Unusual 1H NMR chemical shifts support (His) C(epsilon) 1...O==C H-bond: proposal for reaction-driven ring flip mechanism in serine protease catalysis

13C-selective NMR, combined with inhibitor perturbation experiments, shows that the C(epsilon)(1)H proton of the catalytic histidine in resting alpha-lytic protease and subtilisin BPN' resonates, when protonated, at 9.22 ppm and 9.18 ppm, respectively, which is outside the normal range for such protons and approximately 0.6 to 0.8 ppm further downfield than previously reported. They also show that the previous alpha-lytic protease assignments [Markley, J. L., Neves, D. E., Westler, W. M., Ibanez, I. B., Porubcan, M. A. & Baillargeon, M. W. (1980) Front. Protein Chem. 10, 31-61] were to signals from inactive or denatured protein. Simulations of linewidth vs. pH demonstrate that the true signal is more difficult to detect than corresponding signals from inactive derivatives, owing to higher imidazole pK(a) values and larger chemical shift differences between protonated and neutral forms. A compilation and analysis of available NMR data indicates that the true C(epsilon)(1)H signals from other serine proteases are similarly displaced downfield, with past assignments to more upfield signals probably in error. The downfield displacement of these proton resonances is shown to be consistent with an H-bond involving the histidine C(epsilon)(1)H as donor, confirming the original hypothesis of Derewenda et al. [Derewenda, Z. S., Derewenda, U. & Kobos, P. M. (1994) J. Mol. Biol. 241, 83-93], which was based on an analysis of literature x-ray crystal structures of serine hydrolases. The invariability of this H-bond among enzymes containing Asp-His-Ser triads indicates functional importance. Here, we propose that it enables a reaction-driven imidazole ring flip mechanism, overcoming a major dilemma inherent in all previous mechanisms, namely how these enzymes catalyze both the formation and productive breakdown of tetrahedral intermediates.     

Anti-CS1 humanized monoclonal antibody HuLuc63 inhibits myeloma cell adhesion and induces antibody-dependent cellular cytotoxicity in the bone marrow milieu

Currently, no approved monoclonal antibody (mAb) therapies exist for human multiple myeloma (MM). Here we characterized cell surface CS1 as a novel MM antigen and further investigated the potential therapeutic utility of HuLuc63, a humanized anti-CS1 mAb, for treating human MM. CS1 mRNA and protein was highly expressed in CD138-purified primary tumor cells from the majority of MM patients (more than 97%) with low levels of circulating CS1 detectable in MM patient sera, but not in healthy donors. CS1 was expressed at adhesion-promoting uropod membranes of polarized MM cells, and short interfering RNA (siRNA) targeted to CS1 inhibited MM cell adhesion to bone marrow stromal cells (BMSCs). HuLuc63 inhibited MM cell binding to BMSCs and induced antibody-dependent cellular cytotoxicity (ADCC) against MM cells in dose-dependent and CS1-specific manners. HuLuc63 triggered autologous ADCC against primary MM cells resistant to conventional or novel therapies, including bortezomib and HSP90 inhibitor; and pretreatment with conventional or novel anti-MM drugs markedly enhanced HuLuc63-induced MM cell lysis. Administration of HuLuc63 significantly induces tumor regression in multiple xenograft models of human MM. These results thus define the functional significance of CS1 in MM and provide the preclinical rationale for testing HuLuc63 in clinical trials, either alone or in combination.     

A retrospective analysis of practice patterns in the treatment of methicillin-resistant Staphylococcus aureus skin and soft tissue infections at three Canadian tertiary care centres

BACKGROUND:

Methicillin-resistant Staphylococcus aureus (MRSA) infections are increasingly being encountered and pose an increasing burden to the health care system in Canada.

OBJECTIVE:

To elucidate and characterize the factors influencing the current MRSA treatment patterns in patients with skin and soft tissue infections (SSTIs) before linezolid became available on the Canadian market.

METHODS:

A retrospective study collected demographic, treatment and resource use data on patients hospitalized at one of three geographically distinct tertiary care facilities, where MRSA SSTI treatment was initiated with intravenous (IV) vancomycin. Analysis of opportunities for IV-to-oral switch therapy was based on eligibility criteria.

