Lack of correlation between Treg quantification assays in inflammatory bowel disease patients

AIM:To compare the number of regulatory T-cells( Tregs) measured by flow cytometry with those obtained using a real-time quantitative PCR(q PCR) method in patients suffering from inflammatory bowel disease(IBD).METHODS:Tregs percentages obtained by both flow cytometry and q PCR methods in 35 adult IBD patients,18 out of them with Crohn′s disease(CD)and 17 with ulcerative colitis(UC)were compared to each other as well as to scores on two IBD activity questionnaires using the Harvey Bradshaw Index(HBI)for CD patients and the Simple Colitis Clinical Activity Index(SCCAI)for UC patients.The Treg percentages by flow cytometry were defined as CD4+CD25highCD127lowFOXP3+cells in peripheral blood mononuclear cells,whereas the Treg percentages by q PCR method were determined as FOXP3 promoter demethylation in genomic DNA.RESULTS:We found an average of 1.56%±0.78%Tregs by using flow cytometry,compared to 1.07%±0.53%Tregs by using q PCR in adult IBD patients.There were no significant correlations between either the percentages of Tregs measured by flow cytometry or q PCR and the HBI or SCCAI questionnaire scores in CD or UC patients,respectively.In addition,there was no correlation between Treg percentages measured by q PCR and those measured by flow cytometry(r=-0.06,P=0.73;Spearman Rho).These data suggest that,either Treg-related immune function or the clinical scores in these IBD patients did not accurately reflect actual disease activity.Until the cause(s)for these differences are more clearly defined,the resultssuggest caution in interpreting studies of Tregs in various inflammatory disorders.CONCLUSION:The two methods did not produce equivalent measures of the percentage of total Tregs in the IBD patients studied which is consistent with the conclusion that Tregs subtypes are not equally detected by these two assays.     

Immunoguided Laser Assisted Microdissection Techniques for DNA Methylation Analysis of Archival Tissue Specimens

Altered DNA methylation is a fundamental characteristic of carcinogenesis. The analysis of DNA methylation in tumor cells may help to better understand tumor pathogenesis and more importantly may be used as diagnostic tool with therapeutic consequences. To detect targets relevant in tumorigenesis, it is essential to separate neoplastic cells from nonneoplastic cells. An excellent method for isolating specific cells is laser-assisted microdissection (LAM). Target cell identification for immunoguided LAM (ILAM) requires immunohistochemistry (IHC). Yet, it is unclear whether IHC for ILAM influences DNA methylation. The goals of this study were to establish an optimized protocol for antigen retrieval and IHC of formalin-fixed paraffin-embedded (FFPE) specimens suitable for ILAM and to evaluate its effect on the DNA methylome using a high throughput array. Using ten archival FFPE specimens, we showed specific staining suitable for ILAM. Extracted DNA from microdissected cells of immunohistochemically or H&E-stained tissue sections showed identical DNA quality and a strong correlation (r = 0.94 to 0.98) for CpG target methylation of 1505 analyzed sites in a series of five paired samples. No differential methylation between H&E and IHC was detected in 1501 of 1505 CpG targets (99.7%; P < 0.05). These results demonstrate the validity and utility of the herein described protocol, which allows the application of ILAM for large-scale genomic and epigenetic analyses of archival tissue specimens.Epigenetics is defined as the study of heritable changes in gene expression that are not attributable to alterations in the DNA sequence.¹ The best-known epigenetic marker is DNA methylation. DNA methylation plays an important role in the control of gene activity and the architecture of the nucleus.² During carcinogenesis, DNA of tumor cells is characterized by hypomethylation in gene regions that promote tumor development, whereas hypermethylation occurs among tumor suppressor genes.^3,4 Especially for large-scale analysis of cancer tissue methylation, it is important to investigate the tumor cells and not the intermixed inflammatory cells or other normal host cells to identify targets relevant in tumorigenesis. Therefore, laser-assisted microdissection (LAM) is a helpful tool to isolate cell populations for cell-specific molecular profiling.⁵ Typically, the identification of target cells relies on morphological cell characteristics observed after routine histological staining. The gold standard for histopathological diagnosis is hematoxylin-eosin (H&E) staining.⁶ However, depending on the type of tissue and the cells of interest, H&E staining may be inadequate for a clear identification of some cell types. Optical resolution is especially impaired when using the LAM scope, because LAM is not compatible with use of a cover slide and selection of cells with less distinct morphological characteristics can be hampered under these conditions. In cases where routine staining does not allow discrimination between normal and pathological cell morphology, visualization of cell surface or intracellular structures can be helpful. For example, labeling of cellular proteins by immunohistochemistry (IHC) can assist in identification of specific target cells. The application of IHC to LAM, also called immunoguided LAM (ILAM), is a valuable technique that allows localization of target cells and microdissection of particular cell populations. In addition, IHC enables the use of automatic cell recognition programs included in the advanced series of LAM instruments. By using LAM devices with automatic cell recognition programs it is technically feasible to collect high numbers of target cells in a reasonable time.⁷Currently antigen retrieval (AR) and IHC on positively charged glass slides is a widely established method in routine pathology laboratories. The original laser capture microdissection system was based on an infrared (IR) laser that captured the cells of interest from tissue sections mounted on glass slides.⁵ However, all current microdissection systems incorporate an UV cutting laser that is not suitable for positively charged glass slides but requires special polyethylene naphthalate (PEN) membrane slides. IHC on membrane slides, particularly common AR procedures required for formalin-fixed paraffin embedded (FFPE) tissue samples, usually result in loss of parts of the tissue or the entire tissue section. For that reason regularly used AR methods at high temperature (90°C) are not suitable for ILAM. More importantly, it is unclear whether IHC for ILAM influences DNA integrity or DNA methylation of target cells from archival tissue samples. The goals of this study were to establish an optimized protocol for AR and IHC of FFPE tissue samples suitable for ILAM and to determine whether the DNA methylation profile is affected by AR or IHC. For establishing this protocol, we selected FFPE specimens from primary mediastinal large B-cell lymphoma (PMLBCL) as well as cases of classical Hodgkin''s lymphoma (CHL), the latter representing a model of a highly challenging LAM.     

