Angiotensin converting-enzyme inhibition restores glomerular glycosaminoglycans in rat puromycin nephrosis.

BACKGROUND: Aberrant glomerular polyanionic charge of glycosaminoglycans (GAGs) and sialic acid expression has been observed in proteinuric human and experimental glomerular diseases. Angiotensin-converting enzyme inhibitors (ACEI) lower proteinuria and amend renal function deterioration via hemodynamic mechanisms. We tested the hypothesis that ACEI modulate proteinuria additionally by modifying glomerular GAGs. METHODS: In this study, we explored the effects of the ACEI enalapril on proteinuria and GAG synthesis in puromycin aminonucleoside (PAN)-treated rats. We employed cationic colloidal gold (CCG) localization in glomerular basement membranes (GBM) to identify GAGs by electron microscopy and determined sialic acid residues by immunohistochemical staining with lectins. To clarify ACEI effects on GAG production in vitro, we studied de novo GAG synthesis into newly synthesized proteoglycans in podocytes and mesangial cells using 35S incorporation. Cells were incubated with or without PAN, and with increasing doses of the ACEI enalaprilat. RESULTS: PAN rats developed severe proteinuria that was significantly improved by enalapril treatment. In non-treated PAN rats GBM GAGs were reduced, whereas in the enalapril-treated group GBM GAGs were significantly increased to control levels. Enalapril did not affect glomerular sialic acid. Furthermore, in cultured podocytes and mesangial cells PAN decreased de novo GAG synthesis, an effect which was significantly ameliorated by enalaprilat treatment. CONCLUSION: Treatment with ACEI improves permselectivity properties of the glomerular capillary wall by maintaining its GAG content. This finding provides an additional new mechanism, whereby ACEI exert anti-proteinuric effects.

Localization of anionic constituents in mast cell granules of brachymorphic (bm/bm) mice by using avidin-conjugated colloidal gold.

We used the egg avidin gold complex as a polycationic probe for the localization of negatively charged sites in the secretory granules of mouse mast cells. We compared the binding of this reagent to mast cell granules in wild-type mice and in congenic brachymorphic mice in which mast cell secretory granules contained undersulfated proteoglycans. We localized anionic sites by post-embedding labeling of thin sections of mouse skin and tongue tissues fixed in Karnovsky's fixative and OsO(4) and embedded in Araldite. Transmission electron microscopy revealed that the mast cell granules of bm/bm mice had a lower optical density than those of wild-type mice (P<0.001) and a lower avidin gold binding density (by approximately 50%, P<0.001). The latter result provided additional evidence that the contents of mast cell granules in bm/bm mice were less highly sulfated than in those of wild-type mice. In both wild-type and bm/bm mast cells, the distribution of granule equivalent volumes was multimodal, but the unit granule volume was approximately 19% lower in bm/bm cells than in wild-type cells (P<0.05). Thus, bm/bm mast cells develop secretory granules that differ from those of wild-type mice in exhibiting a lower optical density and slightly smaller unit granules, however the processes that contribute to granule maturation and granule-granule fusion in mast cells are operative in bm/bm cells.

Designing ageing conditions in tumour microenvironment-a new possible modality for cancer treatment.

While tumour incidence is known to augment with age, paradoxically tumour growth and metastasis were often found to proceed at a slower rate at late ages. This age-related biological behaviour of tumours actually imposes a differential therapeutic approach to the old cancer patient. Several mechanisms of the age-related reduced tumour progression have been demonstrated: decreased tumour cell proliferation, increased apoptotic cell death, decreased angiogenesis and anti-tumoural immune response changes. We postulated that it might be possible to design age-adjusted treatment modalities based on the mechanisms responsible for the reduced tumour progression rate in the aged. Based on these mechanisms, we compared the effect of different treatments (apoptosis-inducing agents, Hydrocortisone and Adriamycin, anti-angiogenic agent, TNP-470, and immunomodulators-Levamisole and BCG) on two experimental tumours (B16 melanoma and AKR lymphoma) growing in young and old mice. Most treatments showed, in both tumours, a higher inhibitory effect on tumours growing in old mice than on those developing in young ones, to our knowledge, a feature not described before for anti-tumoural agents. We suggest that designing ageing conditions in tumours of young patients might possibly alleviate neoplastic aggressiveness in these patients as well.

