In vitro metabolic study of temsirolimus: preparation, isolation, and identification of the metabolites.

The in vitro metabolism of temsirolimus, (rapamycin-42-[2,2-bis-(hydroxymethyl)]-propionate), an antineoplastic agent, was studied using human liver microsomes as well as recombinant human cytochrome P450s, namely CYP3A4, 1A2, 2A6, 2C8, 2C9, 2C19, and 2E1. Fifteen metabolites were detected by liquid chromatography (LC)-tandem mass spectrometry (MS/MS or MS/MS/MS). CYP3A4 was identified as the main enzyme responsible for the metabolism of the compound. Incubation of temsirolimus with recombinant CYP3A4 produced most of the metabolites detected from incubation with human liver microsomes, which was used for large-scale preparation of the metabolites. By silica gel chromatography followed by semipreparative reverse-phase high-performance liquid chromatography, individual metabolites were separated and purified for structural elucidation and bioactivity studies. The minor metabolites (peaks 1-7) were identified as hydroxylated or desmethylated macrolide ring-opened temsirolimus derivatives by both positive and negative mass spectrometry (MS) and MS/MS spectroscopic methods. Because these compounds were unstable and only present in trace amounts, no further investigations were conducted. Six major metabolites were identified as 36-hydroxyl temsirolimus (M8), 35-hydroxyl temsirolimus (M9), 11-hydroxyl temsirolimus with an opened hemiketal ring (M10 and M11), N- oxide temsirolimus (M12), and 32-O-desmethyl temsirolimus (M13) using combined LC-MS, MS/MS, MS/MS/MS, and NMR techniques. Compared with the parent compound, these metabolites showed dramatically decreased activity against LNCaP cellular proliferation.     

Activation of the NLRP3 Inflammasome Is Associated with Valosin-Containing Protein Myopathy

Aberrant activation of the NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome, triggers a pathogenic inflammatory response in many inherited neurodegenerative disorders. Inflammation has recently been associated with valosin-containing protein (VCP)-associated diseases, caused by missense mutations in the VCP gene. This prompted us to investigate whether NLRP3 inflammasome plays a role in VCP-associated diseases, which classically affects the muscles, bones, and brain. In this report, we demonstrate (i) an elevated activation of the NLRP3 inflammasome in VCP myoblasts, derived from induced pluripotent stem cells (iPSCs) of VCP patients, which was significantly decreased following in vitro treatment with the MCC950, a potent and specific inhibitor of NLRP3 inflammasome; (ii) a significant increase in the expression of NLRP3, caspase 1, IL-1β, and IL-18 in the quadriceps muscles of VCP^R155H/+ heterozygote mice, an experimental mouse model that has many clinical features of human VCP-associated myopathy; (iii) a significant increase of number of IL-1β⁽⁺⁾F4/80⁽⁺⁾Ly6C⁽⁺⁾ inflammatory macrophages that infiltrate the muscles of VCP^R155H/+ mice; (iv) NLRP3 inflammasome activation and accumulation IL-1β⁽⁺⁾F4/80⁽⁺⁾Ly6C⁽⁺⁾ macrophages positively correlated with high expression of TDP-43 and p62/SQSTM1 markers of VCP pathology in damaged muscle; and (v) treatment of VCP^R155H/+ mice with MCC950 inhibitor suppressed activation of NLRP3 inflammasome, reduced the F4/80⁽⁺⁾Ly6C⁽⁺⁾IL-1β⁽⁺⁾ macrophage infiltrates in the muscle, and significantly ameliorated muscle strength. Together, these results suggest that (i) NLRP3 inflammasome and local IL-1β⁽⁺⁾F4/80⁽⁺⁾Ly6C⁽⁺⁾ inflammatory macrophages contribute to pathogenesis of VCP-associated myopathy and (ii) identified MCC950 specific inhibitor of the NLRP3 inflammasome with promising therapeutic potential for the treatment of VCP-associated myopathy.     

Attitudes of applicants for surgical residency toward work hour limitations

BACKGROUND: There is an ongoing debate regarding the merits of resident work-hour limitations. We postulated that this issue would be a factor in the decision-making process of applicants to surgical residency. METHODS: Candidates for surgical residency at a university-based program completed an anonymous survey during their visit. Data was analyzed by analysis of variance and the chi-square test. RESULTS: Most candidates viewed work-hour limitations as being favorable to their future training. Nevertheless, work-hour limitations ultimately were not a critical factor in the decision-making process compared with issues such as quality of training and program reputation. Candidates ranked "reading in surgery" the most likely way they would spend the leisure time afforded by work-hour limitations. CONCLUSIONS: Most applicants for surgical residency consider work hour-limitations as being favorable to their training and view the extra free time as an opportunity for furthering their education. However, other issues take precedence when choosing a residency.     

