Functional considerations of stem cell transplantation therapy for spinal cord repair

Stem cells hold great promise for therapeutic repair after spinal cord injury (SCI). This review compares the current experimental approaches taken towards a stem cell-based therapy for SCI. It critically evaluates stem cell sources, injury paradigms, and functional measurements applied to detect behavioral changes after transplantation into the spinal cord. Many of the documented improvements do not exclusively depend on lineage-specific cellular differentiation. In most of the studies, the functional tests used cannot unequivocally demonstrate how differentiation of the transplanted cells contributes to the observed effects. Standardized cell isolation and transplantation protocols could facilitate the assessment of the true contribution of various experimental parameters on recovery. We conclude that at present embryonic stem (ES)-derived cells hold the most promise for therapeutic utility, but that non-neural cells may ultimately be optimal if the mechanism of possible transdifferentiation can be elucidated.     

IL-36R ligands are potent regulators of dendritic and T cells

IL-36α (IL-1F6), IL-36β (IL-1F8), and IL-36γ (IL-1F9) are members of the IL-1 family of cytokines. These cytokines bind to IL-36R (IL-1Rrp2) and IL-1RAcP, activating similar intracellular signals as IL-1, whereas IL-36Ra (IL-1F5) acts as an IL-36R antagonist (IL-36Ra). In this study, we show that both murine bone marrow-derived dendritic cells (BMDCs) and CD4(+) T lymphocytes constitutively express IL-36R and respond to IL-36α, IL-36β, and IL-36γ. IL-36 induced the production of proinflammatory cytokines, including IL-12, IL-1β, IL-6, TNF-α, and IL-23 by BMDCs with a more potent stimulatory effect than that of other IL-1 cytokines. In addition, IL-36β enhanced the expression of CD80, CD86, and MHC class II by BMDCs. IL-36 also induced the production of IFN-γ, IL-4, and IL-17 by CD4(+) T cells and cultured splenocytes. These stimulatory effects were antagonized by IL-36Ra when used in 100- to 1000-fold molar excess. The immunization of mice with IL-36β significantly and specifically promoted Th1 responses. Our data thus indicate a critical role of IL-36R ligands in the interface between innate and adaptive immunity, leading to the stimulation of T helper responses.     

Systemic and vascular markers of inflammation in relation to metabolic syndrome and insulin resistance in adults with elevated atherosclerosis risk

ObjectiveIn recent years high sensitive C-reactive protein (hsCRP), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular cell adhesion molecule-1 (sICAM-1), fibrinogen, and plasminogen activator inhibitor-1 (PAI-1) were recognized as risk factors for cardiovascular disease (CVD). The aim of the present study was to investigate the relationship between these vascular and systemic markers of low-grade inflammation and traditional risk factors, the metabolic syndrome (MetS) or insulin resistance (IR).Methods and resultsIn 137 adults (41–78 years) with at least 2 risk factors for atherosclerosis the following parameters were determined: hsCRP, sVCAM-1, sICAM-1, PAI-1, fibrinogen, waist circumference (WC), blood pressure, Body Mass Index (BMI), fasting serum glucose (FSG), insulin, triglycerides (TG), total cholesterol (TC), LDL, and HDL. The presence or absence of MetS according to the AHA/NHLBI Scientific Statement criteria was assessed. IR was defined using the homeostasis model (HOMA-IR). Subjects with MetS had significantly higher values of hsCRP, sICAM-1, sVCAM-1, PAI-1, fibrinogen (each P < 0.05) and lower HDL-levels (P < 0.05) compared with subjects without MetS. Similar results were found using HOMA-IR-quartiles. Subjects in the bottom quartile (HOMA-IR ≤ 1.32) had significantly lower levels of hsCRP, sVCAM-1, sICAM-1, and PAI-1 (each P < 0.05) than subjects in the top quartile (HOMA-IR ≥ 5.03). HDL was significantly higher (P < 0.05) in subjects in the lowest quartile versus those in the highest quartile. Incidentally we found no significant differences in total and LDL cholesterol among MetS, HOMA, and traditional CVD risk factor groups, respectively.ConclusionSystemic and vascular markers of inflammation showed significant associations with IR and the MetS and may be incorporated into traditional CVD risk prediction models. Such models should be established and validated in forthcoming large scale prospective studies on CVD risk.     

Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) in Specimens from Various Body Sites: Performance Characteristics of the BD GeneOhm MRSA Assay,the Xpert MRSA Assay,and Broth-Enriched Culture in an Area with a Low Prevalence of MRSA Infections

Universal surveillance upon patient admission is important in reducing the transmission of methicillin-resistant Staphylococcus aureus (MRSA) and associated disease in hospitals. High costs for the health care system in conjunction with MRSA have promoted the development of rapid screening methods to detect MRSA carriers. This study compared two real-time PCR methods, the BD GeneOhm MRSA assay (BDGO) and the Xpert MRSA assay, with broth-enriched culture to define their performance characteristics and rapidity in an area with low MRSA prevalence. In total, 414 swabs from the nose and 389 swabs from the groin from 425 patients were tested. Of those 425 patients, 378 had swabs from both the nose and groin in parallel. Two hundred thirty-one and 194 patients were randomly assigned to the BDGO group and the Xpert MRSA group, respectively. In general, sensitivity, specificity, and negative predictive value (NPV) were high for the BDGO (100%, 98.5%, and 100%, respectively) and the Xpert MRSA (100%, 98.2%, and 100%, respectively), irrespective of whether or not nasal and inguinal specimens were considered alone or combined. In contrast, the positive predictive value (PPV) was lower: before the resolution of discrepant results, the PPVs for nasal and inguinal specimens alone and combined were 87.5%, 86.7%, and 82.4% for the BDGO and 91.7%, 66.7%, and 92.9% for the Xpert MRSA, respectively. After the resolution of discrepant results, PPVs were 93.8%, 93.3% and 94.1% for the BDGO and 91.7%, 88.9% and 92.9% for the Xpert MRSA, respectively. With the BDGO, 4 of 16 carriers were each identified by nasal or inguinal swabs alone, whereas in the Xpert MRSA group, 4 of 13 carriers were exclusively identified by nasal swabs and 2 of 13 were identified by inguinal swabs alone. Both PCR methods showed no significant difference in the number of discrepant results (odds ratio, 0.70 [P = 0.789]), but specimens from wounds and other body sites (axilla, vagina, and throat) produced discrepancies more often than nasal and groin specimens (odds ratios, 4.724 [P = 0.058] and 12.163 [P < 0.001], respectively). The facts that no false-negative PCR results were detected and increased PPVs were found after the resolution of discrepant results point to PCR as the actual gold standard. Since both sensitivity and NPV were exceptionally high for PCR, backup cultures may, therefore, be unnecessary in an area with low prevalence and with a preemptive isolation strategy but may still be useful for PCR-positive specimens because of the lower PPV for both methods and the possibility of susceptibility testing. The median time for analysis, including extraction, hands-on time, and actual PCR was 2 h 20 min for the Xpert MRSA versus 5 h 40 min for the BDGO. Concerning reporting time, including administration and specimen collection, the Xpert MRSA was faster than the BDGO (7 h 50 min versus 17 h).Methicillin-resistant Staphylococcus aureus (MRSA) strains have become a major concern for health care systems. Prevention of the spread of MRSA has, therefore, become a main goal in the past decade, and active screening programs have been established worldwide (4, 27). Compared to infections caused by methicillin-susceptible S. aureus (MSSA), the organism causes severe infections with increased morbidity and mortality and prolonged hospitalization (9, 17). Unlike countries facing a high prevalence of MRSA, such as the United States and Japan, the prevalence in Switzerland has remained low to date (5, 13, 21, 32). In most parts of our country, prevalence rates between 4% and 7% are observed (19). Apart from its spread in the hospital environment, MRSA carriage in our community, as well as in other countries, seems to be more prevalent than previously assumed (31, 32, 37).To facilitate the rapid detection of colonized patients, real-time PCR assays have been developed. The first method to directly detect MRSA from clinical specimens was developed by Huletsky et al. (20). The principle of this method is used in two commercially available tests, the BD GeneOhm MRSA assay (BDGO) (BD, San Diego, CA) and the Xpert MRSA assay (Cepheid, Inc., Sunnyvale, CA).Recent studies have shown that universal admission surveillance for MRSA was associated with a reduction in MRSA disease (18, 28). Likewise, Cunningham et al. have reported a reduction in MRSA transmissions in a critical care unit. The authors attributed these findings, at least partially, to the availability of rapid PCR screening tests, apart from other measures like improved hygiene measures (10). PCR screening methods are cost efficient, especially in an area of low prevalence where high-risk patients are subjected to preemptive contact isolation (6). Our facility is a 1,000-bed tertiary care teaching hospital with a known low prevalence (<5%) of MRSA colonization of patients and follows a surveillance policy similar to that of the University Hospital of Berne, Switzerland (6). As reported in other studies, this means preemptive isolation on admission of all patients who (i) came from or had traveled to countries with known high prevalence rates for MRSA, (ii) were transferred from long-term care facilities, (iii) were transferred from another health care facility, (iv) were hospitalized within the previous 6 months, and/or (v) had a history of MRSA colonization or infection (6, 8, 23). As soon as PCR is negative for MRSA, patient isolation is ended. Under these circumstances, a rapid screening method with a high negative predictive value (NPV) is desirable, because the bulk of costs emerge mainly from noncolonized patients being unnecessarily isolated. In this study, we compared two real-time PCR methods, the BDGO and Xpert MRSA assays, with broth-enriched culture to assess their performance characteristics and rapidity in an area with a low prevalence of MRSA.     

