Specific antisera produced by immunization with precipitin lines

Individual plasma proteins were precipitated, identified and isolated on the basis of line immunoelectrophoresis. A monospecific antibody response was induced by immunization of rabbits with less than 50 ng of precipitated antigen. Preservation of monospecificity was obtained by reimmunization with precipitates developed against the specific antisera.     

The ability of three global plasma assays to recognize thrombophilia

Three global assays, the Calibrated Automated Thrombogram (CAT), the ProC Global (PCG), and the Coagulation Inhibitor Potential (CIP) were performed in frozen plasma samples from 24 normal controls and 24 patients with inherited thrombophilia. Six patients had inherited antithrombin (AT) deficiency; 18 patients had abnormalities in the protein C/S anticoagulant system (protein C deficiency (n=3), protein S deficiency (n=10), homozygous FV Leiden mutation (n=5)). Nine of these twenty four patients carried additionally the heterozygous FV Leiden mutation. All three assays separated the thrombophilia group and the control group (P=0.083 for CAT, P<0.0001 for the other two assays) but there was considerable overlap, particularly in the CAT assay. The CAT assay separated all plasma samples with AT deficiency but was less sensitive to abnormalities in the protein C/S system. In contrast, ProC Global was more sensitive to abnormalities in the protein C system than to AT deficiency. The CIP assay was approximately equally sensitive to defects in both systems. Receiver operator characteristic (ROC) curves confirmed that the ProC Global and the CIP assays performed better than the CAT assay (P=0.0179 and P=0.0003, respectively). With the CIP assay ROC analysis showed that with a sensitivity of 100% the specificity was 87.5%. With the PCG assay, optimal threshold resulted in both a sensitivity and a specificity of 79.2%. Although our material is relatively small, the data suggest that at a cut-off value with a specificity of >80%, the CIP assay should be evaluated as a screening test for severe thrombophilia.     

Development of putative GABAergic neurons in the appendicularian urochordate Oikopleura dioica

Studying the developing brain of urochordates can increase our understanding of brain evolution in the chordate lineage. To begin addressing regional patterns of neuronal differentiation in appendicularian urochordates, we examined the development of putative GABAergic neurons in Oikopleura dioica using GABA immunohistochemistry and in situ hybridization for the GABA-synthesizing enzyme GAD. First, we assessed the developmental dynamics of neuron number and organization in the cerebral and caudal ganglia. We then identified and mapped the positions of putative GABAergic neurons using confocal microscopy. We found GAD mRNA-positive and GABA-immunopositive neurons in the first brain nerves and the cerebral and caudal ganglia, but not in the caudal nerve cord. In both ganglia GAD mRNA-positive and GABA-immunopositive neurons are found in the same characteristic intraganglionic locations. The differentiation of these GABAergic markers occurs first in the first brain nerves and the cerebral ganglion and then with a several-hour delay in the caudal ganglion. In all three structures GAD mRNA expression appears 2-3 hours prior to GABA expression. In general, GABA is expressed by the same number of neurons as express GAD. Several discrepancies suggest differential regulation of the GABAergic phenotype in different neurons, however. Our results show that the GABAergic phenotype has a stereotyped pattern of expression along the anteroposterior axis of the CNS. Given recent genome sequencing and developmental patterning gene studies in this species, the GABAergic neurons in O. dioica provide a good model for assessing, at the invertebrate-vertebrate transition, the molecular mechanisms that specify the GABAergic phenotype.     

Higher Preimplantation Opioid Doses Associated With Long‐Term Spinal Cord Stimulation Failure in 211 Patients With Failed Back Surgery Syndrome

ObjectiveSpinal cord stimulation (SCS) is an effective treatment in failed back surgery syndrome (FBSS). We studied the effect of preimplantation opioid use on SCS outcome and the effect of SCS on opioid use during a two-year follow-up period.Materials and methodsThe study cohort included 211 consecutive FBSS patients who underwent an SCS trial from January 1997 to March 2014. Participants were divided into groups, which were as follows: 1) SCS trial only (n = 47), 2) successful SCS (implanted and in use throughout the two-year follow-up period, n = 131), and 3) unsuccessful SCS (implanted but later explanted or revised due to inadequate pain relief, n = 29). Patients who underwent explantation for other reasons (n = 4) were excluded. Opioid purchase data from January 1995 to March 2016 were retrieved from national registries.ResultsHigher preimplantation opioid doses associated with unsuccessful SCS (ROC: AUC = 0.66, p = 0.009), with 35 morphine milligram equivalents (MME)/day as the optimal cutoff value. All opioids were discontinued in 23% of patients with successful SCS, but in none of the patients with unsuccessful SCS (p = 0.004). Strong opioids were discontinued in 39% of patients with successful SCS, but in none of the patients with unsuccessful SCS (p = 0.04). Mean opioid dose escalated from 18 ± 4 MME/day to 36 ± 6 MME/day with successful SCS and from 22 ± 8 MME/day to 82 ± 21 MME/day with unsuccessful SCS (p < 0.001).ConclusionsHigher preimplantation opioid doses were associated with SCS failure, suggesting the need for opioid tapering before implantation. With continuous SCS therapy and no explantation or revision due to inadequate pain relief, 39% of FBSS patients discontinued strong opioids, and 23% discontinued all opioids. This indicates that SCS should be considered before detrimental dose escalation.     

