Ilan Hammel

Pathways of Macromolecular Extravasation Across Microvascular Endothelium in Response to VPF/VEGF and Other Vasoactive Mediators

Objective: The goal of these studies was to define the anatomic pathways by which circulating macromolecules extravasate from the hyperpermeable microvessels that supply tumors and from normal venules that have been rendered hyperpermeable by vasoactive mediators.

Different activation signals induce distinct mast cell degranulation strategies

Mast cells (MCs) influence intercellular communication during inflammation by secreting cytoplasmic granules that contain diverse mediators. Here, we have demonstrated that MCs decode different activation stimuli into spatially and temporally distinct patterns of granule secretion. Certain signals, including substance P, the complement anaphylatoxins C3a and C5a, and endothelin 1, induced human MCs rapidly to secrete small and relatively spherical granule structures, a pattern consistent with the secretion of individual granules. Conversely, activating MCs with anti-IgE increased the time partition between signaling and secretion, which was associated with a period of sustained elevation of intracellular calcium and formation of larger and more heterogeneously shaped granule structures that underwent prolonged exteriorization. Pharmacological inhibition of IKK-β during IgE-dependent stimulation strongly reduced the time partition between signaling and secretion, inhibited SNAP23/STX4 complex formation, and switched the degranulation pattern into one that resembled degranulation induced by substance P. IgE-dependent and substance P-dependent activation in vivo also induced different patterns of mouse MC degranulation that were associated with distinct local and systemic pathophysiological responses. These findings show that cytoplasmic granule secretion from MCs that occurs in response to different activating stimuli can exhibit distinct dynamics and features that are associated with distinct patterns of MC-dependent inflammation.

Histopathology of the Inferior Turbinate With Compensatory Hypertrophy in Patients With Deviated Nasal Septum

Objective To measure the dimensions, composition, and possible structural and/or histopathological changes of the compensatory hypertrophic inferior turbinate in patients with deviated nasal septum.

Synaptotagmin III is a critical factor for the formation of the perinuclear endocytic recycling compartment and determination of secretory granules size

Early endosomes and a perinuclear, Rab-11-positive compartment have been implicated in the recycling of internalized receptors. In this study, we show that synaptotagmin III (Syt III), a member of the Syt family of proteins, is required for the formation and delivery of cargo to the perinuclear endocytic recycling compartment (ERC). We demonstrate that rat basophilic leukemia (RBL-2H3) mast cells endogenously express Syt III, and >70% of this protein colocalizes with early endosomal markers, such as EEA1, annexin II and syntaxin 7, and the remaining protein colocalizes with secretory granule (SG) markers such as β-hexosaminidase, histamine and serotonin. To study the functional role of Syt III, we stably transfected RBL cells with Syt III antisense cDNA and monitored the route of transferrin (Tfn) internalization in cells that displayed substantially reduced (<90%) levels of Syt III (RBL-Syt III-). In these cells, Tfn binding and internalization into early endosomes were unaltered. However, whereas in the mock-transfected cells Tfn was subsequently delivered to the ERC, in the RBL-Syt III- cells, Tfn remained associated with dispersed peripheral vesicles and Rab 11 remained cytosolic. Nevertheless, the rates of Tfn internalization and recycling were not affected. RBL-Syt III- cells also displayed enlarged SGs, reminiscent of the SGs present in Chediak-Higashi (beige) mice. However, morphometric analyses suggested that granule formation was unaltered and that the calculated unit granule volume is the same in both cell lines. Therefore, our results implicate Syt III as a critical factor for the generation and delivery of internalized cargo to the perinuclear endocytic recycling compartment and suggest a possible link between ERC and recycling from immature SGs during the process of SG maturation.

Regulation of secretory granule size by the precise generation and fusion of unit granules.

•  Introduction •  The ‘unit granule’ and evidence of granule–granule fusion •  Evidence for two models of secretory granule formation, unit addition and random fusion •  Formation of unit granules: progranule fusion, immature granule maturation and membrane conservation ‐  Progranule fusion and immature granule maturation ‐  Membrane conservation •  Functional implications •  Conclusions and future directions

Combining an EGFR directed tyrosine kinase inhibitor with autophagy-inducing drugs: A beneficial strategy to combat non-small cell lung cancer

The potential therapeutic value of combinatorial regimens based on an EGF receptor tyrosine kinase inhibitor (TKI) and autophagy inducing drugs was evaluated by comparing their molecular impacts on H1299 and A549 non-small cell lung cancer (NSCLC) cells, which overexpress wild type EGF receptor, but are either deficient or have wild type p53 alleles, respectively. We show that H1299 cells display a considerably lower sensitivity to erlotinib treatment, which can be restored by combining erlotinib with rapamycin or with imatinib, though to a lesser extent. Cytotoxicity was associated with increased autophagy and hyperpolarization of the mitochondrial membrane potential. Therefore, combining an EGF receptor directed TKI with an autophagy-inducing drug, preferably, rapamycin, might be beneficial in treating poor responding NSCLC patients.