Glycoconjugate expression of sinus mucosa in chronic rhinosinusitis: a lectin histochemical study.

BACKGROUND: Glycoproteins are responsible for the rheological properties of the mucus. Changes in mucus viscosity may interfere with mucociliary clearance, leading to stasis and aggravation of chronic rhinosinusitis (CRS). The purpose of this study was to assess the composition and concentration of various sinus mucosa glycoproteins in normal and chronically inflamed sinuses. METHODS: A semiquantitative scoring system was used to express the binding intensity of 10 lectins to 6 carbohydrates (galactose, sialic acid, N-acetylglucosamine, N-acetylgalactosamine [GalNac] [corrected], fucose, mannose) residing in the sinus mucosa of patients with CRS (N = 20) and normal controls (N = 10). RESULTS: The mean CT score, compatible with extensive sinus disease, was 13.9 +/- 6.7. All six carbohydrates were found in the epithelium and submucosal glands of patients and controls. Peanut agglutinin staining showed significantly higher concentration of disaccharide galactose beta 1,3 GalNac [corrected] in epithelial goblet cells and in submucosal mucous and mixed glands of patients compared with that of controls (p = 0.01, p = 0.03, and p = 0.018, respectively). The epithelial glycocalyx of patients was also significantly more abundant in fucose and in GalNac [corrected] (p = 0.015, and p < 0.001, respectively). CONCLUSION: Glycoconjugate expression of the sinus mucosa in CRS is markedly altered in extensive inflammatory conditions. The increased concentration of galactose beta 1,3 GalNac [corrected] in CRS probably contributes to the high viscosity of the nasal mucus and interferes with normal mucociliary clearance. Exposed peripheral galactose residues may serve as attachment receptor sites for pathogenic bacteria. Means interfering with the ability of bacteria to adhere to specific receptors on host tissues warrant additional research.

Decreased DNA ploidy may constitute a mechanism of the reduced malignant behavior of B16 melanoma in aged mice.

Numerous data demonstrate a lower aggressiveness of tumors in aged as compared to young patients. The mechanisms underlying this phenomenon have not yet been completely elucidated. Several mechanisms have been shown, such as reduced tumor cell proliferation, increased apoptosis, immune response modifications and reduced angiogenesis in aged organism tumors. In the present study we report an incidentally found, not yet described mechanism, of the age-related reduced tumor progression, namely a decreased ploidy in B16 melanoma growing in old (near diploidy) as compared to young mice (tetraploidy). We surprisingly observed that tumor cells from aged mice were of smaller cell and nuclear size than those of young animals. Flow cytometry forward scatter data also showed a smaller cell size of melanoma cells from old mice. DNA flow cytometry profile comparison demonstrated that while B16 melanoma cells from young animals contained a high percentage of tetraploid cells, those derived from old animals were mostly close to diploid. A high importance has recently been attributed to aneuploidy as being at the origin of the genetic instability of neoplasia. Our results may support this notion. The transit from tetraploidy to near euploidy in melanoma cells growing in aged mice might avoid the genetic instability inherent to tumor progression.

Glycoconjugate expression in perennial allergic rhinitis: a lectin histopathological study.

CONCLUSIONS: The data show that differences in the concentrations of glycoconjugates of patients with perennial allergic rhinitis (PAR) and normal controls are modest, thus indicating that the composition of the mucus in allergic patients largely resembles that of healthy individuals. The findings may point to the need for volume reduction methods controlling mucus production in patients with PAR. OBJECTIVES: We aimed to study the composition and concentration of inferior turbinate glycoconjugates of patients with PAR. MATERIALS AND METHODS: Six specific oligosaccharides found in the inferior turbinate mucosa were stained with a battery of 10 lectins. The samples recruited for study were 15 sections from patients with PAR and 17 from healthy individuals who had no nasal disease and underwent rhinoplasty surgery for cosmetic reasons. Both groups were matched for age (p = 0.208). Results. No significant difference in the concentration of galactose, fucose, sialic acid, N-acetylglucosamine, and N-acetylgalactosamine in the epithelium and submucosal glands of the inferior turbinate was found between the groups. Likewise, neuraminidase digestion of peripheral sialic acid revealed similar concentration of the penultimate galactose residue. The only significant difference was a higher concentration of mannose in submucosal serous glands of patients with PAR compared with normal controls (p = 0.04).