Norwegian General Practitioners’ Perspectives on Implementation of a Guided Web-Based Cognitive Behavioral Therapy for Depression: A Qualitative Study

Background

Previous research suggests that Internet-based cognitive behavioral therapy (ICBT) has a positive effect on symptoms of depression. ICBT appears to be more effective with therapist support, but it is unclear what this support should comprise. General practitioners (GPs) have positive attitudes toward ICBT. However, ICBT is rarely used in regular care in general practice. More research is warranted to integrate the potential of ICBT as part of regular care.

Objective

The aim of this study was to explore aspects perceived by GPs to affect the implementation of guided ICBT in daily practice. Understanding their perspectives may contribute to improving the treatment of depression in the context of general practice.

Methods

A training package (3-day course) introducing a Norwegian translation of the ICBT program MoodGYM was developed and presented to GPs in Norway. Following training, GPs were asked to include guided ICBT in their regular care of patients with symptoms of depression by providing brief, face-to-face follow-up consultations between modules. We interviewed 11 GPs who had taken the course. Our interview guide comprised open questions that encouraged GPs to frame their responses using examples from their experiences when implementing ICBT. Thematic analysis was chosen to explore patterns across the data.

Results

An overall belief that ICBT would benefit both the patients’ health and the GPs’ own work satisfaction prompted the GPs to take the ICBT course. ICBT motivated them to invest time and effort in improving treatment. The most important motivating aspects in MoodGYM were that a program based on cognitive behavioral therapy could add a structured agenda to their consultations and empower depressed patients. Organizational aspects, such as a lack of time and varied practice, inhibited the use of ICBT. Inadequate knowledge, recalling the program, and changing own habits were also challenging. The GPs were ambivalent about whether ICBT had a negative impact on the doctor–patient interaction in the module follow-ups. Generally, GPs made an effort to recommend MoodGYM, but the expected module follow-ups were often not provided to patients and instead the GPs returned to standard treatment.

Conclusions

GPs’ feedback in the present study contribute to our understanding of the challenges of changing treatment for depression. Our findings indicated that recommending ICBT could add to the GP’s toolkit. Offering training and highlighting the following aspects may increase recommendation of ICBT by GPs: (1) ICBT is theory-based and credible, (2) ICBT increases the GPs’ work satisfaction by having a tool to offer, and (3) ICBT facilitates empowerment of patients in their own health. In addition, the present study also indicated that complex aspects must be accommodated before module follow-ups can be incorporated into GPs’ treatment of depression.     

Increased Inflammation, Reduced Plasma Phospholipid Eicosapentaenoic Acid and Reduced Antioxidant Potential of Treated Hypertensive Patients with Metabolic Syndrome

Purpose

In the present study, we tested whether the presence of metabolic syndrome (MetS) would worsen the features of inflammation, plasma omega 3 fatty acid levels and antioxidant potential in treated hypertensive patients.

Materials and Methods

Two groups were classified by the components of MetS: a reference group of treated hypertensive subjects: hypertension (HTN) group (n = 39) and with more than two additional MetS components: HTN with Mets group (n = 40). We further compared the parameters between HTN group and HTN with MetS group.

Results

The results showed that age (p < 0.001) and body mass index (BMI) (p < 0.001) were significantly different between HTN group and HTN with MetS group. Age- and BMI-adjusted total radical trapping antioxidant potential (TRAP) (p < 0.01) was significantly lower, whereas age- and BMI-adjusted CD (p < 0.05) and interleukin (IL) 6 (p < 0.05) were significantly higher in HTN with MetS group than in HTN group. Moreover, HTN with MetS group had significantly lower levels of age- and BMI-adjusted plasma phospholipid eicosapentaenoic acid (EPA) than HTN group (p < 0.05). On the other hand, the levels of age- and BMI-adjusted intracellular cell adhesion molecule-1 (ICAM-1), adiponectin and high molecular weight (HMW)-adiponectin were not significantly different between the groups.

