Analysis of minor hemoglobins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

BACKGROUND: Hemoglobin (Hb) heterogeneity arises mainly from posttranslational modifications of the globin chains, and cation-exchange chromatography reveals falsely increased concentrations of some minor Hbs in the presence of abnormal Hbs. Here we describe a method for identification of the globin chains and their posttranslational modifications contained in the Hb fractions. METHODS: We used cation-exchange HPLC (PolyCAT A column) for separation of Hb fractions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for analysis of the separated globin chains. Globin chains were identified by their molecular masses. Posttranslational modifications of globin chains were identified by digestion of the proteins with endoproteinase V8 before MALDI-TOF MS of the resulting peptides. RESULTS: Analysis of the HbA2 fractions of patients with HbS revealed 4 different globin chains. We found, in addition to the expected alpha- and delta-chains, the carbamylated alpha- and the betaS-chains. Additionally, we analyzed HbH, Hb Barts, HbA 1b, pre-HbA 1c, HbA 1c, HbF1, HbF, HbA 1d3a, HbA 1d3b, HbA2, and HbC1 fractions from control and pathologic blood samples. We identified several posttranslational modifications of the globin chains, such as pyruvatization, glycation, acetylation, carbamylation, and acetaldehyde adduct formation. CONCLUSIONS: The native and posttranslationally modified globin chains in minor and major Hbs are unambiguously identified by MALDI-TOF MS. A minor Hb containing the carbamylated alpha- and the betaS-chain elutes at the same time as normal HbA2 (alpha2delta2) and thus leads to falsely increased HbA2 values in patients with HbS when blood is analyzed with PolyCAT A chromatography.     

Dementia considered? Safety-relevant communication between health care settings: a systematic review

Aim

To identify literature on safety-relevant handover communication, as operationalized by an adapted Lasswell communication model, and to reveal the extent to which this literature is relevant to patients with dementia (PwD). Furthermore, the study identifies interventions that could be applicable in dementia handover communication.

Methods

A systematic review was performed. MEDLINE, EMBASE, Cochrane Library, CINAHL, PsycInfo and GeroLit were searched for publications indexed up to and including April 2011. Forward citation tracking was conducted in January 2012 using the SCOPUS database. The inclusion criteria followed an adapted Lasswell communication model. Evidence was excluded that did not meet more than three-quarters of the critical appraisal’s 27 quality criteria. A content analysis and a critical appraisal were performed to eliminate ineligible publications.

Results

Out of 3,918 positive search results, a total of 5 publications were ultimately eligible for inclusion. Only one of them included PwD in the intervention group. However, since dementia was not the focus of the study, the paper contained no discussion of the results specifically for PwD.

Conclusion

There are several options for improving safety-relevant handover communication between settings, but strong evidence is still lacking, particularly for PwD. Research involving PwD should include topics such as the role of advanced nurse practitioners (APN), the information needs of everyone involved in the handover and appropriate involvement of informal caregivers. Responsibilities in care should be defined and specific communication processes implemented. Further investigation and application of Naylor et al.’s (2007) transitional care model could be particularly helpful in dementia research.     

Differentiated control of deranged nitric oxide metabolism: a therapeutic option in sepsis?

Derangement of nitric oxide (NO) metabolism represents one of the key mechanisms contributing to macro- and microcirculatory failure in sepsis. Sepsis-related therapy combining fluid resuscitation with administration of vasopressor and inotropic agents, however, does not guarantee correction of maldistributed nutritive perfusion between and within organs. Therefore, the differentiated and selective pharmacologic modulation of NO-mediated vascular function could play a useful role in hemodynamic management of patients with sepsis. This viewpoint carefully evaluates the potential role of intentionally using partially opposing effects of NO donors and NO synthase inhibitors to complement current therapy of hemodynamic stabilization in patients with sepsis.     

