The role of intraoperative cystoscopy in prolapse and incontinence surgery.

OBJECTIVE: Unrecognized ureteral and bladder injury are a known source of morbidity and mortality in gynecologic surgery. The objective of this study was to determine the frequency that intraoperative cystoscopy during prolapse and incontinence procedures produced a change in intraoperative management to prevent ureteric and bladder injury. STUDY DESIGN: This study reviewed the charts and operative reports of 235 cases of routine intraoperative cystoscopy during prolapse and incontinence surgery during a 2-year period in a tertiary care urogynecology unit. Demographic data and potential risk factors for intraoperative urinary tract injury were recorded. Cases that involved a change in management brought about by intraoperative cystoscopy with intravenous indigo carmine were compared with cases in which intraoperative cystoscopy was normal. Variables were compared with use of the Student t test and the chi(2) test. RESULTS: Of 235 cases, 11 were excluded. Of the 224 remaining cases, 12 (5.3%) underwent changes in intraoperative management as a result of cystoscopic findings. Eight cases involved ureteric blockage. Patients with abnormal cystoscopies did not differ from patients with normal intraoperative findings with regard to age, weight, parity, maximum grade of prolapse, estimated blood loss, or previous surgery. In 58% of patients with abnormal cystoscopies, there was no suspicion of technical difficulty on the basis of previous surgical history. Preoperative renal imaging did not predict cases with abnormal cystoscopy. There were no cases of complications caused by the intraoperative cystoscopy. CONCLUSION: Intraoperative cystoscopy with intravenous injection of indigo carmine is a safe technique that can detect otherwise undetected intraoperative compromise of the urinary tract during prolapse and incontinence surgery. It is recommended that cystoscopy be used liberally to reduced the frequency of serious sequelae from urinary tract injury.

Urge incontinence and detrusor instability.

Detrusor instability is a syndrome of urinary frequency, urgency and urge incontinence which can be demonstrated using urodynamic studies to document uninhibited bladder contractions. Idiopathic cases account for 90% and 10% are related to neurologic disorders. Several different treatment modalities are available, including bladder training/drill, electrical stimulation, medical and surgical therapies.

Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells.

The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4(+) and CD8(+) T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4(+) T cells. First, CD4(+) cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4(+) cells and production of IFN-gamma, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4(+) T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4(+) T cells produced more IFN-gamma and less IL-4 and IL-10 than CD4(+) cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.

Autoantigen-responsive T cell clones demonstrate unfocused TCR cross-reactivity toward multiple related ligands: implications for autoimmunity.

It has been suggested that the cross-reaction of a single T cell receptor with multiple different peptide ligands is a mechanism for maintaining a diverse yet compact immune repertoire. In the context of autoimmune disease it is important to understand how this property is balanced against the maintenance of self-tolerance. Specifically, whether the cross-reactivity inherent in the immune system is focused or unfocused will have important consequences for the development of autoimmune disease. If cross-reactivity is "focused," then in an immune response to a foreign antigen all T cell receptors that recognize the foreign antigen will cross-react with a specific autoantigenic peptide. However, if cross-reactivity is "unfocused," an immune response to a foreign antigen will result in the activation of a small number of self-reactive cells within a larger pool of cells specific for the foreign antigen. We have tested whether cross-reactivity is focused or unfocused by generating a panel of T cell clones that respond to two closely related ligands. W144 is an autoantigenic peptide of myelin proteolipid protein, PLP 139-151 (HSLGKWLGHPDKF), and Q144 is an altered peptide of PLP 139-151 bearing a glutamine for tryptophan substitution at position 144. The Q144-responsive clones have a broad degree of cross-reactivity with other position 144 substituted peptides. We find that despite their characteristic responses to Q144 and W144, the patterns of responses of these clones to other structurally related ligands are random, demonstrating that cross-reactivity is unfocused in the absence of selection. Maintaining a diverse range of cross-reactive interactions may limit nonspecific responses to autoantigens.

Studies in B7-deficient mice reveal a critical role for B7 costimulation in both induction and effector phases of experimental autoimmune encephalomyelitis.

