Influence of Nitric Oxide-Cyclic GMP and Oxidative STRESS on Amyloid-ß Peptide Induced Decrease of Na,K-ATPase Activity in Rat Hippocampal Slices.

Amyloid-ß peptide (Aß) has been shown to cause synaptic dysfunction and can render neurons vulnerable to excitotoxicity and oxidative stress. Na,K-ATPase plays an important role to maintain cell ionic equilibrium and it can be modulated by N-methyl-D-aspartate (NMDA)-nitric oxide (NO)-cyclic GMP pathway. Disruption of NO synthase (NOS) activity and reactive oxygen species (ROS) production could lead to changes in Na,K-ATPase isoforms' activities that may be detrimental to the cells. Our aim was to evaluate the signaling pathways of Aß in relation to NMDA-NOS-cyclic GMP versus oxidative stress on α1-/α2,3-Na,K-ATPase activities in rat hippocampal slices. Aß1-40 induced a concentration-dependent increase of NOS activity and increased cyclic guanosine monophosphate (cGMP), TBARS (thiobarbituric acid reactive substances), and 3-Nitrotyrosine (3-NT)-modified protein levels in rat hippocampal slices. The increase in NOS activity and cyclic GMP levels induced by Aß1-40 was completely blocked by MK-801 (inhibitor of NMDA receptor) and L-NAME (inhibitor of NOS) pre-treatment but changes in TBARS levels were only partially blocked by both compounds. The Aß treatment also decreased Na,K-ATPase activity which was reverted by N-nitro-L-arginine methyl ester hydrochloride (L-NAME) but not by MK-801 pre-treatment. The decrease in enzyme activity induced by Aß was isoform-specific since only α1-Na,K-ATPase was affected. These findings suggest that the activation of NMDA-NOS signaling cascade linked to α2,3-Na,K-ATPase activity may mediate an adaptive, neuroprotective response to Aß in rat hippocampus.

Clinical efficacy, safety and anti-inflammatory activity of two sevelamer tablet forms in patients on low-flux hemodialysis.

Sevelamer hydrochloride is an ionic exchange resin with high affinity for phosphate. This phosphate-binding agent has few serious adverse reactions with the advantage of reducing total and low density lipoprotein (LDL) cholesterol levels. However, it is controversial as to whether sevelamer hydrochloride can modulate the inflammatory response via endotoxin reduction. Therefore, a single-center, open-label, prospective and randomized study was performed to compare the clinical efficacy, safety and anti-inflammatory activity of two sevelamer hydrochloride tablet forms a branded tablet form, Renagel (Genzyme manufacturer) and its generic equivalent (EMS manufacturer). Twenty-eight chronic kidney disease volunteer patients at stage 5 (CDK 5D), on chronic low-flux hemodialysis carried out in 4-hour sessions, three times a week, were studied. The serum phosphorus, ionic calcium, total cholesterol and fractions, bicarbonate, blood pH, interleukin (IL)-6, IL-10, IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) levels were collected prior to dialysis at mid-week. The incidence of gastrointestinal adverse effects were determined at the end of the phosphate-binder washout period as well as at the end of the fourth and eighth weeks of use of both tablet forms. The same magnitude of reduction in serum phosphorus was observed with both sevelamer tablet forms. Only the Renagel group showed lower total cholesterol and lower LDL cholesterol levels at the fourth and eighth week versus baseline. No significant differences in serum cytokine levels were identified in either drug group. However, the incidence of intestinal obstipation was higher among patients who used the generic equivalent form. In conclusion, Renagel and its EMS generic equivalent tablet forms have a similar clinical efficacy in reducing phosphorus in CKD 5D patients on low-flux hemodialysis and a similar safety profile.

Curcumin requires tumor necrosis factor α signaling to alleviate cognitive impairment elicited by lipopolysaccharide.