Human oocyte cryopreservation and the fate of cortical granules.

OBJECTIVE: To examine the effect of the commonly used oocyte cryopreservation protocol on the cortical granules (CGs) of human immature germinal vesicle (GV) and mature metaphase II (MII) oocytes. DESIGN: Laboratory study. SETTING: IVF unit. INTERVENTION(S): Unfertilized, intracytoplasmic sperm injected (ICSI) oocytes, and immature oocytes were cryopreserved using a slow freezing-rapid thawing program with 1,2-propanediol (PROH) as a cryoprotectant. MAIN OUTCOME MEASURE(S): Cortical granule exocytosis (CGE) was assessed by either confocal microscopy or transmission electron microscopy (TEM). RESULT(S): The survival rates of frozen-thawed oocytes (mature and immature) were significantly lower compared with zygotes. Both mature and immature oocytes exhibited increased fluorescence after cryopreservation, indicating the occurrence of CGE. Mere exposure of oocytes to cryoprotectants induced CGE of 70% the value of control zygotes. The TEM revealed a drastic reduction in the amount of CGs at the cortex of frozen-thawed GV and MII oocytes, as well as appearance of vesicles in the ooplasm. CONCLUSION(S): The commonly used PROH freezing protocol for human oocytes resulted in extensive CGE. This finding explains why ICSI is needed to achieve fertilization of frozen-thawed human oocytes.

Evaluation of the pro-angiogenic effect of factor XIII in heterotopic mouse heart allografts and FXIII-deficient mice.

Thrombin-activated Factor XIII (FXIIIa), a plasma transglutaminase, stabilizes fibrin clots by crosslinking fibrin chains. FXIIIa was previously shown by us to exhibit proangiogenic activity associated with downregulation of thrombospondin-1, phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2), and upregulation of c-Jun. In the current study, we evaluated the proangiogenic effect of FXIIIa in two murine models: a neonatal heterotopic cardiac allograft model in normal mice, and a Matrigel plug model in FXIII-deficient mice. In the neonatal cardiac allograft model, the number of new vessels as well as graft viability (contractile performance) was significantly higher in FXIIIa-injected animals than in controls. A significant increase in the level of c-Jun mRNA and a significant decrease in the level of TSP-1 mRNA were observed in heart allografts treated with FXIIIa. A marked decrease in TSP-1 protein expression was observed within the endothelial cells of hearts treated with FXIIIa. In the Matrigel plug model, FXIII-deficient mice showed a significantly decreased number of new vessels compared to that of the control mice, and the number of vessels almost reached normal levels following addition of FXIIIa. The results of this study provide substantial in vivo evidence for the proangiogenic activity of FXIIIa.

Efficacy of anti-angiogenic treatment of tumors in old versus young mice.

Cancer treatment in the older population, the most afflicted by the disease, is as yet, inefficient. A reduced aggressiveness of tumors is often observed in the elderly, implying the necessity for therapeutic modalities adjusted to age. A rational design of age-related cancer therapy could be based on the mechanisms of this phenomenon. It is suggested that, in addition to the patient's old age-specific health problems (which prohibit the use of the aggressive cancer treatments now in use), the age-related differential tumor biology (apparently beneficial to the old) should also be considered for the design of treatment modalities suitable for the aged. Based on one mechanism of the reduced aggressiveness of tumors in the old (age-dependent decreased angiogenesis), we compared the effect of an anti-angiogenic treatment in young and old mice. TNP-470 treatment resulted in an inhibitory effect on B16 melanoma in both young and old mice but the effect was more pronounced in old animals. Moreover, a high percentage of long-term surviving animals was observed only in the old-treated mice. Treatment with TNP-470 of the AKR lymphoma produced similar results. We thus found a differential age-dependent therapeutic efficiency of an anti-angiogenic agent on two tumors. Importantly, the anti-angiogenic drug was more efficient against tumors of old animals.

Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells.

Shortage in tissue availability from cadaver donors and the need for life-long immunosuppression severely restrict the large-scale application of cell-replacement therapy for diabetic patients. This study suggests the potential use of adult human liver as alternate tissue for autologous beta-cell-replacement therapy. By using pancreatic and duodenal homeobox gene 1 (PDX-1) and soluble factors, we induced a comprehensive developmental shift of adult human liver cells into functional insulin-producing cells. PDX-1-treated human liver cells express insulin, store it in defined granules, and secrete the hormone in a glucose-regulated manner. When transplanted under the renal capsule of diabetic, immunodeficient mice, the cells ameliorated hyperglycemia for prolonged periods of time. Inducing developmental redirection of adult liver offers the potential of a cell-replacement therapy for diabetics by allowing the patient to be the donor of his own insulin-producing tissue.

Glycoconjugate expression in normal human inferior turbinate mucosa: a lectin histochemical study.

BACKGROUND: Further study on the composition and concentration of normal nasal mucosal glycoconjugates is needed. METHODS: A battery of ten lectins was used to stain six carbohydrates residing in the inferior turbinate mucosa of 21 healthy individuals. RESULTS: The concentrations of glycoconjugates of the glycocalyx exceeded that of epithelial goblet, ciliated, and basal cells, and also of mucous, serous, and mixed submucosal glands. Epithelial goblet cells and submucosal mucous glands contained relatively high concentrations of glycoconjugates, whereas in epithelial ciliated and basal cells, similar to serous and mixed submucosal glands, concentrations were scanty. The concentrations of N-acetylgalactosamine and galactose were higher in goblet cells than in mucous glands (p = 0.006, and p = 0.056, respectively). Differences in the concentrations of N-acetylglucosamine, fucose, mannose, and sialic acid were insignificant. CONCLUSIONS: The findings provide a baseline for comparison of the composition and concentration of carbohydrates of the normal and pathologic inferior turbinate mucosa.

Ontogenesis of peripheral electromagnetic receptors in hornets.

This article traces the ontogenesis of peripheral electromagnetic receptors (PER) in the cuticle of the Oriental hornet (Vespa orientalis). In the abdominal cuticle of adult hornets, the PERs are densely distributed throughout, but there are even more than 30 at the margins of the segments. These organelles develop as a network in the hornet cuticle immediately upon its completion. Briefly, from each basic cell of a PER grows a bulge towards the exterior, that is, towards the illuminated region of the cuticle. This bulge develops rapidly and as it grows it starts to push out and lift up the various layers of the cuticle, the while pressing them together. By a spiraling movement, the bulge insinuates itself between the layers, whereupon it dissolves and punctures its way through all the layers of the hypocuticle, via the endocuticle up to the exocuticle. The only cuticular layer that remains intact is the epicuticle, but even that undergoes change, assuming the shape of a smooth surface with a depression at its center. The indented part in the epicuticle is circular, approximately 2.5 microm in diameter and enables the entry of radiation (illumination) from the outside into the PER, which is located half-way down the cuticle, with the distance from the exterior to the base of the PER being approximately 25 microm. The numerous lamellae of the cuticle run parallel to one another, but in the region of the bulge they are either perpendicular or directed upwards. This ontogeny of the PERs lends the cuticle a sandwich-like shape, being radically perforated by the PERs bulges, yet covered at the top by the epicuticle and at the bottom by basal cells. The PERs also extend shoots into the cuticular layer and these further perforate the cuticle but also interlink the various PERs. From all the above, it is clear that the cuticle forms first and only subsequently does the network of PERs develop and interpenetrate its various layers.

Age-dependent differences in the efficacy of cancer immunotherapy in C57BL and AKR mouse strains.