The importance of B7 costimulation in regulating T cell expansion and peripheral tolerance suggests that it may also play a significant regulatory role in the development of autoimmune disease. It is unclear whether B7 costimulation is involved only in the expansion of autoreactive T cells in the periphery, or if it is also required for effector activation of autoreactive T cells in the target organ for mediating tissue injury and propagating autoimmune disease. In this study, the role of B7-CD28 costimulation and the relative importance of B7 costimulators for the induction and effector phases of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) peptide were examined. Wild-type, B7-1/B7-2-deficient mice, or CD28-deficient C57BL/6 mice were immunized with MOG 35-55 peptide. Mice lacking both B7-1 and B7-2 or CD28 showed no or minimal clinical signs of EAE and markedly reduced inflammatory infiltrates in the brain and spinal cord. However, mice lacking either B7-1 or B7-2 alone developed clinical and pathologic EAE that was comparable to EAE in wild-type mice, indicating overlapping functions for B7-1 and B7-2. Resistance to EAE was not due to a lack of induction of T helper type 1 (Th1) cytokines, since T cells from B7-1/B7-2(-/-) mice show reduced proliferative responses, but greater interferon gamma production compared with T cells from wild-type mice. To study the role of B7 molecules in the effector phase of the disease, MOG 35-55-specific T lines were adoptively transferred into the B7-1/B7-2(-/-) and wild-type mice. Clinical and histologic EAE were markedly reduced in B7-1/B7-2(-/-) compared with wild-type recipient mice. These results demonstrate that B7 costimulation has critical roles not only in the initial activation and expansion of MOG-reactive T cells, but also in the effector phase of encephalitogenic T cell activation within the central nervous system.

Plasma levels of high-energy compounds compared with severity of illness in critically ill patients in the intensive care unit.

BACKGROUND: Plasma metabolic changes have been shown to reflect deterioration of the energy state of tissue in studies in animals. This study evaluates whether high-energy compounds and their metabolites in plasma reflect the clinical condition and predict outcome in critically ill patients. METHODS: Thirteen critically ill patients with major trauma, severe septic shock, or cardiogenic shock (initial Acute Physiology and Chronic Health Evaluation [APACHE] II score > or = 16) were studied. The APACHE II score was recorded daily until discharge from the intensive care unit or death. The plasma contents of adenosine triphosphate, adenosine diphosphate, adenosine, inosine, hypoxanthine, creatine phosphate, creatine, uric acid, and lactic acid were determined daily. Fifteen healthy volunteers were used as control subjects. RESULTS: All patients with an APACHE II score of 12 or less at some time during their stay in the intensive care unit survived (n = 4); all patients with a score of 26 or higher died (n = 5). The initial APACHE II median score for survivors was 21 (range 16 to 25; n = 7) and for nonsurvivors 24 (range 17 to 28; n = 6) (difference not significant). The final APACHE II score for the survivors was 11 (range 3 to 16) and for nonsurvivors 29 (range 20 to 47) (p < 0.01). The plasma metabolites were grouped according to the patients' APACHE II score of the day. There was a positive correlation between the severity of metabolic derangement and the APACHE II score. The plasma contents of adenosine triphosphate and creatine phosphate were depleted with higher APACHE II scores (p < 0.01), whereas creatine and uric acid levels increased progressively (p < 0.001). The levels of adenosine, inosine, hypoxanthine, and lactic acid were elevated significantly in critically ill patients. CONCLUSIONS: Grouping patients with successively higher APACHE II scores revealed specific patterns of altered plasma metabolism, possible reflecting different levels of tissue adenylate energy charge. However, neither the initial individual APACHE II score nor any initial plasma metabolic level had any prognostic value in this group of critically ill patients, although the deterioration of the physiologic parameters was coexistent with specific metabolic changes.

Breakdown of adenine nucleotides, formation of oxygen free radicals, and early markers of cellular injury in endotoxic shock.

