The Stickland Reaction Precursor trans-4-Hydroxy-l-Proline Differentially Impacts the Metabolism of Clostridioides difficile and Commensal Clostridia.

An intact gut microbiota confers colonization resistance against Clostridioides difficile through a variety of mechanisms, likely including competition for nutrients. Recently, proline was identified as an important environmental amino acid that C. difficile uses to support growth and cause significant disease. A posttranslationally modified form, trans-4-hydroxyproline, is highly abundant in collagen, which is degraded by host proteases in response to C. difficile toxin activity. The ability to dehydrate trans-4-hydroxyproline via the HypD glycyl radical enzyme is widespread among gut microbiota, including C. difficile and members of the commensal Clostridia, suggesting that this amino acid is an important nutrient in the host environment. Therefore, we constructed a C. difficile ΔhypD mutant and found that it was modestly impaired in fitness in a mouse model of infection, and was associated with an altered microbiota when compared to mice challenged with the wild-type strain. Changes in the microbiota between the two groups were largely driven by members of the Lachnospiraceae family and the Clostridium genus. We found that C. difficile and type strains of three commensal Clostridia had significant alterations to their metabolic gene expression in the presence of trans-4-hydroxyproline in vitro. The proline reductase (prd) genes were elevated in C. difficile, consistent with the hypothesis that trans-4-hydroxyproline is used by C. difficile to supply proline for energy metabolism. Similar transcripts were also elevated in some commensal Clostridia tested, although each strain responded differently. This suggests that the uptake and utilization of other nutrients by the commensal Clostridia may be affected by trans-4-hydroxyproline metabolism, highlighting how a common nutrient may be a signal to each organism to adapt to a unique niche. Further elucidation of the differences between them in the presence of hydroxyproline and other key nutrients will be important in determining their role in nutrient competition against C. difficile. IMPORTANCE Proline is an essential environmental amino acid that C. difficile uses to support growth and cause significant disease. A posttranslationally modified form, hydroxyproline, is highly abundant in collagen, which is degraded by host proteases in response to C. difficile toxin activity. The ability to dehydrate hydroxyproline via the HypD glycyl radical enzyme is widespread among gut microbiota, including C. difficile and members of the commensal Clostridia, suggesting that this amino acid is an important nutrient in the host environment. We found that C. difficile and three commensal Clostridia strains had significant, but different, alterations to their metabolic gene expression in the presence of hydroxyproline in vitro. This suggests that the uptake and utilization of other nutrients by the commensal Clostridia may be affected by hydroxyproline metabolism, highlighting how a common nutrient may be a signal to each organism to adapt to a unique niche. Further elucidation of the differences between them in the presence of hydroxyproline and other key nutrients will be important to determining their role in nutrient competition against C. difficile.

Successful treatment of a chronic oroantral fistula infected with extensively drug resistant bacteria using long-term oesophageal tube feeding and several non-conventional treatments in a horse.

BACKGROUND: Chronic oroantral fistulae (OAF) with secondary sinusitis can occur following repulsion of cheek teeth in horses. CASE REPORT: An 8-year-old Andalusian cross gelding presented with an iatrogenic clinical crown fracture of tooth 209, which underwent repulsion of its apical portion (day 0). The horse was treated with intramuscular penicillin and intravenous gentamicin (5 days), followed by oral trimethoprim-sulphonamide (10 days) and then oral doxycycline (14 days). The acute iatrogenic OAF created during the initial repulsion persisted; a chronic OAF was identified on day 24. On day 48, septic sinusitis with multidrug-resistant (MDR) Escherichia coli was confirmed. Although susceptible to enrofloxacin in vitro, 30 days of therapy was unsuccessful. Subsequent serial cultures grew multiple MDR and extensively drug-resistant (XDR) gram-negative microorganisms. Whole-genome sequencing (WGS) revealed multiple sequence types of E. coli, with a range of resistance and virulence genes. The orientation of the OAF, regional osteomyelitis and septic sinusitis were confirmed with computed tomography on day 70. On day 74, enteral nutrition was provided through a cervical oesophagostomy tube for 3 months for prevention of oral feed contamination. The OAF was treated with various alternative therapeutics, including apple cider vinegar, propolis and amikacin impregnated products, until resolution on day 116. CONCLUSION: These non-conventional therapeutics, antimicrobials and long-term oesophagostomy contributed to the successful treatment of a complicated OAF. In the future, WGS may be useful to inform antimicrobial selection when MDR or XDR organisms are identified.

