Sub-micrometric liposomes as drug delivery systems in the treatment of periodontitis.

Periodontitis is a complex disease and bacterial infection is one of the most common factors involved in this disease. Current strategies for the local delivery of antibiotics do not allow a complete clearance of bacteria filling dentinal tubules and this limits their therapeutic efficacy. Therefore, there is a strong need for the development of new delivery strategies aimed at improving the efficacy of antibiotic therapy for periodontitis with special reference to their ability to penetrate into the tubules. The aim of the present study is to develop liposome-based delivery systems of sub-micron dimension, able to diffuse into the dentinal tubules. A further aim of the research is to develop a protocol for enhanced diffusion based on the use of magnetic liposomes and magnetic fields. Liposomes were produced by hydration of a pre-liposomal formulation. The vesicles were stabilised with PEG and their re-sizing was achieved by extrusion. Magnetite nanoparticles were synthesized inside the vesicles, i.e., the chemical reaction involving FeCl2, FeCl3 and NH3 occurred within the core of the newly formed liposomes. Dynamic light scattering analysis was performed for size characterization. A mathematical model was implemented to predict the diffusion of the liposomes in dentinal tubules. Ex-vivo validation was performed on extracted human teeth. We produced PEG-ylated liposomes (average size 204.3 nm) and PEG-ylated magnetic liposomes (average size 286 nm) and an iron content of 4.2 µg/ml. Through mathematical modelling, we deduced that sub-micrometer vesicles are able to penetrate into dentinal tubules. This penetration is considerably more effective when the vesicles are magnetized and subjected to an external magnetic field which accelerates their movement within the tubules. The liposome-based delivery systems developed by the present study are able to penetrate deeply into the tubules, sometimes reaching their terminal ends.

The effects of inhaled house dust mite on airway barrier function and sensitivity to inhaled methacholine in mice.

Asthma is functionally characterized by increased airway sensitivity and reactivity. Multiple mechanisms are believed to underlie these functional disorders, including impairment of airway wall barrier function. One proposed mechanism of impaired barrier function is through the direct consequence of proteolytic properties of inhaled allergens, including house dust mite (HDM). Here, we have observed the direct effects of HDM on airway barrier function and response to nebulized or intravenous methacholine. HDM naïve BALB/c mice were anesthetized, exposed to intranasal or intratracheal HDM (15 or 100 µg), and allowed to recover for 30 min or 2 h before methacholine challenge. A separate group of mice was exposed to intratracheal poly-L-lysine (PLL; 100 µg) for a duration of 30 min. This group served as a positive control for the presence of impaired barrier function and airway hypersensitivity. Negative control mice received saline challenges. Outcomes included assessment of lung mechanics in response to nebulized or intravenous methacholine as well as clearance of intratracheally instilled technetium-labeled ((99m)Tc) DTPA to evaluate airway epithelial barrier function. We found that PLL produced a leftward shift in the dose-response curve following nebulized but not intravenous methacholine challenge. This was associated with a significantly faster clearance of (99m)Tc-DTPA, indicating impairment in airway barrier function. However, HDM exposure did not produce changes in these outcomes when compared with saline-exposed mice. These findings suggest that direct impact on airway barrier function does not appear to be a mechanism by which HDM produces altered airway sensitivity in airway disease.

Glutamatergic innervation of the hypothalamic median eminence and posterior pituitary of the rat.

