Time course of increased plasma cytokines, cortisol, and urea nitrogen in pigs following intraperitoneal injection of lipopolysaccharide

The emerging view is that reduced feed intake, lean muscle accretion, and growth in immunologically challenged pigs is the result of increased cytokine activity, but this has not been directly tested. To begin addressing this issue, 72 crossbred barrows and gilts (11.55 +/- .19 kg BW) were not fed for 12 h and then injected i.p. with 0, .5, or 5 micrograms/kg of Escherichia coli lipopolysaccharide (LPS). Blood was collected by jugular puncture at 0, 2, 4, 8, 12, and 24 h after injection. Plasma levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), cortisol, plasma urea nitrogen (PUN), NEFA, and triglycerides were determined. Immunological stress was induced by LPS as indicated by increased secretion of TNF-alpha, IL-6, and cortisol. In pigs receiving 5 micrograms/kg of LPS, plasma TNF-alpha was increased 10-fold at 2 h after injection and was still elevated (P < .01) at 4 h. In these same pigs, plasma concentration of IL-6 was increased at 2 h and peaked at 4 h with levels exceeding baseline values by 200-fold (P < .01). Cortisol was elevated at 2, 4, and 8 h after injection (P < .01). The increased secretion of cytokines and cortisol in pigs injected with 5 micrograms/kg of LPS was followed by an increase in protein degradation, as evidenced by PUN values that were increased two- and threefold at 8 and 12 h after injection, respectively. However, unlike previous reports in laboratory animal species, plasma glucose, NEFA, and triglycerides were not altered by LPS. Nonetheless, as the period of feed deprivation progressed from 12 to 36 h, plasma NEFA and triglycerides increased (P < .05) and plasma glucose tended to decrease. We believe that immunological challenge induces cytokine synthesis and secretion in swine which, in turn, may induce protein catabolism.     

Photodynamic therapy on rat urinary bladder with intravesical instillation of 5-aminolevulinic acid: light diffusion and histological changes

PURPOSE: Photodynamic therapy (PDT) has the potential to treat extensive premalignant lesions and microinvasive tumors in the bladder, but its use has been hampered by the risk of detrusor muscle damage and prolonged skin photosensitivity. We have shown that the rat urothelium can be sensitized by selectively using a 10% solution of 5-aminolevulinic acid (ALA) at pH 5.5 administered intravesically. This paper evaluates the photodynamic effects on sensitized bladders. MATERIALS AND METHODS: The bladders ofs Wistar rats were instilled with ALA solutions of different concentrations at pH 5.5 and subsequently treated with laser light at 630 nm. Bladders were harvested 1 to 7 days after PDT for histological assessment. RESULTS: Under optimum conditions (10% intralipid diffusion medium, light dose 50J) uniform urothelial necrosis was seen after 1 to 2 days; it healed in 7 days without damage to the underlying muscle layer although some increase in collagen was seen in the lamina propria. Overtreatment or poor light distribution resulted in muscle necrosis and scarring. CONCLUSIONS: Selective urothelial necrosis is possible with PDT using intravesical ALA. There is now sufficient data for pilot clinical trials to start photodynamic therapy for management of superficial bladder cancer or carcinoma in situ.     

Generalized peritonitis caused by spontaneous intraperitoneal rupture of the urinary bladder

We report a case of generalized peritonitis caused by spontaneous intraperitoneal rupture of the urinary bladder. A 74-year-old female was admitted with abdominal pain and biochemical findings of acute renal failure (ARF). She had recently complained of macrohematuria. She had a past history of radiotherapy for uterine cervical cancer and Parkinson's disease treated with levodopa and amantadine. We diagnosed this case as intraperitoneal rupture of the bladder by cystogram. Biochemical findings of ARF might have resulted from urine reabsorption. Intraperitoneal rupture of the bladder should be considered in all cases of peritonitis, especially in patients with urological symptoms and features of ARF.     

