Cultivation of a Synergistetes strain representing a previously uncultivated lineage.

Subgingival plaque samples obtained from human subjects with periodontitis, shown to include previously uncultivable members of the phylum Synergistetes, were used to inoculate Cooked Meat Medium (CMM). The presence of Cluster A (uncultivable) Synergistetes was monitored by fluorescent in situ hybridization (FISH) and quantitative PCR (Q-PCR). Cluster A Synergistetes were found to grow in CMM in co-culture with other plaque bacteria and growth was stimulated by the addition of mucin and serum. Plaque samples were also used to inoculate Blood Agar (BA) plates and growth of Cluster A Synergistetes was revealed after anaerobic incubation, by colony hybridization with specific probes. Surface growth on the plates in regions identified by colony hybridization was harvested and used to inoculate fresh plates, thus enriching for Cluster A Synergistetes. Cross-streaks of other plaque bacteria were also used to stimulate Synergistetes growth. In the early passages, no discrete Synergistetes colonies were seen, but after eight passages and the use of cross-streaks of other bacteria present in the enriched community, colonies arose, which consisted solely of Cluster A Synergistetes cells, as determined by 16S rRNA gene PCR and cloning. This is the first report of the successful culture of a member of the uncultivable branch of this phylum.

Diversity and morphology of members of the phylum "synergistetes" in periodontal health and disease.

Members of the phylum "Synergistetes" have frequently been detected in the human oral cavity at sites of dental disease, but they have rarely been detected in studies of oral health. Only two oral "Synergistetes" taxa are cultivable. The aims of this study were to investigate the diversity of "Synergistetes" in the oral cavity, to establish whether "Synergistetes" taxa are more strongly associated with periodontitis than with oral health, and to visualize unculturable "Synergistetes" in situ. Sixty samples (saliva, dental plaque, and mucosal swabs) were collected from five subjects with periodontitis and five periodontally healthy controls. Using phylum-specific 16S rRNA gene primers, "Synergistetes" were identified by PCR, cloning, and sequencing of 48 clones per PCR-positive sample. Subgingival plaque samples were labeled with probes targeting rRNA of unculturable oral "Synergistetes" using fluorescent in situ hybridization (FISH). Analysis of 1,664 clones revealed 12 "Synergistetes" operational taxonomic units (OTUs) at the 99% sequence identity level, 5 of which were novel. "Synergistetes" OTU 4.2 was found in significantly more subjects with periodontitis than controls (P = 0.048) and was more abundant in subgingival plaque at diseased sites than at healthy sites in subjects with periodontitis (P = 0.019) or controls (P = 0.019). FISH analysis revealed that unculturable oral "Synergistetes" cells were large curved bacilli. The human oral cavity harbors a diverse population of "Synergistetes." "Synergistetes" OTU 4.2 is associated with periodontitis and may have a pathogenic role.

Periodontal tissue engineering and regeneration: Consensus Report of the Sixth European Workshop on Periodontology.

AIM: To review the scientific preclinical background and clinical studies of current methods of periodontal regeneration in the treatment of infrabony defects and soft tissue deficiencies. METHOD: Five commissioned review papers including two systematic reviews were scrutinized by a group of experts in order to derive consensus conclusions, clinical relevance/implications and to propose future research requirements. RESULTS: The following five papers were assessed: 1. Biological mediators and periodontal regeneration: a review of enamel matrix proteins at the cellular and molecular levels. 2. Regeneration of periodontal tissues: combination of barrier membranes and grafting materials - Biological foundation and preclinical evidence. 3. Clinical outcomes with bioactive agents alone or in combination with grafting or GTR 4. Treatment of gingival recession with coronally advanced flap procedures. A systematic review. 5. Soft tissue management at implant sites.

Immediate and early replacement implants and restorations.