Recent studies have localized the glutamatergic cell marker type-2 vesicular glutamate transporter (VGLUT2) to distinct peptidergic neurosecretory systems that regulate hypophysial functions in rats. The present studies were aimed to map the neuronal sources of VGLUT2 in the median eminence and the posterior pituitary, the main terminal fields of hypothalamic neurosecretory neurons. Neurons innervating these regions were identified by the uptake of the retrograde tract-tracer Fluoro-Gold (FG) from the systemic circulation, whereas glutamatergic perikarya of the hypothalamus were visualized via the radioisotopic in situ hybridization detection of VGLUT2 mRNA. The results of dual-labeling studies established that the majority of neurons accumulating FG and also expressing VGLUT2 mRNA were located within the paraventricular, periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area. In contrast, only few FG-accumulating cells exhibited VGLUT2 mRNA signal in the arcuate nucleus. Dual-label immunofluorescent studies of the median eminence and posterior pituitary to determine the subcellular location of VGLUT2, revealed the association of VGLUT2 immunoreactivity with SV2 protein, a marker for small clear vesicles in neurosecretory endings. Electron microscopic studies using pre-embedding colloidal gold labeling confirmed the localization of VGLUT2 in small clear synaptic vesicles. These data suggest that neurosecretory neurons located mainly within the paraventricular, anterior periventricular and supraoptic nuclei and around the organum vasculosum of the lamina terminalis and the preoptic area secrete glutamate into the fenestrated vessels of the median eminence and posterior pituitary. The functional aspects of the putative neuropeptide/glutamate co-release from neuroendocrine terminals remain to be elucidated.

Localization and osmotic regulation of vesicular glutamate transporter-2 in magnocellular neurons of the rat hypothalamus.

In this report we present immunocytochemical and in situ hybridization evidence that magnocellular vasopressin and oxytocin neurons in the hypothalamic supraoptic and paraventricular nuclei express type-2 vesicular glutamate transporter, a marker for their glutamatergic neuronal phenotype. To address the issue of whether an increase in magnocellular neuron activity coincides with the altered synthesis of the endogenous glutamate marker, we have introduced a new dual-label in situ hybridization method which combines fluorescent and autoradiographic signal detection components for vasopressin and vesicular glutamate transporter-2 mRNAs, respectively. Application of this technique provided evidence that 2% sodium chloride in the drinking water for 7 days produced a robust and significant increase of vesicular glutamate transporter-2 mRNA in vasopressin neurons of the supraoptic nucleus. The immunocytochemical labeling of pituitary sections, followed by the densitometric analysis of vesicular glutamate transporter-2 immunoreactivity in the posterior pituitary, revealed a concomitant increase in vesicular glutamate transporter-2 protein levels at the major termination site of the magnocellular axons. These data demonstrate that magnocellular oxytocin as well as vasopressin cells contain the glutamatergic marker vesicular glutamate transporter-2, similarly to most of the parvicellular neurosecretory neurons examined so far. The robust increase in vesicular glutamate transporter-2 mRNA and immunoreactivity after salt loading suggests that the cellular levels of vesicular glutamate transporter-2 in vasopressin neurons are regulated by alterations in water-electrolyte balance. In addition to the known synaptic actions of excitatory amino acids in magnocellular nuclei, the new observations suggest novel mechanisms whereby glutamate of endogenous sources can regulate magnocellular neuronal functions.

Eosinophilia-myalgia syndrome (L-tryptophan-associated neuromyopathy).

Histopathologic study of skeletal muscle biopsy in a patient with eosinophilia-myalgia syndrome following L-tryptophan use showed prominent lymphocytic perineuritis, neuritis, and perimysial fasciitis. The presence of perineuritis and neuritis provides a histopathologic basis for clinical features of neuropathy in eosinophilia-myalgia syndrome and occurred in conjunction with a fasciitis or interstitial myositis that was predominantly perimysial and focally endomysial.

Endothelial morphology and plasma total and high density lipoprotein cholesterol changes in hypothalamically stimulated squirrel monkeys fed a modified atherogenic diet.