OBJECTIVE: To study the influence of shock on muscle and plasma adenine nucleotide and creatine pools and their metabolites, and to identify early markers of cellular injury in shock. SETTING: Surgical research laboratory, Kuwait and UAE. DESIGN: Experimental study. MATERIAL: 19 New Zealand rabbits. INTERVENTIONS: 15 rabbits were injected with Escherichia coli endotoxin, and an additional 4 rabbits acted as controls. MAIN OUTCOME MEASURES: Blood and muscle energy metabolites, platelet count, arterial blood gas tensions, and arterial pressure were followed until the animals died. RESULTS: Five minutes after injection of endotoxin muscle ATP, creatine phosphate, and total adenine purine concentration decreased. This decrease was later reversed, but again decline to a critical level in the terminal phase. Loss of the muscle creatine pool indicated cellular damage after 3 hours. Plasma hypoxanthine, creatine, and lactate concentrations increased continuously throughout the study. CONCLUSION: Hypoxanthine formation is a possible source of oxygen free radicals in shock. The rise of hypoxanthine, creatine, and lactate concentrations in plasma during septic shock may reflect early high energy nucleotide failure, membrane injury, and anaerobic metabolism, respectively.

Crystallization of the calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli K12.

Single crystals of phosphoenolpyruvate carboxykinase from Escherichia coli K12 have been grown in the orthorhombic crystal system. Single crystals developed to a maximum size of 0.25 mm x 0.25 mm x 1.5 mm by the technique of washing and reseeding. The space group is P2(1)2(1)2(1), with a = 77.24 A, b = 89.18 A, c = 93.24 A and Z = 4; there is one enzyme molecule per crystallographic asymmetric unit and the solvent content is estimated to be 59%. The crystals diffract to at least 2.8 A d spacings and decompose in the X-ray beam after approximately two days of exposure.

Adenosine, inosine, and hypoxanthine/xanthine measured in tissue and plasma by a luminescence method.

This simple method for sequentially quantifying hypoxanthine (HYP), inosine (INO), and adenosine (ADN) concentrations exploits the H2O2 peroxidase-catalyzed chemiluminescence of luminol. Though applied here only to tissue and plasma, this method can be adapted to analyze other body fluids. HYP in human plasma was stable for 30 min in 10 mmol/L EDTA reagent, whereas ADN was slowly converted to INO. Analytical recovery of HYP and INO added to plasma was 102% each; that of ADN was 95%. The within-run mean CVs for determinations of HYP, INO, and ADN at 1 mumol/L were 3.46%, 2.65%, and 3.01%; at 10 mumol/L they were 2.16%, 1.88%, and 1.63%, respectively. Corresponding between-run CVs were 5.34%, 4.09%, and 4.17%; and 3.43%, 2.40%, and 2.88%, respectively. Bilirubin at a concentration greater than 50 mumol/L interferes, but this interference is eliminated by bilirubin oxidase. Results for both tissue and plasma are compared with previously published results based on different analytical methods.

Plasma metabolic disturbances and reperfusion injury following partial limb ischaemia in man.

Despite efficient revascularisation procedures for vascular disease, the limb can occasionally be lost following reperfusion. One contributing factor might be the formation of oxygen free radicals. This study attempts to describe the conditions necessary for oxy-radical formation from adenine nucleotide breakdown products and the role of plasma creatine content as a marker of cellular injury. Twelve patients undergoing aortic reconstructive surgery were studied. Only partial ischaemia of the lower limbs was induced by the aortic clamping, since varying degrees of collateral circulation existed. Radial arterial and external iliac venous blood was obtained simultaneously before, during and after cross-clamping of the aorta, and plasma levels of ATP, ADP, hypoxanthine, phosphocreatine, creatine, creatinine and lactate measured using luminescence and spectrophotometry. Venous creatine content increased during ischaemia and was doubled 30 min after recirculation. This increase was possibly due to leakage following cellular injury agreeing with a previously observed decrease in muscle tissue creatine content. The iliac arterio-venous difference of hypoxanthine and lactate markedly increased immediately post-ischaemia, while the phosphocreatine difference decreased. Plasma hypoxanthine was abundant in the leg on reoxygenation. The existence of a xanthine oxidase system in skeletal muscle could produce favourable conditions for oxy-radical formation through hypoxanthine degradation, which may contribute to the known muscle tissue injury.

Plasma creatine determination using a luminescence method.