There have been rapid developments in dental implant treatment protocols to reduce the time between implant placement and restoration. Implants may be placed immediately following tooth extraction or following a period of healing to allow resolution of residual infection or sufficient bone and soft tissue healing. Early restoration and loading of implants has to be carefully controlled to avoid increased failure and complications. Advantages and disadvantages of the various techniques are described.

Effects of inhibition of nitric oxide formation on basal vasomotion and endothelium-dependent responses of the coronary arteries in awake dogs.

The role of nitric oxide in basal vasomotor tone and stimulated endothelium-dependent dilations in the coronary arteries in chronically instrumented awake dogs was studied by examining the consequences of inhibiting endogenous nitric oxide formation with the specific inhibitor of nitric oxide formation, NG-monomethyl-L-arginine (L-NMMA). In four awake dogs, coronary dimension crystals were chronically implanted on the circumflex artery for the measurement of epicardial coronary diameter, and Doppler flow probes were implanted for quantitation of phasic coronary blood flow (vasomotion of distal regulatory resistance vessels). Basal epicardial coronary diameter, acetylcholine-stimulated endothelium-dependent dilation, and flow-induced endothelium-dependent dilation of the epicardial arteries and phasic blood flow were recorded before, and after 5, 15, 50, and 120 mg/kg of L-NMMA. L-NMMA induced a dose-related increase in basal epicardial coronary vasomotor tone. There was an accompanying increase in aortic pressure and a decrease in heart rate. At doses greater than or equal to 50 mg/kg, rest phasic coronary blood flow was also decreased. Left ventricular end-diastolic pressure and contractility were not significantly changed. In contrast, the flow-induced or acetylcholine-stimulated endothelium-dependent responses were attenuated only after infusion of the highest does of L-NMMA (120 mg/kg). The changes in the basal vasomotor tone and acetylcholine-stimulated endothelium-dependent responses returned towards the control states in the presence of L-arginine (660 mg/kg). These data support the view that nitric oxide plays a significant role in modulating basal vasomotion and endothelial-dependent dilation stimulated by acetylcholine or increase in blood flow in epicardial coronary arteries and also influence the regulation of coronary blood flow during physiologic conditions.

Breakaway forces of flat and domed surfaced Magfit implant magnet attachments.

The aim of this in vitro study was to compare the resistance to separation (breakaway force) of flat (Magfit-IP-BF) and domed (Magfit-IP-BD) implant magnet attachments subjected to non-axial forces. The separating forces were applied by an Instron Universal Testing Instrument to single magnet attachments at angles of 0 degrees, 2 degrees, 5 degrees 10 degrees, and 20 degrees from the axial line of the components (angle of pull) and at crosshead speeds of 0.5 mn/min and 5 degrees mm/min. The breakaway forces were significantly (p < 0.0001) inversely related to the angle of pull for both flat magnets and for domed magnets. At the slow crosshead speed, the breakaway forces recorded for the domed magnets were significantly greater than those recorded for the flat magnets for angles of pull greater than 5 degrees. At the faster crosshead speed, the breakaway forces recorded for the domed magnets were significantly greater than those recorded for the flat magnets for angles of pull greater than 2 degrees. This apparent superiority of domed magnets under non-axially directed separating forces could influence the choice of magnet attachment for implant overdentures as intraoral displacing forces are multidirectional. Domed magnets may also be advantageous where implants are not parallel.

Pilot study of the efficacy of renzapride on gastrointestinal motility and symptoms in patients with constipation-predominant irritable bowel syndrome.