Experimental animals fed atherogenic diets show endothelial damage, impairment of endothelial regeneration and plasma lipid changes characterized by elevation of LDL and decrease of HDL cholesterol concentrations. Previous studies in this laboratory disclosed that chronic electrical stimulation of the lateral hypothalamus was associated with electron-microscopic evidence of endothelial injury in rats and squirrel monkeys maintained on basal (low fat/cholesterol-free) diets. In the present investigation squirrel monkeys fed similar diets supplemented with "modest" amounts of caloric fat and cholesterol were subjected to chronic lateral hypothalamic stimulation for periods as long as 20 months with the expectation that endothelial injury would be greater than in the absence of the supplements. The expectations were not substantiated. Endothelium was found to be surprisingly intact by electron microscopy and similar to that of implanted nonstimulated controls. A further observation of interest was the cholesterolemic response, notably in the HDL fraction, observed in both groups, but more striking in experimental animals. The data suggest that an interaction between a modified lipid/cholesterol diet and hypothalamic stimulation may lead to elevation of plasma HDL cholesterol concentration and preservation of endothelial integrity. Further investigation is required to determine whether these two events are causally related.

Effect of hypothalamic stimulation on the endothelial morphology of the aorta in the conscious squirrel monkey.

The role of neurogenic factors in the development of atherosclerosis has not previously been studied in detail. In recent years evidence has accumulated to implicate endothelial injury as a primary stimulus for the proliferation of myo-intimal cells resulting in the formation of the early morphologic lesion. In the present investigation, the effect on aortic endothelial morphology of repetitive electrical stimulation of the lateral hypothalamus in the conscious, unrestrained squirrel monkey, maintained on a cholesterol-free low-fat diet, has been studied. Stimulation was performed with a self-powered, miniaturized electronic stimulator connected to indwelling electrodes. Implanted nonstimulated animals served as controls. Endothelial injury in the form of cell degeneration, denudation, with plasma insudation and partial junctional separation were observed electron-microscopically in stimulated animals compared with controls. These alterations were found to be independent of hypercholesterolemia and/or hypertension. Possible pathways for the induction of injury in this neurogenic model are: (1) direct, through neural circuits from the brain to the vessel wall, and (2) indirect, by elaboration of angiopathic substances inside or outside of the CNS, released into the circulation and transported to the vessel wall where they exert their effects. Reversibility of the endothelial injury progression to established lesions and mechanisms involved remain to be determined in further investigations.

Synthesis of gonadotropin-releasing hormone III analogs. Structure-antitumor activity relationships.

Following the observation that the activity of gonadotropin-releasing hormone III (GnRH-III) in the suppression of growth of MDA-MB-231 and MCF-7 breast cancer cells surpasses that of GnRH and other analogs thereof, analogs of GnRH-III were synthesized to investigate the structural basis for the improved antitumor activity. Compounds synthesized include analogs with changes in the central sequence in which GnRH-III differs from GnRH and in the C- and N-terminal regions. The results indicate that a salt bridge between Asp6 and Lys8 stabilizes the active conformation of GnRH-III and show the importance of the Trp7. Replacement of the C-terminal Gly-NH2 with D-Ala-NH2 was not well tolerated, but replacement with ethylamide was. Replacement of pGlu1 with Ac-D-Trp appears to have a significantly deleterious effect on a unique conformation of GnRH-III which is responsible for its binding to the receptors on cancer cell lines and the resultant antitumor activity.

Muscular ventricular septal defects repaired with left ventriculotomy.

Between 1974 and 1979 nine patients, aged 10 months to 4 years, underwent left ventriculotomy for closure of single or multiple defects in the muscular ventricular septum. The vertical incision paralleled the anterior descending branch of the left coronary artery near the apex of the left ventricle and ranged from 2.5 to 3.5 cm in length. Four patients also had a right ventriculotomy with closure of a high perimembranous ventricular defect in two. Serial electrocardiograms indicated changes of myocardial ischemia or necrosis. Left bundle branch block did not develop in any patient. Three patients died in the early postoperative period. The six surviving patients are living and well 2 to 7 years later. There is apparent complete closure of the ventricular defects, which was documented by cardiac catheterization in four cases. Two patients had cardiomegaly and left ventricular dysfunction as assessed with echocardiographic and angiographic study, whereas four displayed good cardiac function. In three of the latter patients, cardioplegia or deep hypothermia techniques were utilized intraoperatively. The observations indicate that left ventriculotomy of limited size is an acceptable approach to the difficult problem of repair of muscular ventricular defects but may involve some risk of compromise of the coronary circulation.