Impact of aging on rat urinary bladder fatigue

PURPOSE: Little is known about the fatigability of the urinary bladder. In these experiments, we characterized contractile and bioenergetic changes in bladder fatigue and investigated the impact of aging on these changes. MATERIALS AND METHODS: Whole urinary bladders from 3-month-old (n = 17) and 24-month-old (n = 12) SD rats were isolated and individually mounted in organ baths. The bladders were electrostimulated repeatedly (50 volts, 32 Hz, 1 MS; every 2.5 minutes). The pressure generation, rate of pressure generation and the emptying ability (% volume emptied) of the isolated bladders were measured with each stimulation. After the 20th electrostimulation, the bladders were immediately stimulated with 500 microM bethanechol. Upon completion of their series of stimulations, some of the bladders were quickly frozen in liquid nitrogen. Tissue phosphocreatine and ATP content of the frozen bladders and a group (six 3-month-old and six 24-month-old rats) of fresh bladder tissues was determined using high performance liquid chromatography (HPLC). RESULTS: The results can be summarized as follows: (1) Pressure generation, rate of pressure generation and emptying ability were gradually reduced in both young and aged bladders as repeated stimulation proceeded. (2) The final bethanechol stimulation emptied the same intravesical volume as the 20th electrostimulation emptied (in both groups), indicating that bladder fatigue is due to a post-synaptic mechanism. (3) As compared to their own first responses, aged rats exhibited significantly greater rates of reduction in both pressure generation and emptying ability than did young rats. (4) Analysing fresh bladder tissues, the phosphocreatine and ATP concentration of the aged bladders were significantly less than those of the young bladders-13.2 +/- 2.0 and 1.2 +/- 0.3 nmol/mg. protein respectively in the aged bladders vs. 21.2 +/- 1.8 and 7.5 +/- 1.0 nmol/mg. protein respectively in the young bladders. After repeated stimulation, phosphocreatine and ATP concentration were reduced in both groups (1.4 +/- 0.3 and 0.43 +/- 0.1 nmol./mg. protein in the aged bladders, 7.5 +/- 1.4 and 4.1 +/- 0.5 nmol./mg. protein in the young bladders), with a greater degree of reduction in the aged bladders. CONCLUSION: These observations indicate that, in response to repeated electrostimulation, aged rat bladders became fatigued faster than young bladders. Decreased capability in energy production might be one contributing factor for faster fatiguability of the aged urinary bladders.     

Structural characterization of the O-antigenic polysaccharide of Escherichia coli serotype 017 lipopolysaccharide

Transverse nuclear magnetic relaxation and self-diffusion of water were measured in hydrated collagen II. Self-diffusion measurements were conducted by pulsed field gradient NMR (PFG NMR) and weighting of the different species in the signal by variable T2 relaxation in the experiment. Two fractions of water protons were detected, one with a short T2 value but high diffusivity and one with a long T2 value and low, completely restricted diffusion. The distance of the diffusion barriers was determined to be 2.3 microns. Possible reasons for the restriction in the movement of the water molecules in comparison with structural models of collagen II are discussed.     

Enhancement of rat urinary bladder tumorigenesis by lipopolysaccharide-induced inflammation

Chronic inflammation of the urinary tract is a significant risk factor for the development of urinary bladder cancer in humans. We previously demonstrated that weekly treatment with killed Escherichia coli enhanced rat urinary bladder tumorigenesis initiated by the carcinogen N-methyl-N-nitrosourea. We conducted the present study to determine whether lipopolysaccharide (LPS), a major cell wall component of E. coli, had a tumor-enhancing effect. LPS was instilled twice a week at three doses (100, 1.0, and 0.01 microgram/ml) into heterotopically transplanted rat urinary bladders which were treated with a single low dose (0.25 mg) of N-methyl-N-nitrosourea or vehicle. Rats treated with 100 micrograms/ml of LPS showed a significant increase in the incidence and number of tumors in the bladders pretreated with N-methyl-N-nitrosourea. Treatment with LPS alone did not induce tumors. The enhancing effects were associated with a marked increase in the numbers of polymorphonuclear leukocytes and an increase in the H2O2 concentration in the bladder lumen. Oxidative stress by reactive oxygen intermediates and a proliferative response of the carcinogen-exposed urothelium to the inflammatory stimulation appeared to play a significant role in tumor enhancement by LPS.     

Purinergic modulation of rat urinary bladder detrusor smooth muscle

This paper reviews the use of economic evaluations in the Swedish health care system. The most important actors are defined and examples are given how economic evaluations have played a role in the decision making process. The introduction of extracorporal shock-wave lithotripsy (ESWL) is used as an example on how economic evaluation was used for recommendations to the county councils to adopt this technology. Mammography is used as an example of how the evaluation is used in the political process following an initiative in the parliament. A study of the cost-effectiveness of hypertension treatment illustrates how economic evaluations are included as part of a medical technology assessment by SBU, the Swedish Council on Technology Assessment in Health Care. The role of economic evaluations for drug reimbursement and pricing is also reviewed. The main conclusions are that economic evaluations are one of several factors influencing a decision making process that have a strong strive for consensus. It is thus difficult to make a definitive statement of the contribution of such study to the outcome of the decision making process, and there is no evidence that the evaluation was the decisive factor. However, a number of changes in the way resources are allocated in the Swedish health care system speaks for an increasing role for such studies in the future. The county councils are identified as the main target for economic evaluations, and SBU has a key role in supplying the county councils with high quality assessment of new and old technologies.     