AIM: To investigate the efficacy and safety of renzapride, a potent 5-hydroxytryptamine type-4 receptor full agonist and 5-hydroxytryptamine type-3 receptor antagonist in patients with constipation-predominant irritable bowel syndrome. METHODS: In this dose-escalating pilot study, 17 patients with constipation-predominant irritable bowel syndrome received placebo, renzapride 2 mg o.d. and renzapride 2 mg b.d. sequentially for 28 days. Response was determined by radio-opaque marker measurement of overall gastrointestinal and segmental colonic transit and patients' assessment of their irritable bowel syndrome symptoms. RESULTS: Renzapride reduced mean overall gastrointestinal transit time (placebo, 2.9 +/- 1.6 days; renzapride 2 mg o.d., 2.6 +/- 1.4 days; renzapride 2 mg b.d., 1.9 +/- 1.6 days) (P = 0.024) and accelerated segmental colonic transit, with statistically significant differences for renzapride 2 mg b.d. over placebo in caecum/ascending colon (P = 0.019) and descending colon (P = 0.022). Renzapride also reduced abdominal pain, increased the number of pain-free days and improved stool consistency. The frequency of reported adverse events was similar on renzapride and placebo. CONCLUSIONS: Renzapride is well-tolerated, stimulates gastrointestinal transit and improves symptoms in patients with constipation-predominant irritable bowel syndrome, particularly at the 2 mg b.d. dose, where improvements in gastrointestinal symptoms were evident over placebo. This study has established proof of concept and supports further investigation of renzapride in patients with constipation-predominant irritable bowel syndrome.

Atypical anxiolytic-like response to naloxone in benzodiazepine-resistant 129S2/SvHsd mice: role of opioid receptor subtypes.

RATIONALE: Mice of many 129 substrains respond to environmental novelty with behavioural suppression and high levels of anxiety-like behaviour. Although resistant to conventional anxiolytics, this behavioural phenotype may involve stress-induced release of endogenous opioids. OBJECTIVES: To assess the effects of opioid receptor blockade on behavioural reactions to novelty stress in a chlordiazepoxide-resistant 129 substrain. MATERIALS AND METHODS: Experiment 1 contrasted the effects of the broad-spectrum opioid receptor antagonist naloxone (1.0-10.0 mg/kg) in C57BL/6JOlaHsd and 129S2/SvHsd mice exposed to the elevated plus-maze. Experiments 2-4 examined the responses of 129S2/SvHsd mice to the mu-selective opioid receptor antagonist beta-funaltrexamine (2.5-10.0 mg/kg), the delta-selective antagonist naltrindole (2.5-10.0 mg/kg) and the kappa-selective antagonist nor-binaltorphimine (2.5-5.0 mg/kg). RESULTS: 129 mice displayed higher levels of anxiety-like behaviour and lower levels of general exploration relative to their C57 counterparts. Although naloxone failed to alter the behaviour of C57 mice, both doses of this antagonist produced behaviourally selective reductions in open-arm avoidance in 129 mice. Surprisingly, none of the more selective opioid receptor antagonists replicated this effect of naloxone: beta-funaltrexamine was devoid of behavioural activity, naltrindole suppressed rearing (all doses) and increased immobility (10 mg/kg), while nor-binaltorphimine (5 mg/kg) nonspecifically increased percent open arm entries. CONCLUSIONS: Recent evidence suggests differential involvement of opioid receptor subtypes in the anxiolytic efficacy of diverse compounds including conventional benzodiazepines. The insensitivity of 129 mice to the anxiolytic action of chlordiazepoxide, coupled with their atypical anxiolytic response to naloxone (but not more selective opioid receptor antagonists), suggests an abnormality in anxiety-related neurocircuitry involving opioid-GABA interactions.