A decline in cognitive ability is a typical feature of the normal aging process, and of neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases. Although their etiologies differ, all of these disorders involve local activation of innate immune pathways and associated inflammatory cytokines. However, clinical trials of anti-inflammatory agents in neurodegenerative disorders have been disappointing, and it is therefore necessary to better understand the complex roles of the inflammatory process in neurological dysfunction. The dietary phytochemical curcumin can exert anti-inflammatory, antioxidant and neuroprotective actions. Here we provide evidence that curcumin ameliorates cognitive deficits associated with activation of the innate immune response by mechanisms requiring functional tumor necrosis factor α receptor 2 (TNFR2) signaling. In vivo, the ability of curcumin to counteract hippocampus-dependent spatial memory deficits, to stimulate neuroprotective mechanisms such as upregulation of BDNF, to decrease glutaminase levels, and to modulate N-methyl-D-aspartate receptor levels was absent in mice lacking functional TNFRs. Curcumin treatment protected cultured neurons against glutamate-induced excitotoxicity by a mechanism requiring TNFR2 activation. Our results suggest the possibility that therapeutic approaches against cognitive decline designed to selectively enhance TNFR2 signaling are likely to be more beneficial than the use of anti-inflammatory drugs per se.

Influence of N-methyl-D-aspartate receptors on ouabain activation of nuclear factor-κB in the rat hippocampus.

It has been shown that ouabain (OUA) can activate the Na,K-ATPase complex and mediate intracellular signaling in the central nervous system (CNS). Inflammatory stimulus increases glutamatergic transmission, especially at N-methyl-D-aspartate (NMDA) receptors, which are usually coupled to the activation of nitric oxide synthase (NOS). Nuclear factor-κB (NF-κB) activation modulates the expression of genes involved in development, plasticity, and inflammation. The present work investigated the effects of OUA on NF-κB binding activity in rat hippocampus and the influence of this OUA-Na,K-ATPase signaling cascade in NMDA-mediated NF-κB activation. The findings presented here are the first report indicating that intrahippocampal administration of OUA, in a concentration that did not alter Na,K-ATPase or NOS activity, induced an activation of NF-κB, leading to increases in brain-derived neurotrophic factor (Bdnf), inducible NOS (iNos), tumor necrosis factor-α (Tnf-α), and B-cell leukemia/lymphoma 2 (Bcl2) mRNA levels. This response was not linked to any significant signs of neurodegeneration as showed via Fluoro-Jade B and Nissl stain. Intrahippocampal administration of NMDA induced NF-κB activation and increased NOS and α(2/3) -Na,K-ATPase activities. NMDA treatment further increased OUA-induced NF-κB activation, which was partially blocked by MK-801, an antagonist of NMDA receptor. These results suggest that OUA-induced NF-κB activation is at least in part dependent on Na,K-ATPase modulatory action of NMDA receptor in hippocampus. The interaction of these signaling pathways could be associated with biological mechanisms that may underlie the basal homeostatic state linked to the inflammatory signaling cascade in the brain.

Amyloid beta-peptide activates nuclear factor-kappaB through an N-methyl-D-aspartate signaling pathway in cultured cerebellar cells.