Rapid Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) in Diverse Clinical Specimens by the BD GeneOhm MRSA Assay and Comparison with Culture

The efficacy of the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) assay was assessed by analyzing nasal swabs and swabs from other body sites for the presence of MRSA in a low-prevalence area. From 681 patients with a high risk for MRSA carriage, 1,601 specimens were collected and transported in Amies agar. After discordant analysis, the sensitivity, specificity, positive predictive value, and negative predictive value of the BD GeneOhm MRSA assay were 84.3%, 99.2%, 88.4%, and 98.9%, respectively, compared to culture.Rapid availability of laboratory results is paramount for early detection of methicillin-resistant Staphylococcus aureus (MRSA) carriers, implementation of efficient control measures, and adequate therapy. To date, detection of MRSA is conventionally done by culture. Several chromogenic culture media have contributed to a more rapid detection of MRSA (24 versus 72 h with conventional culture [6]). In contrast to former PCR procedures, which required previous isolation of the organism via culture (21), the commercial BD GeneOhm MRSA assay (formerly IDI-MRSA; BD-GeneOhm, San Diego, CA) discriminates mecA-positive Staphylococcus aureus from coagulase-negative staphylococci (CoNS) and detects MRSA in clinical specimens within a few hours. This real-time PCR assay has been used particularly in high-prevalence areas (3, 6, 9). In Switzerland, MRSA prevalence ranges between 4 and 7% with the exception of Geneva (>25% [4, 14]). Surveillance strategies involve screening for MRSA from the nose and other body sites. The BD GeneOhm MRSA assay has been approved by the United States Food and Drug Administration (FDA) for the detection of MRSA from nasal swabs stored in liquid Stuart''s medium. Analyzing specimens from other body sites by this test and transportation of the swab in media other than Stuart''s medium are currently not FDA approved for use with this test.In this study, we aimed at assessing (i) the performance of the BD GeneOhm MRSA assay compared to conventional culture in an extended spectrum of clinical specimens and (ii) the suitability of Amies agar as a transport medium for swabs in a low-prevalence setting.(The study has been presented at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007.)During 18 months, 1,601 single swabs from nose (n = 676), groin (n = 643), wound (n = 153), axilla (n = 61), throat (n = 37), rectum (n = 9), vagina (n = 5), and miscellaneous body sites (n = 17) were collected from 681 patients at high risk for MRSA carriage who were admitted to the Luzerner Kantonsspital, the major teaching hospital of central Switzerland, with approximately 1,300 beds, including a children''s hospital. High-risk patients were individuals (i) admitted from outbound hospitals/health care units of high-MRSA-prevalence areas in Switzerland or from hospitals/health care units of foreign countries; (ii) with skin lesions and intravenous drug abuse; or (iii) with a former positive MRSA test. Swabs were collected