Stimulation of protein synthesis and phospholipase D activity by vasopressin and phorbol ester in L6 myoblasts.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and vasopressin on protein synthesis and phospholipase D (PLD) activity were investigated in L6 myoblasts. TPA stimulated a concentration-dependent increase in protein synthesis (EC50 approx. 10 nM) during a 90 min incubation, but had no effect after 6 h. The maximum increase was about 15% and was mediated through changes in translation, as TPA had no effect on RNA accretion and the response was not prevented by actinomycin D. TPA also stimulated PLD activity as measured by an 8-fold increase in the formation of phosphatidylbutanol (PtdBuOH) and the release of choline (EC50 5-10 nM). In contrast to TPA, vasopressin stimulated protein synthesis (maximum increase 30%, EC50 approx. 10 nM) and RNA accretion after 6 h, but had no effect after 90 min. Vasopressin also increased PtdBuOH production 4-5-fold (EC50 approx. 0.5 nM) and choline release (EC50 approx. 1 nM). The addition of a highly purified preparation of PLD (2-10 units/ml) from Streptomyces sp. to L6 cells stimulated a concentration-dependent increase in choline release and protein synthesis after both 90 min (maximum stimulation 13%) and 6 h (maximum stimulation 12%). PLD also stimulated RNA accretion after 6 h but not 90 min. The data support a role for PLD in the regulation of protein synthesis in L6 cells.

Insulin and insulin-like growth factor-I responsiveness and signalling mechanisms in C2C12 satellite cells: effect of differentiation and fusion.

In proliferating C2C12 myoblasts, serum and physiological concentrations of insulin and IGF-I stimulated protein synthesis and RNA accretion. After fusion, the multinucleated myotubes remained responsive to serum but not to insulin or IGF-I, even though both insulin and type-I IGF receptor mRNAs increased in abundance. Protein synthetic responses to insulin and IGF-I in myoblasts were not inhibited by dexamethasone, ibuprofen or Ro-31-8220, thus phospholipase A2, cyclo-oxygenase and protein kinase C did not appear to be involved in the signalling mechanisms. Neither apparently were polyphosphoinositide-specific phospholipase C or phospholipase D since neither hormone increased inositol phosphate, phosphatidic acid, choline or phosphatidylbutanol production. Only the phosphatidylinositol-3-kinase inhibitor, wortmannin, and the 70 kDa S6-kinase inhibitor, rapamycin, wholly or partially blocked the effects of insulin and IGF-I on protein synthesis. 2-deoxyglucose uptake remained responsive to insulin and IGF-I after fusion and was also inhibited by wortmannin. The results suggest that the loss of responsiveness after fusion is not due to loss of receptors, but to the uncoupling of a post-receptor pathway, occurring after the divergence of the glucose transport and protein synthesis signalling systems, and that, if wortmannin acts at a single site, this is prior to that point of divergence.

Cyclic AMP stimulates protein synthesis in L6 myoblasts and its effects are additive to those of insulin, vasopressin and 12-0-tetradecanoylphorbol-13-acetate. Possible involvement of mitogen activated protein kinase.

The role of cyclic AMP as a second messenger in the stimulation of protein synthesis and the potential involvement of mitogen activated protein (MAP) kinase in this response was studied in L6 myoblasts. Dibutyryl-cAMP (dbt-cAMP) increased protein synthesis at 90 min and 6 h in a concentration-dependent manner. The responses at 90 min were probably mediated by increased translation as they were not blocked by actinomycin D; effects at 6 h were accompanied by increases in RNA content implying a transcriptional component. 100 nM 12-0-tetradecanoylphorbol-13-acetate (TPA), 1 nM Insulin (90 min incubations) and 100 nM vasopressin (6 h incubation) also increased protein synthesis and these responses were additive with those of 500 micron dbt-cAMP. Responses to forskolin were similar to dbt-cAMP whilst 1,9-dideoxyforskolin had no effect. Cell extracts immunoblotted with MAP kinase antibody showed bands corresponding to approx. 42, 44, 54 and 83 kDa. 500 micron dbt-cAMP elicited an increase in activity of both the 42 and 44 kDa bands when assayed by the 'in gel' method and a similar response was also observed with forskolin. TPA and vasopressin also stimulated the activity of these two isoforms, but had no significant additive or inhibitory effects when added in combination with 500 micron dbt-cAMP. In contrast, although 1 nM insulin alone had no effect, a synergistic response in terms of MAP kinase activation was observed in the presence of dbt-cAMP. The data demonstrate that cAMP stimulates protein synthesis in L6 cells and suggest a role for MAP kinase in this event.