While tumor incidence increases with age, tumor growth and metastasis often proceed at a slower rate in aged organisms. The mechanisms underlying this age-related reduced tumor development may suggest therapeutic modalities appropriate for the aged. Decreased tumor aggressiveness in the old was shown to be related to altered immune response. Consequently, the aim of the present study was to assess whether cancer immunotherapy has an age-dependent effect. Only a few studies have compared cancer immunotherapy efficiency as a function of age, most showing lower inhibition in older animals. In the present study, we tested the effect of two immunomodulators, levamisole and BCG, on two tumors, B16 melanoma and AKR lymphoma, in mice of different ages. We demonstrated a higher efficiency of immunotherapy in aged as compared to young mice, particularly at low immunomodulator doses. While decreased T cell function during aging is apparently established, nonspecific immunity is more preserved or even enhanced in later life. We found an increased number of macrophages in tumors of old compared to young mice and an increase in MAC-1+ cells in old levamisole-treated compared to non-treated mice. The stronger therapeutic effect of this immunomodulator in old mice might thus be due to an increased macrophage-mediated anti-tumoral effect.

Age-adjusted antitumoral therapy based on the demonstration of increased apoptosis as a mechanism underlying the reduced malignancy of tumors in the aged.

In view of the constant increase in the aged population, age-adjusted cancer therapy becomes an urgent target. Although cancer incidence rises with age, paradoxically, growth rate and metastasis often proceed at a slower rate in the aged. Determining the mechanism(s) underlying this reduced tumor progression in the old might have implications for a rational design of age-adjusted therapy. Thus far, decreased cell proliferation or immune response modifications were suggested as possible mechanisms. We show here that an increased tendency to apoptotic tumor cell death in the aged could constitute an additional mechanism. Based on this mechanism, we compared the therapeutic efficacy of two apoptosis inducers, hydrocortisone and adriamycin, on AKR lymphoma and B16 melanoma growth in young and old mice. Treatment with hydrocortisone acetate inhibited tumor growth practically only in old mice in the two tumor systems. Similar effects were obtained with adriamycin treatment of AKR lymphoma but opposite results were seen with B16 melanoma. We thus demonstrated, in three of the four tumor-therapeutic modality systems examined, an age-related antitumoral efficacy of two apoptosis-inducing agents, with tendency for a remarkably more pronounced effect in aged mice.

Ageing-apoptosis relation in murine spleen.

Relatively few studies have been published with regard to modification of apoptosis in normal tissues as a function of ageing. The majority of these studies demonstrated an increase in programmed cell death (PCD) with age. However, opposite results, namely loss of apoptotic control with age, have also been reported. In the present study, we examined proliferation and apoptotic cell death in spleens of C57/BL mice of different ages. A tendency towards decrease in cell proliferative capacity was seen with age. By contrast, apoptosis was increased in spleens from aged animals. Moreover, the proliferative cell/apoptotic cell ratio decreased in function of age. Ladder type DNA degradation was much more pronounced in DNA derived from splenocytes of old mice. These results were supported by a decrease of Bcl-2 and an increase in Fas receptor expression as well as by increased activation of caspases 8, 3 and 9 in splenocytes from aged animals. In addition, cell surface molecular markers recognizable by macrophages in apoptotic cells, namely decreased sialic acid concomitant with increased unmasking of galactose residues, were more pronounced on splenocytes from old mice than on those from young animals. In addition to the experimental evidence which supports a role of apoptotic cell death in ageing, a series of theoretical reasoning, which could also favor this possibility, are discussed.

The neuroprotective peptide NAP inhibits the aggregation of the beta-amyloid peptide.

Alzheimer's disease (AD) is characterized by brain plaques containing the beta-amyloid peptide (Abeta). One approach for treating AD is by blocking Abeta aggregation. Activity-dependent neuroprotective protein contains a peptide, NAP that protects neurons in culture against Abeta toxicity. Here, NAP was shown to inhibit Abeta aggregation using: (1) fluorimetry; (2) electron microscopy; (3) high-throughput screening of Abeta deposition onto a synthetic template (synthaloid); and (4) Congo Red staining of neurons. Further assays showed biotin-NAP binding to Abeta. These results suggest that part of the neuroprotective mechanism exerted by NAP is through modulation of toxic protein folding in the extracellular milieu.