in Amies agar (Copanswab 108C; Copan, Brescia, Italy). If processing of the swabs was not possible on the same day, swabs were stored overnight at 4°C. Swabs were then broken off into the sample reagent buffer (blue-capped tubes supplied within the kit) and vortexed at high speed for 60 s. The full amount (approximately 1 ml) of buffer (without the swab) was transferred in another tube for cell lysis and DNA extraction according to the instructions of the manufacturer. PCR was performed with the Smart Cycler II instrument (Cepheid, Sunnyvale, CA). Positive and negative controls were included in each run. In the case of PCR inhibition, the sample was briefly frozen at −20°C to remove inhibitors and the run was repeated. Enrichment broth (1 ml; tryptic soy broth [Becton Dickinson, Allschwil, Switzerland] supplemented with 7.5% NaCl) was added to the sample buffer tube containing the swab and incubated at 35°C in ambient air for 24 h. The incubated enrichment broth (approximately 100 μl) was plated on chromogenic agar (Chrom ID MRSA agar; bioMérieux, Marcy l''Etoile, France) at 35°C in ambient air for 48 h. The chromogenic agar was screened for suspect colonies after 24 h and 48 h. Recovered blue colonies were then tested by the Staphaurex Plus coagulase test (Remel Europe Ltd., Dartford, Kent, United Kingdom) and confirmed as S. aureus by the Vitek 2 system (bioMérieux; GP colorimetric identification card; software version 04.03). Confirmation of methicillin resistance was done by disk diffusion testing with 30-μg cefoxitin (bioMérieux) according to the Clinical and Laboratory Standards Institute (7).Staphylococcal strains with discrepant results for the BD GeneOhm MRSA assay and culture were further studied. At first the BD GeneOhm MRSA assay was repeated from the DNA extract of the same specimen to exclude a confusion of samples. MRSA isolates from PCR-negative but culture-positive specimens were retested from subculture by the BD GeneOhm MRSA assay. For specimens with PCR-positive but culture-negative results, the patients'' medical history was studied (antibiotic therapy and previous carriage of MRSA). For these cases gel electrophoresis was performed to determine the size of amplicons in a 2% agarose gel (AgaroseUltraPured; Invitrogen Corporation, Carlsbad, CA). Discrepancies between the BD GeneOhm MRSA assay and culture were resolved according to at least one of the following criteria: PCR-positive but culture-negative results were considered true positive if (i) patients had been decolonized, (ii) the PCR product had the expected molecular size (as described previously by Huletsky et al. [15]), or (iii) there were PCR-positive and culture-positive results from other body sites of the same patient at the same time (Table ​(Table1).1). Sensitivity and specificity for the BD GeneOhm MRSA assay as well as the positive predictive value (PPV) and negative predictive value (NPV) including confidence intervals (CI; according to Wilson''s method [1]) were calculated and compared to culture (Table ​(Table22).

TABLE 1.

Resolution of discrepancies from PCR-positive and culture-negative cases