Impact of Carotid Artery Angioplasty and Stenting on Management of Recurrent Carotid Artery Stenosis

Citing the higher perioperative risk of redo carotid surgery, balloon angioplasty and stenting of the carotid artery (CAS) has been advocated for recurrent carotid stenosis (RCS). To examine the impact of CAS on the management and outcome of recurrent stenosis, a retrospective review of a prospectively compiled database was performed. From a registry of patients treated for carotid disease, 105 procedures were performed from 1992 to 2002 for RCS. For comparison, two study groups were examined. Time I consisted of 77 reoperations performed through 1998, before CAS was introduced at our institution. Time II included 12 reoperations and 16 CAS procedures performed for RCS from 1999 through 2002. Using perioperative stroke as a measure of outcome, the results for time II were poorer than for time I (7.2% vs. 5.2%, p = NS). Overall, the risk of perioperative stroke was the same for reoperation (5/89) and CAS (1/16) (5.6% vs. 6.3%, p = NS). Although not statistically significant, there was a trend toward a higher risk of perioperative stroke for patients treated with reoperation during the latter time period (8.3% vs. 5.2%, p = NS). This probably relates to the finding that during time II, CAS was most likely to be used in asymptomatic patients (68.6% vs. 41.7%, p = NS) with early (<3 years) RCS (87.5% vs. 41.7%, p = 0.01). No patient with asymptomatic, early RCS had a perioperative stroke with either surgery or CAS (0/35 cases, 0%). The presence of preoperative neurologic symptoms was significantly predictive of a perioperative stroke among all procedures performed for RCS (13.6% vs. 0%, p = 0.004). Contrary to suggestions that CAS might improve the management of RCS, a review of our data shows the overall risk of periprocedural stroke to be no better since CAS has become available. The bias for using CAS for asymptomatic myointimal hyperplastic lesions, and reoperation for frequently symptomatic late recurrent atherosclerotic disease, makes direct comparisons of the two techniques for treating RCS difficult. It is expected that the overall risk for redo carotid surgery will increase, as fewer low-risk patients will be receiving open procedures. However, the increased risk among symptomatic patients undergoing reoperation suggests that endovascular techniques should be investigated among this group of cases as well.Presented at the Twenty-eighth Annual Meeting of the Peripheral Vascular Surgery Society, Chicago, IL, June 7, 2003.     

Successful management of carotid stenosis in a high-risk population at an inner-city hospital

This is a retrospective review of all carotid endarterectomies (CEA) (n=91) done from 1993 to 2002 at an inner-city hospital (Group I). This group was compared to a randomly selected group of patients (n=445) treated at a private hospital (Group II). The same high-volume surgeons performed CEAs at both hospitals. The majority of Group I patients (71.4%) were members of racial minority groups. They were also more likely to be younger (p<0.001), hypertensive (p<0.03), diabetic (p<0.001), and current smokers (p<0.001); have contralateral carotid artery occlusion (p=0.04); and present with stroke (p<0.001) than Group II patients. Despite this, the incidence of postoperative myocardial infarction (2.2% vs 0.2%, p=0.08), stroke (1.1% vs 1.6%, NS), and death (1.1% vs 0%, NS) was comparable between the 2 groups. Aggressive preoperative workup for occult cardiac disease in Group I revealed an incidence of 25.9% (n=15). Of these, 5 (33.3%) were found to have coronary artery disease severe enough to warrant intervention before CEA. In an inner-city population with increased medical comorbidities, more severe cerebrovascular disease, and relatively low volume of carotid surgery, the results of CEA were comparable to those in patients treated at a high-volume private hospital. The presence of high-volume surgeons, operating at the low-volume municipal hospital, may contribute to the low complication rate. Finally, aggressive preoperative cardiac workup in this underserved population revealed a meaningful incidence of occult coronary artery disease requiring intervention before CEA.     