Amyloid beta-peptide (A beta) likely causes functional alterations in neurons well prior to their death. Nuclear factor-kappaB (NF-kappaB), a transcription factor that is known to play important roles in cell survival and apoptosis, has been shown to be modulated by A beta in neurons and glia, but the mechanism is unknown. Because A beta has also been shown to enhance activation of N-methyl-D-aspartate (NMDA) receptors, we investigated the role of NMDA receptor-mediated intracellular signaling pathways in A beta-induced NF-kappaB activation in primary cultured rat cerebellar cells. Cells were treated with different concentrations of A beta1-40 (1 or 2 microM) for different periods (6, 12, or 24 hr). MK-801 (NMDA antagonist), manumycin A and FTase inhibitor 1 (farnesyltransferase inhibitors), PP1 (Src-family tyrosine kinase inhibitor), PD98059 [mitogen-activated protein kinase (MAPK) inhibitor], and LY294002 [phosphatidylinositol 3-kinase (PI3-k) inhibitor] were added 20 min before A beta treatment of the cells. A beta induced a time- and concentration-dependent activation of NF-kappaB (1 microM, 12 hr); both p50/p65 and p50/p50 NF-kappaB dimers were involved. This activation was abolished by MK-801 and attenuated by manumycin A, FTase inhibitor 1, PP1, PD98059, and LY294002. A beta at 1 microM increased the expression of inhibitory protein I kappaB, brain-derived neurotrophic factor, inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-1 beta as shown by RT-PCR assays. Collectively, these findings suggest that A beta activates NF-kappaB by an NMDA-Src-Ras-like protein through MAPK and PI3-k pathways in cultured cerebellar cells. This pathway may mediate an adaptive, neuroprotective response to A beta.

Intermittent fasting uncovers and rescues cognitive phenotypes in PTEN neuronal haploinsufficient mice.

Phosphatase and tensin homolog (PTEN) is an important protein with key modulatory functions in cell growth and survival. PTEN is crucial during embryogenesis and plays a key role in the central nervous system (CNS), where it directly modulates neuronal development and synaptic plasticity. Loss of PTEN signaling function is associated with cognitive deficits and synaptic plasticity impairment. Accordingly, Pten mutations have a strong link with autism spectrum disorder. In this study, neuronal Pten haploinsufficient male mice were subjected to a long-term environmental intervention - intermittent fasting (IF) - and then evaluated for alterations in exploratory, anxiety and learning and memory behaviors. Although no significant effects on spatial memory were observed, mutant mice showed impaired contextual fear memory in the passive avoidance test - an outcome that was effectively rescued by IF. In this study, we demonstrated that IF modulation, in addition to its rescue of the memory deficit, was also required to uncover behavioral phenotypes otherwise hidden in this neuronal Pten haploinsufficiency model.

Ultrastructural characteristics of the external surfaces of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits.

The surface structures of the cells of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits were examined by transmission electron microscopy after ruthenium red staining and polycationic ferritin labelling. P. pneumotropica strains ATCC 35149 and K 79114 had slight extracellular fibrous materials associated with cell walls with ruthenium red staining. Ferritin labelling method revealed thick strands or sparsely ferritin-labelled materials on the cell surface of the strains. P. multocida strains Pm-78 and P-2440 had ferritin-labelled capsules surrounded with the cell wall. Strain Pm-78, which was serotyped as A:12, had a thick capsule, whereas serotype -:3 strain P-2440 had a thin and irregular capsule.

Second primary bronchogenic carcinomas after small cell carcinoma. Report of two cases and review of the literature.

Second primary carcinomas of the lung are well described. However, their occurrence following initial diagnosis of small cell lung carcinoma is rare. The development and antemortem diagnosis of metachronous second primary bronchogenic carcinomas in two long-term (more than four years) survivors of small cell lung cancer is described. The histologic types of the second carcinomas were mucoepidermoid and bronchoalveolar. On the basis of a review of the literature, only eight similar cases have been reported; none of the second primaries was mucoepidermoid or bronchoalveolar. The question of whether second primaries after small cell lung cancer represent true metachronous carcinomas, different degrees of differentiation of the same tumor, or the emergence of a previously unrecognized synchronous tumor is discussed. The need for awareness of this complication and the necessity for life-long follow-up in long-term survivors of small cell lung cancer is emphasized.

Hypercalcemia of acute renal failure. Clinical significance and pathogenesis.