Determination of breath isoprene allows the identification of the expiratory fraction of the propofol breath signal during real-time propofol breath monitoring

Real-time measurement of propofol in the breath may be used for routine clinical monitoring. However, this requires unequivocal identification of the expiratory phase of the respiratory propofol signal as only expiratory propofol reflects propofol blood concentrations. Determination of CO₂ breath concentrations is the current gold standard for the identification of expiratory gas but usually requires additional equipment. Human breath also contains isoprene, a volatile organic compound with low inspiratory breath concentration and an expiratory concentration plateau. We investigated whether breath isoprene could be used similarly to CO₂ to identify the expiratory fraction of the propofol breath signal. We investigated real-time breath data obtained from 40 study subjects during routine anesthesia. Propofol, isoprene, and CO₂ breath concentrations were determined by a combined ion molecule reaction/electron impact mass spectrometry system. The expiratory propofol signal was identified according to breath CO₂ and isoprene concentrations and presented as median of intervals of 30 s duration. Bland–Altman analysis was applied to detect differences (bias) in the expiratory propofol signal extracted by the two identification methods. We investigated propofol signals in a total of 3,590 observation intervals of 30 s duration in the 40 study subjects. In 51.4 % of the intervals (1,844/3,590) both methods extracted the same results for expiratory propofol signal. Overall bias between the two data extraction methods was ?0.12 ppb. The lower and the upper limits of the 95 % CI were ?0.69 and 0.45 ppb. Determination of isoprene breath concentrations allows the identification of the expiratory propofol signal during real-time breath monitoring.     

The CGRP receptor antagonist BIBN4096BS peripherally alleviates inflammatory pain in rats

Calcitonin gene–related peptide (CGRP) is known to play a major role in the pathogenesis of pain syndromes, in particular migraine pain. Here we focus on its implication in a rat pain model of inflammation, induced by injection of complete Freund adjuvant (CFA). The nonpeptide CGRP receptor antagonist BIBN4096BS reduces migraine pain and trigeminal neuronal activity. Here we demonstrate that the compound reduces inflammatory pain and spinal neuronal activity. Behavioural experiments reveal a reversal of the CFA-induced mechanical hypersensitivity and monoiodoacetate (MIA)-induced weight-bearing deficit in rats after systemic drug administration. To further investigate the mechanism of action of the CGRP antagonist in inflammatory pain, in vivo electrophysiological studies were performed in CFA-injected rats. Recordings from wide dynamic range neurons in deep dorsal horn layers of the lumbar spinal cord confirmed a reduction of neuronal activity after systemic drug application. The same amount of reduction occurred after topical administration onto the paw, with resulting systemic plasma concentrations in the low nanomolar range. However, spinal administration of BIBN4096BS did not modify the neuronal activity in the CFA model. Peripheral blockade of CGRP receptors by BIBN4096BS significantly alleviates inflammatory pain.     

Work Ability Index Predicts Application for Disability Pension After Work-Related Medical Rehabilitation for Chronic Back Pain

Objective

To determine whether the Work Ability Index (WAI), a short 7-item self-report questionnaire addressing issues of perceived disability, impairment, and expectations for resuming work, predicts application for disability pension, recommendations for further treatment, and other adverse work-related criteria in patients with chronic back pain after rehabilitation.

Design

Cohort study with 3-month follow-up.

Setting

Seven inpatient rehabilitation centers.

Participants

Patients (N=294; 168 women; mean age, 49.9y) with chronic back pain.

Intervention

The WAI was completed at the beginning of rehabilitation. All patients were treated according to the German rehabilitation guidelines for chronic back pain and work-related medical rehabilitation.

Main Outcome Measure

Application for disability pension, as assessed by a postal questionnaire 3 months after discharge.

Results

Receiver operating characteristic curve analysis of the association between the WAI at baseline and subsequent application for disability pension revealed an area under the curve of .80 (95% confidence interval [CI], .62–.97). Youden index was highest when the WAI cutoff value was ≤20 points (sensitivity, 72.7%; specificity, 82.2%; total correct classification, 81.7%). After adjusting for age and sex, persons with a baseline WAI score of ≤20 points had 15.6 times (95% CI, 3.6–68.2) higher odds of subsequent application for disability pension, 4.9 times (95% CI, 1.5–16.8) higher odds of unemployment, and 6 times (95% CI, 2.4–15.2) higher odds of long-term sick leave at follow-up.

Conclusions

The WAI could help rehabilitation professionals identify patients with back pain with a high risk of a subsequent application for disability pension.     

Choi response criteria for prediction of survival in patients with metastatic renal cell carcinoma treated with anti-angiogenic therapies

Objective

Anti-angiogenic drugs cause a reduction in tumour density (Choi criteria) first and then in size [Response Evaluation Criteria In Solid Tumours (RECIST)]. The prognostic significance of changes in tumour density in metastatic renal cell carcinoma (mRCC) is unknown and was assessed in this study.