RESULTS:

Of 89 patients identified over a 43-month period, the mean (±SD) durations of anti-infective treatment and hospitalization were 22.4±21 days and 28.9±20.8 days, respectively. An infected surgical wound was most common, representing 62.9% of infections. The mean duration of vancomycin treatment was 19.5 days and the mean number of 1 g doses received was 29.0±32.9. The majority of patients (55.1%) initiated vancomycin therapy a mean of 5.4±8.9 days after confirmation of MRSA. Of the 70% of patients meeting criteria for IV-to-oral switch therapy, only 10% received oral treatment. The most common reason cited for not switching was lack of an effective oral alternative. Analysis of switch therapy criteria found that IV treatment continued for a mean of 13 days despite the appropriateness of the oral route.

CONCLUSIONS:

Considerable variation exists in treatment patterns for MRSA infections. Improvements in the initiation of therapy and the use of IV-to-oral switch therapy may improve care and reduce the duration of hospitalization for MRSA SSTIs.Key Words: Methicillin-resistant Staphylococcus aureus, Switch therapy, Treatment patterns, VancomycinAntibiotic resistance is of growing importance to health care systems worldwide, and Canada is no exception (1). Due to the serious nature of emerging antibiotic-resistant pathogens and the limited therapeutic options available to treat them, infections caused by these organisms may be associated with increased morbidity and mortality (2-4) compared with those caused by drug-sensitive organisms, and pose an increasing economic burden to health care systems (5). Among the resistant organisms, methicillin-resistant Staphylococcus aureus (MRSA) is increasingly being encountered in Canadian health care facilities (1). Although the first report of an MRSA isolate in Canada was in 1981 (6), only occasional reports followed. In 1995, the Canadian Nosocomial Infection Surveillance Program (CNISP) began following the incidence of MRSA prospectively and reported an increase from 1% of all S aureus isolates in 1995 to 8.1% in 2000 in the health care facilities participating in the CNISP (7). The majority of the increase in MRSA cases has occurred in Ontario and Quebec, followed by British Columbia (7). Although MRSA was initially a hospital-acquired pathogen, it has also recently been recognized as a community-acquired organism (8-11) in Canada, particularly among First Nations peoples.Because MRSA is often resistant to multiple antibiotics, treatment options may be limited. The usual treatment for serious infections caused by MRSA is vancomycin (12). This antibiotic is available only in the intravenous (IV) form for treatment of these types of infections, and it has the potential for greater toxicity and may be less effective than conventional therapy for infections caused by susceptible staphylococci (5). Furthermore, certain organisms have exhibited increasing rates of resistance to vancomycin, which may limit its usefulness. A Passive Reporting Network established within the CNISP identified 1315 cases of vancomycin-resistant enterococci throughout Canada between 1994 and 1998 (13). In addition, cases of vancomycin-intermediate strains of S aureus have been reported recently in Japan, Europe, Hong Kong and the United States (14-16), although no cases have yet been confirmed in Canada.Other than intravenous vancomycin, several oral MRSA treatment alternatives are available including trimethoprim-sulfamethoxazole, alone or in combination with rifampin; doxycycline; fusidic acid in combination with rifampin; or fluoroquinolones alone or in combination with rifampin, dependent on the susceptibility of the strain. Five years of MRSA surveillance in several hospitals across Canada found resistance rates to ciprofloxacin, trimethoprim-sulfamethoxazole, rifampin and fusidic acid to be 89%, 56%, 3% and 3%, respectively (17). Linezolid, an antibacterial agent available in both IV and oral forms, was approved for use in Canada following the completion of the present study and represents a new option for the treatment of Gram-positive infections, including those caused by MRSA.Although vancomycin IV has been identified as the current drug of choice for the treatment of MRSA infections, very little is known regarding the real-life practice patterns of treating physicians across Canada. In an effort to gain an understanding of how MRSA infections are currently being treated, we undertook a retrospective study of vancomycin treatment patterns for MRSA infections in geographically distinct regions across Canada, focusing on infections of the skin and soft tissue (SSTIs). Emphasis was placed on determining MRSA treatment characteristics including antimicrobial use, duration of therapy, length of hospital stay (LOS) and use of home IV care services. In addition, an analysis of IV-to-oral switch therapy patterns was conducted to understand the current level of acceptance of the practice, its potential benefits and the barriers to switch therapy in MRSA SSTIs.     