Hypocalcemia is a frequent accompaniment of acute renal failure, but paradoxically hypercalcemia also has been described in association with acute renal failure. In this paper we describe two patients who provide some insights into both the potential clinical importance and mechanism of the hypercalcemia associated with acute renal failure. The clinical significance is emphasized by the presence of diffuse metastatic calcification observed at postmortem examination in one patient. In both patients the increase in serum calcium concentration was not coincident with a decrease in serum phosphorus concentration; when measured in one patient, serum levels of parathyroid hormone were undetectable. These findings, along with the consistent association with rhabdomyolosis, support the proposal that the hypercalcemia of acute renal failure is caused by dissolution of dystrophic calcifications in traumatized muscle and may lead to severe metastatic calcifications.

Noncontact, two-dimensional measurement of retinal microcirculation using laser speckle phenomenon.

PURPOSE: To report a new apparatus for noncontact, two-dimensional measurement of retinal microcirculation using the laser speckle phenomenon and to demonstrate that this apparatus can document known or expected changes in retinal blood flow. METHODS: The rabbit fundus was illuminated by an argon (blue) laser spot (0.62 x 0.62 mm), and its image speckle was detected with an image sensor. The difference between the average of the speckle intensity (Imean) and the speckle intensity for successive scannings was calculated, and the ratio of Imean to this difference was defined as normalized blur (NB), a quantitative index of blood velocity in the retinal microcirculation. The results were displayed on a color monitor showing the two-dimensional variation of the NB level in the measurement area. Using this apparatus in the rabbit, the NB in the retinal field free of visible surface vessels was determined and compared with the retinal blood flow rate measured using the microsphere technique in the same eye simultaneously. In addition, the effect of the ocular perfusion pressure (OPP) on NB was studied. In the above experiments, a stepwise reduction in OPP was introduced by elevating the intraocular pressure manometrically. RESULTS: The relative decrease in the average NB (NBav) over the field measured, with the reduction in OPP, showed significant correlation with the relative change in the blood flow rate determined using the microsphere technique (r = 0.59, P < 0.001). Although NBav in the retina was little affected by OPP change when OPP was greater than 50 mm Hg, NB decreased along with OPP at levels less than 50 mm Hg. CONCLUSIONS: The NBav showed significant correlation with the retinal blood flow rate determined with microsphere technique. Retinal microcirculation under various conditions can be studied two dimensionally and noninvasively in the living eye with the present apparatus.

Use of the human tumor stem cell assay to study chemotherapy sensitivity in cancer of the cervix.

Two questions concerning chemotherapy of cervical cancers are addressed in this study. 1) Can the human tumor stem cell assay identify active chemotherapeutic agents?; and 2) Can the human tumor stem cell assay predict which patient's tumors will demonstrate clinical responsiveness to chemotherapy? Sixty-seven of 76 cervical cancer specimens (88.2%) were grown in the human tumor stem cell assay, resulting in 159 separate drug assays. Based on the known activity of standard chemotherapeutic agents demonstrated in clinical trials, cisplatin, bleomycin, vincristine, and mitomycin C were chosen for testing. In vitro sensitivity patterns, seen at one-tenth of the peak plasma drug concentration, were similar to those seen in historic clinical trials. Retrospective correlations between in vitro drug sensitivity data and patient responses to chemotherapy are available from 13 patients, demonstrating clinically helpful predictive capabilities of the assay. Two conclusions were made: 1) The human tumor stem cell assay can determine single agent activity, which is often dose related; and 2) Useful correlations with clinical responses were observed.

Postmastectomy angiosarcoma.

Six cases of postmastectomy angiosarcoma of the upper extremity have been treated at the North Carolina Baptist Hospital/Wake Forest University Medical Center over the past 25 years. Five patients had typical ductal carcinoma; one patient had mammary Paget's disease. Two patients, including the patient with Paget's disease, had bilateral mastectomies. The five patients with ductal carcinoma had received postoperative radiation therapy. The mean interval between mastectomy and the appearance of angiosarcoma was 17.5 years. Significant lymphedema of the upper extremity preceded the appearance of angiosarcoma in all six patients, and it appears important that special care be taken during mastectomy to preserve the integrity of the lymphatic system. The patient treated with interscapulothoracic amputation is alive at 2 years postamputation; the five patients whose angiosarcomas were treated with wide excision or radiotherapy died an average of 4.5 years later. Interscapulothoracic amputation is recommended as the initial treatment of postmastectomy angiosarcoma of the upper extremity.