Telemedicine and pediatric surgery.

The practice of pediatric surgery has become increasingly demanding, requiring longer working hours with less reimbursement. Although manpower in this field is adequate, there are still areas that are underserved. Advances in technology offer the pediatric surgeon tools to improve efficiency of practice, cover wider areas of practice, and service underserved locations. The purpose of this report is to introduce the pediatric surgeon to telemedicine technology and its potential impact on the practice environment. The history, key components, and current applications of telemedicine are presented. The ability to integrate this technology in pediatric surgical practice holds great potential.

Antibiotic update for the surgeon.

Infection remains a significant source of morbidity and expense in the treatment of surgical patients there- fore, antibiotics continue to be an important part of the general surgeon's armamentarium. Unfortunately, physicians, and surgeons in particular, continue to order too many antibiotics too often, and for too long. Optimal use of antibiotics, as for any therapeutic modality, requires consideration of the risks and benefits as- sociated with available agents and regimens. Although the desired benefit is always successful eradication or avoidance of offending pathogens, the best way to acheive that goal may not be obvious. Decisions regarding choice of antimicrobial agent, duration of therapy, and route of administration are primarily based upon an- ticipation of clinical efficacy.

Rolipram and isoproterenol reverse platelet activating factor-induced increases in pulmonary microvascular permeability and vascular resistance.

Platelet activating factor (PAF) is an important mediator of pulmonary microvascular endothelial cell (PMVEC) injury in sepsis. Membrane receptors for PAF have been identified on PMVECs and mediate its actions at least in part by protein kinase C activation. Since rolipram, a family IV cyclic AMP phosphodiesterase inhibitor, and isoproterenol, an adenylate cyclase activator, both reverse ischemia-reperfusion-induced lung permeability, we studied the effects of these agents on PAF-induced pulmonary microvascular permeability. The isolated rat lung model was used in which lungs were ventilated and buffer perfused at constant flow while suspended from a force transducer to monitor lung weight along with arterial (P(a)) and venous (Pv) pressures. Control lungs (n = 6) were infused with PAF (40 nmole/kg) via an arterial port and the capillary permeability coefficient (Kf,c) was determined at 0, 15, and 60 min. The remaining lungs were randomized for infusion with either rolipram (n = 4, 20 mumole/kg) or isoproterenol (n = 4, 5 mumole/kg) via an arterial port 30 min after injury with PAF. In the rolipram- and isoproterenol-treated groups, the Kf,c was determined 15 and 60 min postinfusion with these agents. The control group showed significant elevation in the Kf,c and total pulmonary resistance (Rt). At 15 and 60 min, rolipram and isoproterenol reversed PAF injury as shown by the significant improvement in the Kf,c and Rt. These findings support the concept that increased cyclic AMP is an important mediator in the reversal of PAF-increased PMVEC permeability and pulmonary resistance.

Platelet-activating factor increases vascular resistance in rat hindquarters by thromboxane A2.

The effects of platelet-activating factor (PAF) on vascular resistance and capillary permeability were studied in the isolated rat hindquarter. Six groups were studied (n = 30): control; PAF alone (1.4 microM); and PAF (1.4 microM) pretreated with ibuprofen (30 mg/kg), thromboxane A2 (TxA2)-receptor antagonist (BM-13505, 2 mg/kg), PAF-receptor antagonist (WEB-2086, 5 mg/kg), or dexamethasone (5 mg/kg). The vascular resistance was calculated, and the reflection coefficient (sigma) was determined as an index of capillary permeability. Exogenous PAF caused a threefold increase in vascular resistance peaking at 5 min and a 2.5-fold increase in capillary permeability. The increased vascular resistance caused by PAF alone was significantly attenuated by ibuprofen, BM-13505, and dexamethasone. The PAF-induced permeability was neither attenuated by ibuprofen nor BM-13505. However, both the increased vascular resistance and permeability were blocked and attenuated by WEB-2086 and dexamethasone, respectively. We conclude that TxA2 mediates the PAF-induced increased vascular resistance; however, the increased vascular permeability is independent of the formation of TxA2 in the isolated hindquarter.

