors can be performed by only knowing the direction of travel, behavioral experiments indicate that odor tracking insects are able to estimate the wind direction and control their absolute egomotion (i.e., groundspeed). The egomotion estimation method that we have developed, which we call the opto-aeronautic algorithm, is tested in a variety of wind and ground slope conditions using a video recorded flight of a moth tracking a pheromone plume. Over all test cases that we examined, the algorithm achieved a mean absolute error in height of 7% or less. Furthermore, our algorithm is suitable for the navigation of aerial vehicles in environments where signals from the Global Positioning System are unavailable.