Cardiac fibromatosis: an ultrastructural study.

The fine structure of a surgically resected cardiac fibroma is described. The tumor consisted primarily of fibroblasts set in a matrix composed of acid mucopolysaccharide and collagen. Many of the fibroblasts contained abundant myofilaments and dense bodies, suggesting differentiation or transformation of these cells to myofibroblasts. No striated muscle was identified. This study confirms the fact that the cardiac fibroma is derived from connective tissue elements and should not be considered either a hamartoma or a mesoblastic tumor. Structurally, as well as biologically, the cardiac fibroma closely resembles soft tissue fibromatoses and should be considered an example of cardiac fibromatosis.

Dominant expression of multidrug resistance in intraspecific murine lymphoma hybrid cells.

Cultured P388/VCR mouse lymphoma cells resistant to vincristine (VCR) and to 5-bromodeoxyuridine (BUdR) and deficient in thymidine kinase (TK-) were fused with P388/DAG cells resistant to 1.2:5,6-dianhydrogalactitol (DAG), an anticancer alkylating agent, and to 6-thioguanine (6-TG) and deficient in hypoxanthine phosphoribosyl-transferase (HPRT-). The hybrid cells expressed multidrug resistance (MDR), i.e., resistance to VCR and cross-resistance to Adriamycin (ADM) and actinomycin D (Act. D), in a dominant manner. The presence of glycoprotein p170, the MDR gene product, was detected in the hybrid cells. Resistance to DAG was also expressed dominantly, whereas cross-resistance to dibromodulcitol (DBD), a chemically related anticancer drug, was slight.

Neuroendocrine differentiation in gastric adenocarcinomas. An immunohistochemical study.

BACKGROUND: The stomach contains a wide variety of neuroendocrine cells. Early studies with argyrophilic stains documented the presence of these cells in gastric adenocarcinomas. Immunohistochemical techniques for demonstrating hormones are more sensitive and specific and have been applied only sporadically to gastric adenocarcinomas. Thus, the true incidence of neuroendocrine cells in gastric adenocarcinomas is questionable. METHODS: Formalin-fixed, paraffin-embedded archival tissue specimens from 48 gastric adenocarcinomas were immunostained with antibodies to chromogranin A, synaptophysin, serotonin, gastrin, and neuron-specific enolase. The percentage of cells staining positively was evaluated semiquantitatively. RESULTS: Among 48 gastric adenocarcinomas, 36 (75%) stained positively for chromogranin A, 33 (69%) stained for synaptophysin, 29 (60%) stained for neuron-specific enolase, 17 (36%) stained for gastrin, and 15 (31%) stained for serotonin. The distribution of positivity was highest for chromogranin A (7 cases positive in 26% to 75% of cells) and lowest for serotonin (14 out of 15 cases stained in fewer than 1% of the cells present). CONCLUSIONS: Immunohistochemical evaluation of neuroendocrine markers in gastric adenocarcinomas indicates that a high percentage of tumors contain widely scattered single cells with neuroendocrine differentiation. Most often, however, such cells constitute only a small percentage of the total number of tumor cells present.

Clostridial orthopedic infections: case reports and review of the literature.

Clostridia are anaerobic Gram-positive bacilli that can be isolated from the soil and the intestinal tract of humans. These microorganisms are recognized as the cause of devastating soft tissue infections, such as cellulitis, myositis, and gas gangrene. However, such bacteria may also be involved in various postoperative orthopedic infections, including prosthetic joint infection. We present three clinical cases of clostridial orthopedic infection and review the related medical literature.

Proliferation and morphology of ascitic cells as a function of age in cell culture.