Suture technique and wound-bursting strength.

Despite advances in wound healing, fascial dehiscence continues to be a problem in celiotomy wounds. Experimental and clinical studies on suture material and on patient-related factors in wound disruption are abundant, but little attention has been given to mechanical factors in wound closure, although they may be of greater importance. A midline abdominal wound was made in 120 Harlan Sprague-Dawley rats, and the fascia was closed with Dacron in one of six ways: simple interrupted, interrupted figure-of-eight, and running, each tied loosely in one half of each group and tightly in the other half. One week later, the rats were reanesthetized and wound-bursting strength was measured. In general, the running suture technique resulted in the greatest wound-bursting pressures. A loosely tied figure-of-eight technique was nearly as good as a loosely tied running stitch, but tying figure-of-eight sutures tightly caused a significant decrease in wound-bursting pressure. The simple interrupted technique was unaffected by suture tension but was generally inferior to the running stitch in terms of wound-bursting strength. Histologic studies were performed, but most of the disparity in wound strength among the suture techniques apparently was due solely to mechanical factors. Closing midline abdominal fascial wounds with a running suture may be a superior method of closure in clean, incised wounds.

Abdominal wound closure. A comparison of polydioxanone, polypropylene, and Teflon-coated braided Dacron sutures.

A midline abdominal wound was made in 135 Harlan Sprague-Dawley rats, and the fascia was then closed by the continuous suture technique with 4-0 absorbable monofilament polydioxanone sutures (n = 45 rats), 4-0 permanent monofilament polypropylene sutures (n = 45 rats), or 4-0 Teflon-coated braided Dacron sutures (n = 45 rats). At 1-, 2-, and 6-month intervals, wound-bursting pressures were determined in 15 animals from each group. At 1 month and at 6 months, there was no significant difference in wound-bursting strength among the three types of sutures. At 2 months, wounds closed with Teflon-coated braided Dacron were significantly stronger than wounds closed with the other types of sutures. In most of the braided Dacron suture animals, the fascia burst in areas other than the abdominal wound, indicating that the wound was stronger at that time than was the surrounding fascia. There is no advantage to that added strength, and braided Dacron sutures have been shown to cause greater tissue reaction than monofilament sutures. Therefore, except in patients with poor wound healing who are highly susceptible to dehiscence and in whom permanent low-tissue-reaction sutures may be beneficial, the authors recommend a running suture technique using absorbable monofilament suture, such as polydioxanone, for the routine closure of clean abdominal wounds.

Aspiration and exfoliative cytology, including ultrastructure, of a malignant granular-cell tumor.

The cytopathologic features of a malignant granular cell tumor in both exfoliative and aspiration cytopreparations included isolated cells with low nuclear-cytoplasmic ratios and abundant, diffusely granular cytoplasm. Transmission electron microscopy revealed the diagnostic secondary lysosomes in the neoplastic cells.

Multiple myeloma. The diagnostic role and prognostic significance of exfoliative cytology.

The clinical significance and diverse cytomorphologic spectrum of exfoliative cytology in multiple myeloma are presented from our 20-year retrospective and continuing prospective studies and from an extensive review of the literature. Of 370 myeloma patients studied retrospectively, 126 had at least one exfoliative cytologic specimen but only 6 had one or more specimens positive for myeloma. These included six pleural and two ascitic fluids and one sputum. In Papanicolaou-stained smears, myeoloma cells varied from essentially normal-appearing plasma cells to dispersed large malignant cells with little or no plasmacytoid features. Whereas all 203 cervical or vaginal, cerebrospinal, urine and bronchial specimens were negative for myeloma, 40% and 50% of the pleural and ascitic fluids, respectively, were positive. Four prospectively studied patients produced a total of seven positive serous fluid specimens. Follow-up data was available for eight patients with cytology positive for myeoloma. Six were dead within three months of the first positive specimen.