Regulation of beta-chemokine mRNA expression in adult rat astrocytes by lipopolysaccharide, proinflammatory and immunoregulatory cytokines

Methotrexate (MTX) is one of the most widely prescribed drugs in the treatment of rheumatoid arthritis (RA). The mechanism by which MTX exerts its anti-rheumatic effect has not yet been defined. The aim of the present study was to investigate the effect of MTX treatment (7.5-15 mg/week) on synovial tissue in RA. For this purpose, synovial biopsies were taken from 11 RA patients before and 16 weeks after initiation of MTX therapy. Immunohistochemistry was performed using monoclonal antibodies (MAb) specific for CD3, CD4, CD8, CD22, CD25, CD38, CD68, MAb67, Ki67, interferon gamma (IFN-gamma), interleukin (IL)-1alpha, IL-1beta, tumour necrosis factor alpha (TNF-alpha), E-selectin, ICAM-1 and VCAM-1. All parameters for disease activity improved during the period of treatment. Immunohistochemical analysis revealed a statistically significant decrease in scores for CD3, CD8, CD38, CD68, Ki67, IL-1beta, TNF-alpha and the adhesion molecules E-selectin and VCAM-1. The observed decrease in synovial scores for inflammatory cells, monokines and adhesion molecules suggests that the anti-inflammatory effect of MTX is, in part, dependent on a reduction in monokine-inducible vascular adhesion molecules and subsequent reduction of cell traffic into joints.     

Evaluation of autologous fat implantation in the rat urinary bladder submucosa

OBJECTIVES: Autologous fat has been used as a bulking implant material for stress urinary incontinence. There is considerable controversy as to the ultimate fate of the grafted fat. This study was conducted to determine the fate of autogenous fat implanted into the bladder of rats. METHODS: Two groups of adult female rats were studied. In the test animals (group 1, n = 20), mesenteric adipose tissue (1 mL) was harvested and homogenized with an equal weight of sterile saline. Using a 25-gauge needle, 0.5 g of saline-fat mixture was injected into the dorsal bladder neck submucosa. Control animals (group 2, n = 12) were injected with sterile saline only. A subset of animals from each group were killed after 7, 35, 105, and 1 50 days, and the bladder and urethra were fixed. The fixed tissue was examined microscopically and photographed at each follow-up period. RESULTS: Seven days after injection of fat, there was a pronounced acute inflammatory reaction with numerous polymorphonuclear leukocytes and macrophages at the site of fat injection. There was minimal inflammatory reaction at the site of saline injection. By day 35, most of the fat had been eliminated by these phagocytes because of severe acute and chronic inflammation. By day 105, the submucosa tissue of the experimental rats had returned to normal visually and to a flat surface, lacking the appearance of a sizable "bulge" as shown at days 0 and 7. Histopathologic findings were also similar to the control rats. Inflammatory cells were no longer present by day 105. CONCLUSIONS: The implantation of homogenized, autologous fat in the rat urinary bladder submucosa causes acute and chronic inflammation and fat necrosis. The severe phagocytosis at the implant sites eliminates the vast majority of the devitalized implanted fat during the first month.     

Ten-year survey of quinolone resistance in Escherichia coli causing urinary tract infections

Two patients with central nervous system manifestations of Langerhans cell histiocytosis, both with brain stem involvement, are reported. The onset of symptoms was at an age when the diagnosis might not have been considered.     

Recombinant expression of rat and human Gro proteins in Escherichia coli

A full-length rat gro cDNA containing the signal sequence was inserted to a plasmid/phage vector pTD-lacs which had the Escherichia coli alkaline phosphatase leader sequence down-stream of the lac promoter. After removal of the gro signal sequence by site-directed mutagenesis, the vector was introduced to E. coli JM109. The cells grown in the presence of isopropyl beta-D-thiogalactopyranoside were found to contain the recombinant mature rat Gro protein in the periplasmic space. The protein was released from the cells by osmotic shock, and could be purified to homogeneity from the periplasmic fluid by a single-step procedure using reverse phase high performance liquid chromatography. By similar procedures, recombinant human Gro alpha could be obtained. In each case, about 10 mg of purified cytokine were obtained from 1 litre of bacterial culture.     