Induced sputum inflammatory mediator concentrations in chronic cough

Previous studies have shown evidence of airway inflammation in patients with chronic cough and have suggested that the cough may be due to release of tussive mediators and activation of afferent sensory nerve endings. We measured the concentration of various proinflammatory and tussive mediators in induced sputum supernatants from 20 patients with cough variant asthma or eosinophilic bronchitis, 20 patients with nonasthmatic chronic cough, 22 patients with idiopathic chronic cough, and 18 healthy control subjects. We measured histamine, cysteinyl-leukotrienes, prostanoids (prostaglandin D2 and prostaglandin E2), and interleukin-8 by enzyme immunoassay. The median sputum histamine concentrations were significantly higher in patients with idiopathic chronic cough (8.0 ng/ml) and cough variant asthma/eosinophilic bronchitis (10.2 ng/ml) than in normal subjects (2.6 ng/ml; 95% confidence interval of difference from idiopathic chronic cough, 0.8 to 25.8 [p = 0.009] and 95% confidence interval of difference from cough variant asthma/eosinophilic bronchitis, 1.1 to 20.1 [p = 0.01]). Median sputum prostaglandin D2 and prostaglandin E2 concentrations were significantly higher in all categories of chronic cough. Our findings support the view that there is release of inflammatory and tussive mediators in patients with chronic cough and suggest that there might be similarities in the mechanism of cough in a diverse range of conditions.     

Alcohol in a social context: findings from event-contingent recording studies of everyday social interactions

Background:  Data concerning the effects of alcohol on social interaction in everyday life are limited.
Methods:  Healthy volunteers in 4 studies of social behaviors and mood were instructed to complete record forms immediately after a social interaction had occurred, a method known as event-contingent recording. Record forms asked questions about quarrelsome, agreeable, dominant, and submissive behaviors; about aspects of mood; and, in 3 studies, about perceptions of others. Each form also contained a question about alcohol consumption prior to a social interaction. For the present report, only social interactions taking place in the evening and outside the work setting were included. Only individuals who consumed alcohol at least once in these circumstances were included ( n  = 171).
Results:  Social interactions involving alcohol were primarily characterized by higher levels of agreeable behaviors, by perceptions of greater agreeableness in others, and by more positive mood. Alcohol consumption was not associated with higher levels of quarrelsomeness.
Conclusions:  Alcohol consumption in a social context may have predominantly positive effects, an observation which is at odds with most alcohol-induced aggression experiments performed in laboratory settings. Drinking in everyday life may be less likely to result in aggression because, unlike in most laboratory experiments, individuals can choose among a variety of behaviors in response to social cues and the alcohol dose consumed is usually lower. Event-contingent recording provides a new approach for the study of alcohol's effects in everyday life and the conditions in which alcohol might result in interpersonal aggression.     

A clinical trial to investigate the effect of silver nylon dressings on mediastinitis rates in postoperative cardiac sternotomy incisions

Mediastinitis is a rare but serious postoperative complication of cardiac surgery that increases mortality rates, hospital length of stay, and medical costs. A clinical trial was conducted to investigate whether the type of postoperative surgical dressing (silver nylon or standard gauze) affects the rate of mediastinal infections. The sample consisted of 1,600 surgical cardiac patients. Infection rates in the standard gauze group (control, n = 1,235) were collected retrospectively from 24 months of infection control records. In the prospective treatment arm of the study, the wounds of all consecutive surgical patients (n = 365) were covered with a silver nylon dressing and patients were assessed during the 3-week postoperative visit. Thirteen (13) patients in the control group (1%) and none of the patients in the treatment group developed mediastinitis (chi2 [1, N = 1,600] = 3.88, P <0.05). Study findings support the need for a large, prospective, controlled clinical study to confirm the effects of these dressings on mediastinitis, resultant morbidity, and costs of care.