Characteristics of palatal wound healing in desalivated rats

The healing of excisional wounds in the palate of desalivated rats was evaluated. Experimental rats became desalivated after extirpation of the submandibular and sublingual glands and ligation of the parotid ducts. Small or large circular wounds, 3 or 5 mm in diameter, were produced in the palate. The wound area, area of inflammation, area of connective tissue formation and the number of myofibroblasts were determined at 0, 3, 7, 14, 21 and 28 days after surgery. The area of the small wound (3 mm) was similar in experimental and control groups; however, the area of the large wound (5 mm) was greater in the experimental group (p < 0.05-0.01). The area of inflammation was greater in the experimental group with small or large wounds (p < 0.05-0.01). Connective tissue formation was less (p < 0.01) in desalivated rats with a small wound at day 14 and with a large wound at days 21 and 28. There were fewer myofibroblasts in the large wound of desalivated rats (p < 0.01) than in controls between days 3 and 14. The results indicate that palatal wound healing is delayed in desalivated rats and that larger wounds are more sensitive to desalivation than smaller wounds.

The rigidity of bacterial flagellar filaments and its relation to filament polymorphism

We determined and correlated the rigidity of Salmonella typhimurium, Escherichia coli, and Rhizobium lupini flagellar filaments representing various structural and polymorphic states (plain, complex, straight, superhelical, and right- and left-handed). Persistence length, from which the filaments rigidity and other parameters (Youngs modulus, bending force constant, buckling persistence length, flexural deformation, and flexural time) were derived, was determined from electron micrographs of isolated, negatively stained filaments. Outer diameters and radii of strong intersubunit connectivity were determined from three-dimensional image reconstructions and radial mass density profiles from scanning transmission electron microscopy. All filaments appear to be highly rigid with no evident correlation with their helical sense or superhelicity. The complex filament of R. lupini is rigid to the extent that it becomes brittle. The overall flexibility of the flagellum seems to stem mainly from the hook and not from the filament. Polymorphism is probably related to the propelling properties and hydrodynamic shape of the filament rather than to its rigidity.

Regional and developmental variations of blood vessel morphometry in the chick embryo chorioallantoic membrane

SUMMARY Avian eggs contain all the necessary materials for embryonic development except for oxygen, which diffuses in from the environment via pores in the hard, calcified eggshell to the chorioallantoic membrane (CAM), the respiratory organ, which is rich in blood vessels. An air cell is formed at the blunt pole of the egg between the two membranes of the eggshell and enlarges during incubation due to water vapor loss. In this study of the CAM of chicken eggs, we compared blood vessel numerical density [NA(v)], area fraction of blood vessels [AA(v)], CAM thickness (DCAM), total length of blood vessels (L) and surface area of the CAM attached to the eggshell (CAMre) with those under the air cell (CAMac) during incubation. We found that NA(v), AA(v), DCAM and L of the CAM increase with embryonic age and development. The NA(v), AA(v) and L under the air cell were higher in relation to the rest of the CAM at all ages tested, while the DCAM under the air cell was always lower than around the rest of the egg. Since the eggshell over the air cell has a relatively greater porosity, and the respiratory gas exchange ratio there is higher than at other areas of the egg, there is a correlation between all the above morphometric data and the eggshell porosity. This suggests optimization of embryonic gas exchange in the chicken egg. We would like to propose that, during natural incubation, an increased gas diffusion under the air cell, together with increased blood vessel numerical density, may compensate for covering of the central part of the eggshell by the incubating parent.

Histopathology of the Uvula and the Soft Palate in Patients With Mild, Moderate, and Severe Obstructive Sleep Apnea

Objective To study morphometric and qualitative histopathologic changes of the soft palate and uvula in patients with mild, moderate, and severe obstructive sleep apnea.