Signalling pathways regulating protein turnover in skeletal muscle.

The protein content of skeletal muscle is determined by the relative rates of synthesis and degradation which must be regulated coordinately to maintain equilibrium. However, in conditions such as fasting where amino acids are required for gluconeogenesis, or in cancer cachexia, this equilibrium is disrupted and a net loss of protein ensues. This review, utilising studies performed in several situations, summarizes the current state of knowledge on the possible signalling pathways regulating protein turnover in skeletal muscle and highlights areas for future work.

Paraneoplastic cerebellar degeneration. Case report and literature review.

Paraneoplastic cerebellar degeneration (PCD) presents with acute or subacute onset of ataxia, dysarthria, and intention tremor. In patients older than 50 years, acute or subacute cerebellar degeneration is paraneoplastic in origin in 50% of cases. Paraneoplastic cerebellar degeneration most often precedes a potentially curable remote malignancy. Less often, PCD occurs in a patient with a known malignancy or heralds the onset of a recurrence. The presence of specific antibodies in serum samples helps to guide identification of the occult underlying malignancy. Physicians should entertain the diagnosis of PCD when older patients present with signs of cerebellar degeneration without an obvious cause. A systematic evaluation, including the selection of appropriate imaging and laboratory studies, will often enable physicians to identify the responsible cancer. However, because PCD can precede a cancer by months to years, periodic reevaluation is needed when the cancer remains occult.

The impact of the prospective payment system on the treatment of hip fractures in the elderly.

A review of 386 Medicare patients with hip fractures admitted to a private, suburban, teaching hospital from 1981 through 1987 revealed that since the implementation of the prospective payment system in 1984, average hospital stays declined from 17.0 days to 12.9 days (24.1%). Although the mean number of physical therapy sessions declined from 11.1 to 9.8 (11.7%), the average number of treatments per day during the physical therapy phase actually increased from 1.2 before to 1.4 after the prospective payment system. The proportion of patients discharged to nursing homes remained the same (52.9% vs 53.6%); the proportion of patients remaining in a nursing home 6 months after hospital discharge did not differ significantly (22.6% vs 19.9%). Furthermore, there were no differences in the 6-month ambulation status. Total adjusted average hospital charges for the pre- and post-prospective payment system groups did not increase significantly ($7295 vs $7565). These findings do not support the contention that the quality of care provided Medicare patients with hip fractures has deteriorated in this hospital environment.

A retrospective study of periodontal disease severity in smokers and non-smokers.

BACKGROUND: Smoking has been associated with increased risk of periodontitis. The aim of the present study was to compare the periodontal disease severity of adult heavy smokers and never-smokers referred for assessment and treatment of chronic periodontitis. METHODS: A random sample of patients with at least 20 teeth, stratified for smoking and age (5-year blocks, 35 to 55 years), was selected from an original referral population of 1,221 subjects with chronic adult periodontitis. Adequate records for 59 never-smokers and 44 subjects who smoked at least 20 cigarettes per day were retrieved. The percentage of alveolar bone support was measured from dental panoramic radiographs with a Schei ruler at x3 magnification with the examiner unaware of the smoking status. Probing depths at six sites per tooth were obtained from the initial consultation. RESULTS: There was no significant difference in age between groups. Smokers had fewer teeth (p<0.001), fewer shallow pockets (p<0.001) and more deep probing depths (p<0.001). The differences were greater in subjects 45 years of age and over. In this age group, smokers had approximately 13% more bone loss, 15% more pockets in the 4-6 mm category and 7% more pockets in the >/= 7 mm category than the never-smokers. CONCLUSIONS: This study confirmed that smokers had evidence of more severe periodontal disease than never-smokers. The differences increased with age confirming an exposure-related response.