Eicosanoids. Critical agents in the physiological process and cellular injury.

Eicosanoids were discovered as "prostaglandins" in the mid-1930s. The discovery that eicosanoids were ubiquitous in mammalian cells and that nonsteroidal anti-inflammatory drugs worked by inhibiting enzymes that synthesized these chemicals heralded their extensive investigation in all fields of biology. Precursor fatty acids (arachidonic acids) are stored in cell phospholipids, acted on by two enzymes (cyclooxygenase and lipooxygenase) that yield prostaglandins, thromboxane, prostacyclin, and leukotrienes. Knowledge of their biochemical processes continue to unfold, but it is now believed that eicosanoids are part of a larger group of agents termed phospholipid mediators. Eicosanoids are intimately involved with cardiovascular function as well as central and peripheral vascular disease processes and ischemia. In the gastrointestinal tract, these potent lipids not only participate in many normal functions (eg, acid secretion and motility) but also in disease states (eg, inflammatory bowel disease and peptic ulcer disease). In shocklike states of sepsis and/or endotoxemia, eicosanoids have assumed a major role in many events that occur. Recently, discoveries have demonstrated that platelet-activating and tumor necrosis factors exert their effects in part through eicosanoids. The future will demonstrate these compounds to be critical not only in intracellular (molecular) events but also in the effects they produce that are far from the source of origin.

Tumor necrosis factor-induced mortality is reversed with cyclooxygenase inhibition.

OBJECTIVE: The authors hypothesized that TNF would induce eicosanoid synthesis, and a cyclooxygenase inhibitor would attenuate both eicosanoid synthesis and improve survival in an LD90 TNF-induced (150 ng/kg/i.v./5 min) mortality model. SUMMARY BACKGROUND DATA: Tumor necrosis factor is a cardinal mediator in sepsis; however, little is known about its effects on arachidonate metabolism. METHODS: Conscious male rats with carotid arterial and jugular venous catheters were randomized for mortality: group I, TNF alone (150 kg/i.v./15 min, n = 30); group II, ibuprofen (30 mg/kg/i.v. at t = -20 and +240 min), plus TNF, (n = 28); and for hemodynamics, eicosanoid synthesis, blood gases: group III, TNF alone, (n = 8); group IV, ibuprofen + TNF (n = 8); group V, monoclonal antibody to TNF plus TNF (n = 8). Mortality was determined at 4-72 hr. Other parameters determined over 4 hours (0, 5, 60, 120, 240 min). RESULTS: TNF stimulated synthesis of (a) TXB2 (71 +/- 30 pg/ml, mean +/- SE at base vs. 117 +/- 18 at 4 hr, p < 0.02); (b) PGE2 (70 +/- 6 pg/ml at base vs. 231 +/- 68 at 4 hr, p < 0.02); (c) 6PGF (52 +/- 6 pg/ml at base vs. 250 +/- 80 at 4 hr, p < 0.02). Ibuprofen significantly (p < 0.05) inhibited eicosanoid synthesis from TNF. TNF-induced mortality (87%, 26/30) was dramatically decreased with ibuprofen (11%, 3/28), at 4, 24, and 72 hr (p < 0.01). Monoclonal antibody to TNF prevented all abnormalities and had 100% survival. Hemodynamic events were similar in both groups, but metabolic acidosis was attenuated with ibuprofen. CONCLUSIONS: TNF stimulates arachidonic acid metabolism in vivo. A cyclooxygenase inhibitor attenuates eicosanoid synthesis and dramatically improves survival. TNF appears to have different effect on tissues that synthesize certain eicosanoids. Hypotension from TNF is not mediated via the eicosanoids. TNF-induced mortality, like endotoxemia/sepsis may be mediated, in part, via arachidonic acid metabolites. These new findings support the notion that cyclooxygenase inhibitors may be used as adjunctive therapy in clinical sepsis.