An application of laser speckle phenomenon for noninvasive two-dimensional evaluation of microcirculation in ocular fundus--a preliminary report.

A new instrument has been developed which uses the laser speckle phenomenon for noninvasive two-dimensional analysis of the microcirculation in the optic nerve head (ONH). The ONH is illuminated by a diode laser spot and its image speckle is detected by an area sensor. The difference between a pair of output data from successive scannings of the image speckles at the sensor plane is calculated and integrated for each pixel to obtain the average difference (AD) value, which is a quantitative index of blood flow velocity. The results are displayed in color graphics showing the two-dimensional variation of the AD value in the area of interest. In an in vitro model, the AD value correlated well with the speed of rotation of a ground glass in a range between 0 and 5 mm/second. Using this instrument, the effect of intraocular pressure (IOP) on the circulation was studied in the area of the rabbit ONH free of surface vessels. The coefficient of reproducibility was 4.7% when measured twice in this area at 5-minute intervals. A stepwise reduction in the ocular perfusion pressure (OPP) was introduced by elevating the IOP manometrically. The AD value was little affected by a change in OPP when the OPP was above 40 mmHg; while when the OPP was below 40 mmHg, a linear correlation was found between the AD value and the OPP. This finding agreed well with those of previous studies where the effect of the OPP on the ONH blood flow were studied using various invasive methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of indirect haemagglutination test, gel-diffusion precipitin test, and enzyme-linked immunosorbent assay for detection of serum antibodies to Pasteurella multocida in naturally and experimentally infected rabbits.

Enzyme-linked immunosorbent assay (ELISA), gel-diffusion precipitin test (GDPT), and indirect haemagglutination test (IHAT) were evaluated for the detection of antibodies to Pasteurella multocida in both naturally and experimentally infected rabbits. A total of 285 rabbit serum samples from 7 rabbit colonies were tested by ELISA, GDPT, and IHAT, and nasal cultures were taken coincidentally to use as the standard in the serological tests. There was better correlation (98.0%) between the results of ELISA and positive nasal culture than between the GDPT (86.3%) or IHAT (23.5%) and positive nasal culture. In addition, ELISA and GDPT were positive in 26 (11.1%) and 21 (9.0%) of 234 serum samples from nasal culture negative rabbits, respectively. In experimentally infected rabbits, antibodies detected by the ELISA and GDPT began to rise one to 3 weeks post-inoculation. IHAT did not detect antibodies. These results are discussed in terms of value to serodiagnosis of rabbit pasteurellosis.

[An apparatus using the laser speckle phenomenon for non-invasive two-dimensional analysis of retinal microcirculation].

A new instrument using the laser speckle phenomenon for non-invasive two-dimensional analysis of the retinal microcirculation was developed. The fundus was illuminated with an argon laser spot and its image speckle was detected with an area sensor. The difference between the average of output data (Imean) and output data for successive scannings of the image speckles at the sensor plane was calculated and integrated to give Dmean, and Imean/Dmean is defined as normalized blur (NB), which is a quantitative index of blood flow velocity. The results were displayed in color graphics showing the two-dimensional variation of the NB level in the area of interest. The NB had a good correlation with the speed of rotation of the ground glass in a range between 5 and 120 mm/sec. Using this instrument, the effect of intraocular pressure (IOP) on the microcirculation of the rabbit retina was studied. The coefficient of reproducibility was 9.2% when measured in the same area twice at 5-minute intervals. A stepwise reduction in the ocular perfusion pressure (OPP) was introduced by elevating the IOP manometrically. The NB in the retina was little affected by OPP change when OPP was above 50 mmHg, but below this value the NB decreased as the OPP decreased.