Role of nitric oxide synthesis in the regulation of coronary vascular tone in the isolated perfused rabbit heart.

OBJECTIVE: The aim was to study the effects on coronary vascular tone of three inhibitors of nitric oxide (NO) synthesis. METHODS: Studies were performed on isolated perfused hearts of 74 male New Zealand White rabbits fed normal laboratory diet. Resting coronary perfusion pressure was increased to 40-60 mm Hg with the thromboxane mimetic 9,11-dideoxy-9 alpha,11 alpha-methanoepoxy prostaglandin F2 alpha (U46619). The effects of NG-monomethyl-L-arginine (L-NMMA), N-iminoethyl-L-ornithine (L-NIO), and NG-nitro-L-arginine methyl ester (L-NAME) (0.3-300 microM) on resting coronary perfusion pressure were determined. The effects of these compounds, at concentrations that increased the resting perfusion pressure to a similar extent, on the fall in perfusion pressure induced by acetylcholine (0.1 microM) and glyceryl trinitrate (1 microM) were also investigated. In these studies the resting perfusion pressure was maintained at 40-60 mm Hg by reducing the concentration of U46619. RESULTS: L-NMMA, L-NIO, and L-NAME induced concentration dependent increases in resting coronary perfusion pressure (n = 3-9, p < 0.05). L-NAME had the greatest potency and efficacy, increasing the resting pressure by 48.0(SEM 9.6) mm Hg at 30 microM. L-NIO and L-NMMA increased perfusion pressure by 27.3(3.0) and 19.5(5.8) mm Hg respectively at the maximum concentration studied (300 microM). However, at concentrations that were equieffective on resting perfusion pressure (15 mm Hg increase), L-NMMA (100 microM), but not L-NIO (25 microM) or L-NAME (4 microM), significantly inhibited the fall in pressure induced by acetylcholine by 57.2(5.0)%, n = 6, p < 0.05. This effect of L-NMMA++ was attributed to a shorter duration of fall and was reversed by L-arginine (300 microM). L-NMMA (100 microM) and L-NIO (25 microM) potentiated the effect of glyceryl trinitrate by increasing the peak fall in perfusion pressure by 75.6(11.0)% and 68.8(24.1)% respectively (n = 6 for each, p < 0.05). CONCLUSIONS: The differential effects of the three inhibitors on resting coronary perfusion pressure and the acetylcholine induced fall in coronary perfusion pressure suggest that basal and stimulated NO synthesis may be differentially regulated. Reduction in the synthesis of endogenous NO by these compounds potentiates the glyceryl trinitrate induced fall in perfusion pressure, which may have important clinical implications.

The discovery of nitric oxide as the endogenous nitrovasodilator.

Endothelium-derived relaxing factor (EDRF) is a labile humoral agent released by vascular endothelium that mediates the relaxation induced by some vasodilators, including acetylcholine and bradykinin. EDRF also inhibits platelet aggregation, induces disaggregation of aggregated platelets, and inhibits platelet adhesion to vascular endothelium. These actions of EDRF are mediated through stimulation of the soluble guanylate cyclase and the consequent elevation of cyclic guanosine 3',5'-monophosphate. EDRF has been identified as nitric oxide (NO). The pharmacology of NO and EDRF is indistinguishable; furthermore, sufficient NO is released from endothelial cells to account for the biological activities of EDRF. Organic nitrates exert their vasodilator activity following conversion to NO in vascular smooth muscle cells. Thus, NO may be considered the endogenous nitrovasodilator. NO is synthesized by vascular endothelium from the terminal guanido nitrogen atom(s) of the amino acid L-arginine. This indicates the existence of an enzymic pathway in which L-arginine is the endogenous precursor for the synthesis of NO. The discovery of the release of NO by vascular endothelial cells, the biosynthetic pathway leading to its generation, and its interaction with other vasoactive substances opens up new avenues for research into the physiology and pathophysiology of the vessel wall.