Methods

The prognostic significance of partial response (PR) as opposed to non-response [stable disease (SD) + progressive (PD)] to anti-angiogenic therapy was assessed in patients with mRCC separately for both criteria using the log-rank test and Cox regression models.

Results

Both criteria were applied to 35 patients. The response was identical for all eight patients with PR and most patients with PD (10/12) when using the RECIST and Choi criteria. Adding tumour density information, 14 patients with SD were re-categorised as having PR (7), SD (4), and PD (3). Patients with PR (Choi) were progression free significantly longer [hazard ratio (HR) 0.24; 95 % CI 0.10–0.57; P?=?0.001] and had better overall survival (HR 0.36; 95 % CI 0.15–0.89; P?=?0.026) compared to patients with SD or PD. The predictive value of PR according to RECIST was not statistically significant.

Conclusions

In mRCC, the Choi criteria separate prognostic groups better when compared with RECIST. This may allow early discrimination of patients benefiting from continued treatment.

Key Points

? CT is widely used to assess patients with metastatic renal cell carcinoma. ? Various algorithms can be applied for tumour therapy control in patients with mRCC. ? Follow-up should be based on evaluation of the tumour size and density. ? RECIST is based only on tumour shrinkage and might lead to wrong conclusions.     

Mucosal application of cationic poly(d,l-lactide-co-glycolide) microparticles as carriers of DNA vaccine and adjuvants to protect chickens against infectious bursal disease

Infectious bursal disease virus (IBDV) is an immunosuppressive virus of chickens. The virus protein (VP) 2 induces neutralizing antibodies, which protect chickens against the disease. The aim of this study was to develop a cationic poly(d,l-lactide-co-glycolide) (PLGA) microparticle (MP) based IBDV-VP2 DNA vaccine (MP-IBDV-DNA) for chickens to be delivered orally and by eye drop route. The tested IBDV-VP2 DNA vaccines were immunogenic for specific-pathogen-free chickens and induced an antibody response after intramuscular application. Co-inoculation with a plasmid encoding chicken IL-2 (chIL-2) or CpG-ODN did not significantly improve protection against IBDV challenge. However, the application of a MP-IBDV-DNA vaccine alone or in combination with a delayed oral and eye drop application of cationic MP loaded with CpG-ODN or chIL-2 improved protection against challenge. The MP-IBDV-DNA-vaccinated chickens showed less pathological and histopathological bursal lesions, a reduced IBDV antigen load as well as T-cell influx into the bursa of Fabricius (BF) compared to the other groups (p < 0.05). The addition of chIL-2 loaded MP improved challenge virus clearance from the BF as demonstrated by lower neutralizing antibody titers and reduced IL-4 and IFN-α mRNA expression in the bursa at 7 days postchallenge compared to the other challenged groups. Overall, the efficacy of the IBDV-DNA vaccine was improved by adsorption of the DNA vaccine onto cationic PLGA-MP, which also allowed mucosal application of the DNA vaccine.     

Ultraviolet irradiation induces apoptosis in human immature, but not in skin mast cells

As diverse pruritic cutaneous diseases respond to ultraviolet treatment, we have examined whether ultraviolet light is capable of inducing apoptosis in mast cells. Human mast cell line 1 (HMC1) derived from a patient with malignant mastocytosis and purified skin mast cells were irradiated with single doses of ultraviolet B or ultraviolet A1, or pretreated with 8-methoxypsoralen prior to ultraviolet A1 exposure. After 0 to 48 h of incubation, the percentage of apoptotic and dead cells was assessed. In HMC1 cells, morphologic features of apoptosis were further evaluated by electron microscopy. All ultraviolet treatment induced apoptosis of HMC1 cells in a time- and dose-dependent manner. Apoptosis was associated with activation of caspase-3, release of cytochrome C, cleavage of poly(ADP-ribose)-polymerase, and nuclear accumulation of p53. In contrast, resting skin mast cells were resistant to ultraviolet light induced apoptosis. After incubation with stem cell factor and interleukin-4 for 2 wk, however, slowly proliferating skin mast cells also underwent apoptosis in response to ultraviolet light. In conclusion, these data demonstrate that ultraviolet light directly affects mast cells, but mainly aims at the proliferating mast cells as found in mastocytosis and mast cell dependent pruritic diseases, where increased numbers are observed due to the recruitment mast cell precursors from the blood.