Frequency of pap and pil operons in Escherichia coli strains associated with urinary infections

The influence of reproductive variables on cervical cancer incidence, controlling for other sociodemographic factors, was estimated in Norwegian register and census data, using Poisson regression models. Among the 1.3 million women under observation, a total of 2,870 cases of cervical cancer were diagnosed. According to models restricted to parous women, parity level had no independent impact on cervical cancer incidence, but a clear effect of age at first birth was noted. It was most pronounced in the squamous cell carcinomas, where the incidence was reduced by 48 percent from age at first birth < 21 years to age at first birth 27+ years. Women without children had the same cervical cancer incidence as parous women with a first birth after age 24. The sociodemographic variables controlled for exerted a strong net effect on the cervical cancer incidence. Educational level was related inversely to the cancer risk. Moreover, an increased risk was seen for women who had given birth when they were still single (never married) and for those who were divorced/separated at the time of the last previous census. A fairly small excess risk was found to be associated with living in non-rural compared with rural areas.     

Lysosomal cysteine and aspartic proteinases and ubiquitin in rat and human urinary bladder epithelium

To examine localization of cysteine and aspartic proteinases, and ubiquitin in rat and human urinary bladders, immunocytochemistry was applied to the tissues. In semi-thin sections, immunoreactivity for cathepsins B and D was densely localized throughout epithelial layers of rats and humans, while that for cathepsins H and L was mainly localized in rat superficial and human intermediate cells. Immunoreactivity for cathepsin C was relatively high in rat and human epithelia, especially in humans. Immunoreactivity for ubiquitin was detected in rat and human epithelial cells. By electron microscopy, vesicular or heterogeneously dense lysosomes labeled with immunogold particles indicating cathepsin B were seen in rat and human epithelial cells; particularly, they often appeared near fusiform vesicles in rat superficial cells and in human intermediate and superficial cells. By double immunostaining, lysosomes with or without vesicular structures were co-labeled with immunogold particles showing both cathepsin B and ubiquitin. The results suggest that cathepsins B, C, H, and L, and cathepsin D are involved in the lysosomal system of rat and human bladder epithelia. Moreover, considering that ubiquitin is a cofactor in the soluble ATP-dependent proteolysis, the results may also indicate that epithelial cells actively form autophagolysosomes.     

Hypertrophic neurons innervating the urinary bladder and colon of the streptozotocin-diabetic rat

Female rats were made diabetic with an intravenous injection of streptozotocin (STZ) producing bladder hypertrophy. Using fluorescent dyes injected into the bladder or the colon, we have measured the size of neurons in various ganglia associated with these organs in control and STZ-diabetic rats. These include (1) postganglionic neurons in the pelvic ganglion, (2) postganglionic neurons in the inferior mesenteric ganglion, (3) dorsal root ganglion neurons, (4) sympathetic chain ganglion neurons, (5) preganglionic neurons in the sacral parasympathetic nucleus, (6) motor neurons in Onuf's nucleus innervating the external urethral sphincter. In addition we have measured neurons in some of these groups for rats which have been maintained on a 5% sucrose in water and restricted food diet. In the STZ-diabetic animals only those neurons which make direct contact with the bladder or the colon were found to be hypertrophied (15-70%). In the diuretic animals, only neurons directly innervating the bladder exhibited hypertrophy. We speculate that a trophic factor transported from the organ to the neuron is responsible for this effect.     

Light and electron microscopic observations on rat molar periodontal ligament after intraperitoneal injection of vinblastine

Suramin is a polyanionic compound with potent antineoplastic properties. Because polymorphonuclear leukocytes (PMNs) are a crucial component of host defenses against bacteria and fungi, the effects of suramin on PMN function were studied in vitro. PMNs from healthy donors were incubated with concentrations of suramin of 1 to 1,000 micrograms/ml (within and exceeding the therapeutic range) for 30 min, and PMN functional parameters were subsequently assessed. Suramin had no effect on viability, chemotaxis to N-formylmethionyl leucyl phenylalanine, phagocytosis of Candida albicans, or superoxide anion production in response to phorbol myristate acetate and formylmethionyl leucyl phenylalanine. Fungicidal activity against C. albicans blastoconidia was unaffected at a suramin concentration of < 500 micrograms/ml, whereas at higher concentrations a slight suppression was observed (P = 0.04). Bactericidal activity against Staphylococcus aureus was significantly suppressed by concentrations of > or = 100 micrograms/ml (P < 0.01). Phagocytosis of S. aureus was also significantly impaired at > or = 10 micrograms/ml (P < 0.05). The presence of 10% human serum during pretreatment did not abrogate the suramin-induced suppression of bactericidal activity. Treatment of PMNs with granulocyte colony-stimulating factor (4,000 U/ml) for 30 min prior to the addition of suramin (250 micrograms/ml) improved the bactericidal defect (P = 0.02). The PMN functional impairment may be related to increased susceptibility to bacterial infections, and granulocyte colony-stimulating factor may improve the defect.