Control of regional blood flow by endothelium-derived nitric oxide.

The regional hemodynamic consequences of inhibiting vascular endothelial nitric oxide generation with NG-monomethyl-L-arginine (L-NMMA) were studied in conscious Long-Evans rats. Experiments were carried out in groups of chronically instrumented rats with intravascular catheters and pulsed Doppler probes to monitor regional blood flow. L-NMMA (0.3-300 mg/kg) caused a dose-dependent, long-lasting (5-90 minutes), and enantiomerically specific increase in mean blood pressure and also caused bradycardia. The increase in blood pressure was accompanied by a dose-dependent and long-lasting vasoconstriction in the internal carotid, mesenteric, renal, and hindquarters vascular beds that could be attenuated, in a concentration-dependent manner, by L-arginine but not by D-arginine. In contrast, L-arginine did not affect the pressor or vasoconstrictor effects of vasopressin. These results indicate that nitric oxide production by vascular endothelial cells contributes to the maintenance of blood pressure and to the control of the resting tone of different vascular beds in the conscious rat.

Plasma concentrations of reputed tumor-associated soluble CD44 isoforms (v5 and v6) in smokers are dose related and decline on smoking cessation.

There is some evidence to suggest that smoking may affect circulating levels of CD44 (sCD44) molecules. Therefore, we investigated the effect of smoking on the circulating level of sCD44 by comparing the change in total sCD44, sCD44v5, and sCD44v6 concentrations over 1 year in a group of people who quit smoking (n = 30) and a control group of people who continued to smoke (n = 30). Smoking status and compliance were monitored by analysis of plasma cotinine and expired CO levels and also by self-reported tobacco use. We show a dose-dependent relationship between smoke intake and baseline plasma concentrations of reputed tumor-associated CD44 variant isoforms (sCD44v5 and sCD44v6) in smokers (n = 60). There was a significant decline in the level of both sCD44v5 and sCD44v6 in quitters as compared with continuing smokers [-13.2 (95% confidence interval, -7.6 to -18.8; P < 0.001) and -62.2 ng/ml (95% confidence interval, -33.9 to -90.6; P < 0.001), respectively], but not in the total sCD44 concentration. These results show that the increased concentrations of sCD44v5 and sCD44v6 in smokers are dose related and reversible and suggest that the attributed diagnostic specificity and prognostic value of sCD44 molecules in malignant and inflammatory disease may be affected by smoking status.

Nitric oxide generation. A predictive parameter of acute allograft rejection.

The L-arginine:nitric oxide (NO) biosynthetic pathway has been proposed as an important mediator in host defense mechanisms and may therefore play a role in the acute allograft response. We have studied NO generation in liver allograft rejection and determined its value in immunological monitoring. Stable end products of this pathway have been determined serially in 50 primary liver recipients and compared with 2 known mediators and markers of acute allograft rejection (IL-2R positive lymphocytes and circulating TNF alpha). Plasma concentrations of acid-labile nitrosocompounds (NOx), which increased during acute allograft rejection (P < 0.0001), correlated with rejection severity and were reduced after administration of supplemental high dose glucocorticoids. Concentrations were significantly lower in nonrejection graft complications but were elevated during episodes of sepsis. Correlations between plasma NOx levels and circulating TNF-alpha (r = 0.451, P < 0.001) and IL-2R-positive lymphocytes in peripheral blood (r = 0.781, P < 0.001) were demonstrated. In a logistic analysis of these variables, plasma NOx was the most predictive parameter of an episode of acute cellular rejection. Nitric oxide generation in FK506-treated patients was lower compared with patients receiving a CsA-based immunosuppression regimen and was associated with a reduced frequency of acute rejection in the FK506 group. These data are consistent with a role for NO in the cellular alloantigen immune response and indicate that monitoring of plasma levels of NOx may be useful in the detection of acute allograft rejection.