Antimicrobial prophylaxis for surgical wounds. Guidelines for clinical care.

Prophylactic administration of antibiotics can decrease postoperative morbidity, shorten hospitalization, and reduce the overall costs attributable to infections. Principles of prophylaxis include providing effective levels of antibiotics in the decisive interval, and, in most instances, limiting the course to intraoperative coverage only. Use in The National Research Council clean contaminated operations is appropriate and, in many instances, has been proven beneficial. Antibiotic prophylaxis is also indicated for clean operations, such as those involved with insertion of prosthetic devices, that are associated with low infection risk and high morbidity. Extension of antibiotic prophylaxis to other categories of clean wounds should be limited to patients with two or more risk factors established by criteria in the study of the efficacy of nosocomial infection control (SENIC) because the baseline infection rate in these patients is high enough to justify their use. Cefazolin (or cefoxitin when anaerobic coverage is necessary) remains the mainstay of prophylactic therapy. Selection of an alternate agent should be based on specific contraindications, local infection control surveillance data, and the results of clinical trials. Newer criteria for determining the risk of "site infection" (wound and intracavitary) are in evolution and may lead to modification of these recommendations over the next several years.

Anastigmatic tilted Mangin mirror for long-focus imaging systems.

A tilted Mangin mirror can be designed to be free of coma and astigmatism and to cancel the chromatic aberrations of refracting components.

Platelet-activating factor augments tumor necrosis factor and procoagulant activity.

Infusion of platelet activating factor (PAF) reproduces the host physiologic response to endotoxemia and sepsis. Tumor necrosis factor (TNF) and procoagulant activity (PCA) are two other potentially deleterious central inflammatory mediators produced in large quantities by tissue-fixed macrophages (M phi). The relationship, if any, between PAF and TNF or PCA production is unknown. Rabbit alveolar M phi were treated in vitro with PAF alone and prior to endotoxin (LPS). PAF alone had no effect on M phi PCA or TNF. PAF (10(-9)-10(-6) M) cotreatment enhanced M phi PCA and TNF levels in a dose response from two- to sixfold above that of LPS treatment alone. PAF (10(-6) M) pretreatment of M phi at T -4 to -6 hr produces an eight- to ninefold enhancement in both TNF and PCA levels. Thus, both coincubation and pretreatment or "priming" of the M phi with PAF prior to LPS stimulation greatly increase M phi production of PCA and TNF. The ability to augment the production of these two potent inflammatory mediators may explain in part the mechanism of action of PAF in vivo.

Nadolol to treat aggression and psychiatric symptomatology in chronic psychiatric inpatients: a double-blind, placebo-controlled study.

BACKGROUND: Considerable evidence indicates that the lipophilic beta-blocker propranolol is useful in treating organically based aggression. This study looked at the efficacy of a more hydrophilic beta-blocker, nadolol, to treat aggression in chronic psychiatric inpatients. METHOD: Forty-one chronic psychiatric inpatients with an average of one aggressive outburst per week (defined by the Overt Aggression Scale [OAS]) were entered into a double-blind, placebo-controlled study lasting 17 weeks. The OAS was used to track aggression on a per-incident basis, while the Brief Psychiatric Rating Scale (BPRS) and the Clinical Global Impressions scale (CGI) were used to track clinical status. RESULTS: Nadolol subjects showed a significant decline in frequency of aggression compared with controls (p = .026) and a significant decline in the BPRS total score (p = .007) and in the subfactors "hostility and suspicion," "negative symptoms," and "signs of hyperarousal/tension." There was no significant change in CGI "severity of illness" ratings between groups, although the nadolol group was significantly improved from baseline at every subsequent time period while the placebo group was unchanged throughout the study. CONCLUSION: Nadolol is of significant benefit in the treatment of aggression in chronic psychiatric inpatients. This drug does penetrate the brain over time, but the success of a drug whose primary locus of action is peripheral may implicate a bimodal mechanism of action, i.e., a role for the CNS and the soma in the maintenance of aggression.