Activation of peroxisome proliferator-activated receptor (PPAR)-gamma by 15S-hydroxyeicosatrienoic acid parallels growth suppression of androgen-dependent prostatic adenocarcinoma cells

Although dietary gamma-linolenic acid (GLA) and its 15-lipoxygenase metabolite, 15S-hydroxyeicosatrienoic acid (15S-HETrE), have been reported to exert antiproliferative activities in other systems, their role in prostatic carcinogenesis is unknown. To evolve a possible mechanism for the suppressive effect on growth of prostatic cells, we incubated GLA and 15S-HETrE with androgen-dependent prostatic adenocarcinoma cells. 15S-HETrE but not GLA markedly inhibited [(3)H]thymidine uptake in parallel with the upregulation of peroxisome proliferator-activated receptor-gamma expression (a growth modulating nuclear receptor). The data, taken together, suggest that dietary GLA via its in vivo metabolite 15S-HETrE could serve as an endogenous adjunct to attenuate prostatic tumorigenesis.     

Morphological studies on periodontal disease in the cynomolgus monkey

Electron microscopic observations were made on naturally occurring gingivitis, on gingivitis at non-ligated sites of an experimental animal, and on ligature-induced periodontitis in a periodontal disease model using female cynomolgus monkeys.
For both types of gingivitis specimens, a plaque associated chronic inflammatory reaction was observed, comparable to that described for the human established lesion. Bacterial accumulations on tooth surfaces were separated from the epithelium by neutrophils which exhibited variations in fine structure and extent of bacterial phagocytosis related to their proximity to the plaque. In the inflamed connective tissue of the papillae, collagen was reduced to strands extending between the cellular elements. The plasma cell was the most common inflammatory cell and had three major variations in form.
With ligature-induced periodontitis, similar inflammatory features were observed in the gingiva. A complex flora like that encountered in human periodontitis was observed around and within the ligature as well as more apically in the pocket. As compared to the flora in gingivitis, more spirochetes were encountered, a larger proportion of the microorganisms appeared to be in a living state, and bacteria were in contact with the epithelial surface, rather than being walled off by the neutrophils. This seemingly more aggressive plaque was associated with an advanced lesion characterized by changes that indicated sequential destruction of the collagenous attachment to cementum and concommitant apical migration of the epithelial attachment. No bacteria were observed in the soft tissues, even in the wide intercellular spaces between the epithelial cells.     

Perioperative outcomes of anterior lumbar surgery in obese versus non-obese patients

Background contextAnterior lumbar surgery is a common procedure for anterior lumbar interbody fusion and disc replacement but the impact of obesity on this procedure has not been determined.PurposeTo assess the perioperative outcomes of anterior retroperitoneal lumbar surgery in obese versus non-obese patients.Study design/settingProspective review of patients with anterior retroperitoneal lumbar disc proceduresPatient sampleSeventy-four patients with anterior retroperitoneal lumbar disc procedures performed were evaluated.Outcome measuresAccess-related parameters included tissue depth (skin-to-fascia and fascia-to-spine depths), length of incision, estimated blood loss during the anterior procedure, the duration of the anterior exposure, and the duration of the entire anterior procedure. Outcome measures included complications attributable to the anterior procedure, analgesic use, length of time to ambulation, and length of hospitalization.MethodsSeventy-four anterior retroperitoneal lumbar disc procedures were prospectively analyzed. Patient age, sex, body mass index, comorbidities, diagnosis, and operative parameters were collected. Access-related parameters and outcome measures were compared between obese and non-obese patients. Obesity was defined as body mass index greater than or equal to 30.ResultsThere were 35 males and 39 females. Mean age was 46.6 years. The main diagnosis (63.5%) was discogenic back pain. Forty-one (55%) patients were non-obese and 33 were obese. The two patient groups were comparable in terms of age, sex, diagnosis, mean number of anterior levels operated, and previous abdominal surgery (all p>.05). In obese patients, there were two iliac vein lacerations (major complication rate, 6.1%), one superficial infection, and one urinary tract infection (minor complication rate, 6.1%). In non-obese patients, there were two iliac vein lacerations, one intestinal serosal tear (major complication rate, 7.3%), and two urinary tract infections (minor complication rate, 4.9%). There was no significant difference in the complication rates between obese and non-obese patients (p=.6). Obese patients have significantly longer duration of anterior exposure, duration of entire anterior surgery, longer length of anterior incision, and more depth from skin to fascia and from fascia to spine compared with non-obese patients. However, obesity does not affect blood loss, analgesic use, length of time to ambulation, and length of hospitalization.ConclusionPerioperative outcomes in obese and non-obese patients were comparable and obesity is not related to an increased risk of morbidity in anterior lumbar surgery.