A new luminescence procedure based on the creatine kinase reaction was developed for measuring creatine in plasma. The method is highly applicable to small animal work where the amount of blood volume is critical. Only 20 microliter of sample is necessary for creatine analysis. Deproteinizing the plasma sample with ethanol at room temperature is convenient. This extraction method is adaptable to a clinical setting. The ethanol used in the extraction is compatible with the luminescence method but precipitated enzymes in the NADH spectrophotometric method because of the greater sample volume needed for analysis. The creatine concentration is stable in plasma for at least 1 hr in a final anticoagulant concentration of 10 mM EDTA. The correlation between the new luminescence method with the established NADH spectrophotometric method was excellent (r = 0.99). The accuracy of the within-run precision is high, with a mean coefficient of variation, 2-3%. Plasma creatine levels could be an important indicator denoting early cellular damage and of potential prognostic value. Preliminary studies in human muscle ischemia and early shock in rabbits revealed a significant increase in plasma creatine levels. Further investigations are necessary to evaluate its clinical importance.

Comparative study of blood clotting factors in anaphylactic and primary and secondary endotoxin shock.

Cinephotomicrography shows emboli are formed in the lung during stages of anaphylactic and endotoxin shock and platelet counts are decreased. Rabbits in anaphylactic shock had a 50 percent mortality while 100 percent mortality was associated with endotoxin shock. During the terminal phase, the platelet count recovers in anaphylactic shock but not in endotoxin shock. This suggests a difference in the emboli formed during these two forms of shock. This study was directed at determining what might be the difference. Periodic measurement of electrocardiographic tracings, fibrinogen, factor VIII and fibrin degradation products (FDP) were made using standard equipment and test kits. The results of the study showed no changes in any of the measured clotting factors during anaphylactic shock. However, in endotoxin shock FDP appeared after seven to 10 hours, fibrinogen levels decreased from 221 mg per dl to 85 mg per dl and factor VIII time increased significantly. From these results, it is concluded that platelet aggregation in anaphylactic shock does not involve fibrin deposition, whereas in endotoxin shock platelet aggregation and fibrin deposition are both a factor in the formation of the emboli, possibly explaining the irreversible aggregation observed in terminal endotoxin shock.

Plasma changes in endotoxin and anaphylactic shock (ATP, ADP and Creatine phosphorus).

Decisive patterns have been demonstrated in plasma adenosine 5' triphosphate (ATP) levels in both endotoxin and anaphylactic shock which correlate with periods of low platelet counts, low arterial pressures and abnormal electrocardiograms. When these irregularities were occurring, the plasma ATP level was low; when improvement occurred, the plasma ATP level rose. Plasma ATP levels appear to be an index to the metabolic state of the animal. The plasma creatine phosphate (CP) level showed a tendency to decrease when the ATP level dropped in anaphylactic shock, although the CP level did not recover to the same extent as the ATP level. In endotoxin shock, the plasma CP level increased on an average of six-fold. It is proposed that this rise resulted from either CP mobilization from tissues, for the purpose of replenishing the energy deficient myocardial muscle, or possible leakage from damaged cells. Adenosine diphosphate (ADP) plasma values were measured in both anaphylactic and endotoxin shock. High initial ADP values were prone towards a more severe anaphylactic reaction and a shorter survival time in the endotoxin shock experiments.

Plasma ADP levels: direct determination with luciferase luminescence using a biometer.

A method is described for the determination of low plasma levels of adenosine-5'-diphosphate (ADP) using a Dupont Biometer to measure luminescence produced by the luciferin-luciferase reaction. Endogenous ATP is removed by incubation with luciferase. The remaining ADP is then quantitated, following its conversion to ATP, after incubation with creatine phosphate and creatine kinase. The mean coefficient of variation for 0.02 and 2.2 micromol/liter ADP standards were 2.1 and 1.8% respectively. The method has been applied to human and rabbit plasma. Human plasma ADP concentrations were found to be 0.13 +/- 0.025 (10) micromol/liter and rabbit plasma concentration were 0.07 +/- 0.05 (5) micromol/liter. Several other possible applications of the method are discussed.

Microdetermination of plasma ATP and creatine phosphate concentrations with a luminescence biometer.

We optimized conditions for determination of adenosine-5'-triphosphate (ATP) and creatine phosphate from plasma extracted with ethanol/water (96/4 by vol). The procedures utilize the firefly luciferin/luciferase reaction, the bioluminescence being measured with a Du Pont Biometer. ATP is quantitated directly and creatine phosphate is quantitated by reaction with creatine kinase and ADP, after plasma ATP is removed by incubation with the enzyme apyrase. The method is applied to plasma from humans, rabbits, and rats, and possible clinical applications are discussed.