A PAF receptor antagonist, BN 52021, attenuates thromboxane release and improves survival in lethal canine endotoxemia.

Platelet-activating (PAF) is a putative mediator in endotoxemia and sepsis. Administration of a PAF receptor antagonist prior to endotoxin improves survival in rats and attenuates the hypotension of endotoxemia. Both PAF and endotoxin stimulate eicosanoid production. We hypothesized that a PAF receptor antagonist, BN 52021, would alter the hemodynamic events, improve the survival and attenuate the eicosanoid release associated with endotoxemia in a resuscitated, but lethal, canine model. Male dogs were randomzied to two groups (n = 10 each). Group I received only E. coli endotoxin, 1 mg/kg IV, at time 0, while group II received BN 52021, 5 mg/kg IV, 30 min before and again 240 min after endotoxin treatment. During the 4-h study period, hemodynamics were measured and blood samples were taken at 0, 2, 60, 120, and 240 min. Survival was determined at 24, 48, and 72 h. All group I animals died before 24 h; all group II lived longer than 72 h (P less than 0.05). In group I, plasma TXB2 values increased from a baseline value of 0.26 +/- .04 ng/ml to 4.38 +/- 1.56 ng/ml at 120 min and then decreased to 2.64 +/- .96 ng/ml by 240 min. For group II, respective plasma TXB2 values were 0.35 +/- 0.13 ng/ml at baseline, 0.58 +/- 0.14 ng/ml at 120 min, and 0.39 +/- .09 ng/ml at 240 min. At the 120-min and 240-min time points, the groups differed at P less than 0.05. Heart rate tended to be less in group II, but MAP was unaffected. In group I, pH values were more acidotic than those observed in group II.

Platelet activating factor receptor antagonist improves survival and attenuates eicosanoid release in severe endotoxemia.

Exogenous platelet activating factor (PAF) causes hypotension, plasma extravasation, metabolic acidosis, and death. These effects are similar to those of endotoxin as well as the eicosanoids. A specific PAF receptor antagonist, BN52021, was used to determine its effects on the hemodynamic events, the eicosanoid production, and on survival in severe rat endotoxemia. Endotoxin alone significantly produced hypotension, prostaglandins (TxB2, PGE2) release, and death. In contrast pretreatment with BN52021, a specific PAF receptor antagonist, significantly altered the hypotension, significantly attenuated the eicosanoid release, and improved the survival rate (p less than 0.01). These findings suggest that PAF receptor activation is an early event in endotoxemia. Eicosanoid release in endotoxemia could be related to PAF synthesis and PAF receptor activation. These findings support the hypothesis that there may be an intimate relationship between PAF and the eicosanoids and that in endotoxemia some of the effects of PAF may be mediated via the cyclo-oxygenase pathway.

Evaluation of cis-platinum and DTIC combination chemotherapy in disseminated melanoma. A Southwest Oncology Group Study.

The development of disseminated melanoma is associated with an extremely poor prognosis. Use of a variety of chemotherapy agents alone and in combination has yielded response rates of only 10-30%. Surgery and radiation therapy play a useful role in palliative treatment, but have little or no value in the treatment of disseminated disease. In order to evaluate the response of disseminated malignant melanoma to combination chemotherapy with cis-platinum and 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide (DTIC), a phase II pilot study was developed and conducted at Oregon Health Sciences University. Thirty patients were treated from January 1983 to October 1986 and evaluated. There were two complete responses (7%) and nine partial responses (30%) for a total response rate of 37%. The median duration of response was 31 weeks. This response rate is higher than has been previously achieved with DTIC and cis-platinum, or with other drugs alone or in combination. Severe (grade 3) renal, neurologic, and hematologic toxicity was seen in four of 30, three of 30, and ten of 30 patients, respectively.