Ascitic cells in the logarithmic growth phase increase the accumulation of glycogen particles in the course of explantation into suspension culture, probably due to the increasing arrest of glycogenolytic enzymes. At this age, a part of the cells are capable of restitution by exopinocytosis of the glycogen-containing vacuoles ia formation of cytoplasmic buds. Older cells, taken from the plateau-phase, pass atypical differentiation and ageing, are less capable of hindering the abnormal accumulation of glycogen and the hypervacuolisation. As a consequence, the cells finally degenerate and die.

Utility of cytokeratin 7 and 20 subset analysis as an aid in the identification of primary site of origin of malignancy in cytologic specimens.

This study was undertaken to assess the utility of combined cytokeratin (CK) 7/20 immunoprofile determination in malignant cytologic cell blocks as an aid to the identification of tumor primary site of origin. Fifty-one cases in which CK 7/20 immunocytochemistry was performed as part of the initial workup were retrieved. Their contribution to the final cytologic diagnosis of tumor primary site of origin was analyzed. CK reactivity patterns were 7+/20- (n = 34), 7-/20+ (n = 9), 7-/20- (n = 7), and 7+/20+ (n = 1). The CK 7+/CK 20- immunophenotype was the most common one obtained, and due to its wide expression in a number of common carcinomas, the least informative. The second most common immunophenotype was CK 7-/20+, which is associated with colorectal origin, and as such was very useful when obtained. The CK immunoprofile was more useful in the setting of a prior carcinoma, being a major diagnostic determinant in 13 cases (55%) from group 1 (those with a prior history of malignancy), compared to 8 cases (29%) from group 2 (those with no prior history of malignancy). In the setting of prior carcinoma, the CK immunoprofile is most useful when carcinomas under consideration have different expected immunoprofiles (e.g., CK 7+/CK20- carcinomas, including lung, breast, ovary, endometrium, and others, vs. CK 7-/CK 20+ carcinomas, primarily colorectal). When similar immunoprofiles are obtained, their usefulness is greater if they are immunoprofiles other than the most common 7+/20- pattern. Similarly, in newly diagnosed carcinomas, the CK immunoprofile either helps to narrow the differential diagnosis or points to a specific diagnosis.

K+ depolarization evokes ATP, adenosine and glutamate release from glia in rat hippocampus: a microelectrode biosensor study.

BACKGROUND AND PURPOSE: This study was undertaken to characterize the ATP, adenosine and glutamate outflow evoked by depolarization with high K(+) concentrations, in slices of rat hippocampus. EXPERIMENTAL APPROACH: We utilized the microelectrode biosensor technique and extracellular electrophysiological recording for the real-time monitoring of the efflux of ATP, adenosine and glutamate. KEY RESULTS: ATP, adenosine and glutamate sensors exhibited transient and reversible current during depolarization with 25 mM K(+) , with distinct kinetics. The ecto-ATPase inhibitor ARL67156 enhanced the extracellular level of ATP and inhibited the prolonged adenosine efflux, suggesting that generation of adenosine may derive from the extracellular breakdown of ATP. Stimulation-evoked ATP, adenosine and glutamate efflux was inhibited by tetrodotoxin, while exposure to Ca(2+) -free medium abolished ATP and adenosine efflux from hippocampal slices. Extracellular elevation of ATP and adenosine were decreased in the presence of NMDA receptor antagonists, D-AP-5 and ifenprodil, whereas non-NMDA receptor blockade by CNQX inhibited glutamate but not ATP and adenosine efflux. The gliotoxin fluoroacetate and P2X7 receptor antagonists inhibited the K(+) -evoked ATP, adenosine and glutamate efflux, while carbenoxolone in low concentration and probenecid decreased only the adenosine efflux. CONCLUSIONS AND IMPLICATIONS: Our results demonstrated activity-dependent gliotransmitter release in the hippocampus in response to ongoing neuronal activity. ATP and glutamate were released by P2X7 receptor activation into extracellular space. Although the increased extracellular levels of adenosine did derive from released ATP, adenosine might also be released directly via pannexin hemichannels.