Conclusion

In conclusion, our results showed increased inflammatory marker, reduced antioxidant potential and EPA levels in treated hypertensive patients in the presence of MetS, suggesting the importance of changes of therapeutic lifestyle to modify the features of MetS.     

Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms

Whereas the stress-inducible heat-shock protein 70 (Hsp70) has gained plenty of attention as a putative target for tumor therapy, little is known about the role of other Hsp70 proteins in cancer. Here we present the first thorough analysis of the expression and function of the cytosolic Hsp70 proteins in human cancer cells and identify Hsp70-2, a protein essential for spermatogenesis, as an important regulator of cancer cell growth. Targeted knock-down of the individual family members by RNA interference revealed that both Hsp70 and Hsp70-2 were required for cancer cell growth, whereas the survival of tumorigenic as well as nontumorigenic cells depended on Hsc70. Cancer cells depleted for Hsp70 and Hsp70-2 displayed strikingly different morphologies (detached and round vs. flat senescent-like), cell cycle distributions (G2/M vs. G1 arrest) and gene expression profiles. Only Hsp70-2 depletion induced the expression of macrophage inhibitory cytokine-1 that was identified as a target of P53 tumor-suppressor protein and a mediator of the G1 arrest and the senescent phenotype. Importantly, concomitant depletion of Hsp70 and Hsp70-2 had a synergistic antiproliferative effect on cancer cells. Thus, highly homologous Hsp70 proteins bring about nonoverlapping functions essential for cell growth and survival.     

An integrative approach to CTL epitope prediction: a combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions

Reverse immunogenetic approaches attempt to optimize the selection of candidate epitopes, and thus minimize the experimental effort needed to identify new epitopes. When predicting cytotoxic T cell epitopes, the main focus has been on the highly specific MHC class I binding event. Methods have also been developed for predicting the antigen-processing steps preceding MHC class I binding, including proteasomal cleavage and transporter associated with antigen processing (TAP) transport efficiency. Here, we use a dataset obtained from the SYFPEITHI database to show that a method integrating predictions of MHC class I binding affinity, TAP transport efficiency, and C-terminal proteasomal cleavage outperforms any of the individual methods. Using an independent evaluation dataset of HIV epitopes from the Los Alamos database, the validity of the integrated method is confirmed. The performance of the integrated method is found to be significantly higher than that of the two publicly available prediction methods BIMAS and SYFPEITHI. To identify 85% of the epitopes in the HIV dataset, 9% and 10% of all possible nonamers in the HIV proteins must be tested when using the BIMAS and SYFPEITHI methods, respectively, for the selection of candidate epitopes. This number is reduced to 7% when using the integrated method. In practical terms, this means that the experimental effort needed to identify an epitope in a hypothetical protein with 85% probability is reduced by 20-30% when using the integrated method.The method is available at http://www.cbs.dtu.dk/services/NetCTL. Supplementary material is available at http://www.cbs.dtu.dk/suppl/immunology/CTL.php.     

Requests for euthanasia or physician-assisted suicide from older persons who do not have a severe disease: an interview study

OBJECTIVE: To determine how often requests are made for euthanasia and physician-assisted suicide (EAS) in the absence of severe disease and how such requests are dealt with in medical practice in The Netherlands. METHOD: Retrospective interview study. Participants: 125 general practitioners (GPs), 77 nursing home physicians (NHPs), and 208 clinical specialists. RESULTS: In The Netherlands, each year approximately 400 people request EAS, because they are 'weary of life'. Thirty per cent of all physicians have at some time received an explicit request for EAS in the absence of severe disease; 3% of all physicians had granted a request for EAS in such a case. Most requests for EAS to GPs in the absence of severe disease (n = 29) were made by single people aged 80 years and over. While their problems were most frequently of a social nature, 79% had one or more non-severe illnesses. Most GPs refused the request; half of them proposed an alternative treatment, which the patient often refused. Nineteen people who did not receive any treatment persisted in their wish to die; the request for EAS from 5 out of 10 patients who received one or more types of treatment was withdrawn or became less explicit. CONCLUSIONS: Most physicians in The Netherlands refuse requests for EAS in the absence of severe disease. Most patients persist in their request. In an ageing population more research is needed to provide physicians with practical interventions to prevent suicide and to make life bearable and satisfactory for elderly people who wish to die.