The monoclonal CD4 antibody M-T413 inhibits cellular infection with human immunodeficiency virus after viral attachment to the cell membrane: an approach to postexposure prophylaxis.

Infectious cellular uptake of human immunodeficiency virus (HIV) is initiated by a complex sequence of interactions between the viral envelope gp120/gp41 complex and the cellular CD4 receptor resulting in the exposure of a hydrophobic region of gp41 that mediates the irreversible fusion of the virus with the cell membrane. Here we show that viral penetration into a susceptible cell can be inhibited by the high-affinity monoclonal CD4 antibody (CD4 mAb) M-T413 even when it is added as late as 30-120 min after the initial contact of virus with the cell membrane. Inhibition of infection was assessed by monitoring cultures for 34 days after exposure to virus using four different methods simultaneously, including detection of viral DNA by PCR. The interval during which HIV remains sensitive to postbinding neutralization by CD4 mAb depends on strain of virus and type of target cell. Preparations of recombinant soluble CD4 (and the immunoadhesin CD4-IgG1) were much less efficient when compared with mAb M-T413, particularly in blocking infection by fresh HIV-1 isolates. Also cellular transmission of HIV, as determined by syncytia formation within 24 hr, was prevented by mAb M-T413 when added within 45 min of contact of infected H9 cells with uninfected C8166 cells. Together with the favorable clinical experience obtained with CD4 mAbs as immunomodulatory drugs, these data suggest that infusion of CD4 mAb M-T413 may be a therapeutic modus for immediate prophylactic intervention after occupational exposure to HIV and for prevention of intrapartum mother-to-infant HIV transmission.     

Gunshot Injuries in the Elderly: Patterns and Outcomes. A National Trauma Databank Analysis

Background  

Trauma in the elderly (≥55 years) accounts for a significant proportion of admissions to trauma centers. Our understanding of the epidemiology and outcomes associated with penetrating injury in this age segment of the population, however, is severely limited. The aim of the present study therefore was to investigate the incidence and type of injuries sustained by elderly patients from firearms and the impact of age on outcomes.     

Chronic thromboembolic pulmonary hypertension — assessment by magnetic resonance imaging

Chronic thromboembolic pulmonary hypertension (CTEPH) is a severe disease that has been ignored for a long time. However, with the development of improved therapeutic modalities, cardiologists and thoracic surgeons have shown increasing interest in the diagnostic work-up of this entity. The diagnosis and management of chronic thromboembolic pulmonary hypertension require a multidisciplinary approach involving the specialties of pulmonary medicine, cardiology, radiology, anesthesiology and thoracic surgery. With this approach, pulmonary endarterectomy (PEA) can be performed with an acceptable mortality rate. This review article describes the developments in magnetic resonance (MR) imaging techniques for the diagnosis of chronic thromboembolic pulmonary hypertension. Techniques include contrast-enhanced MR angiography (ce-MRA), MR perfusion imaging, phase-contrast imaging of the great vessels, cine imaging of the heart and combined perfusion-ventilation MR imaging with hyperpolarized noble gases. It is anticipated that MR imaging will play a central role in the initial diagnosis and follow-up of patients with CTEPH. This article is dedicated to Manfred Thelen, MD, Professor of Radiology, our teacher in clinical radiology. This study was supported by the Deutsche Forschungsgemeinschaft (grant FOR 474/1).     

Thromboembolic Prophylaxis with Heparin in Patients with Blunt Solid Organ Injuries Undergoing Non-operative Treatment

Background

Patients with blunt solid organ injuries (SOI) are at risk for venous thromboembolism (VTE), and VTE prophylaxis is crucial. However, little is known about the safety of early prophylactic administration of heparin in these patients.

Methods

This is a retrospective study including adult trauma patients with SOI (liver, spleen, kidney) undergoing non-operative management (NOM) from 01/01/2009 to 31/12/2014. Three groups were distinguished: prophylactic heparin (low molecular weight heparin or low-dose unfractionated heparin) ≤72 h after admission (‘early heparin group’), >72 h after admission (‘late heparin group’), and no heparin (‘no heparin group’). Patient and injury characteristics, transfusion requirements, and outcomes (failed NOM, VTE, and mortality) were compared between the three groups.

Results

Overall, 179 patients were included; 44.7% in the ‘early heparin group,’ 34.6% in the ‘late heparin group,’ and 20.8% in the ‘no heparin group.’ In the ‘late heparin group,’ the ISS was significantly higher than in the ‘early’ and ‘no heparin groups’ (median 29.0 vs. 17.0 vs. 19.0; p < 0.001). The overall NOM failure rate was 3.9%. Failed NOM was significantly more frequent in the ‘no heparin group’ compared to the ‘early’ and ‘late heparin groups’ (10.8 vs. 3.2 vs. 1.3%; p = 0.043). In the ‘early heparin group’ 27.5% patients suffered from a high-grade SOI; none of these patients failed NOM. Mortality did not differ significantly. Although not statistically significant, VTE were more frequent in the ‘no heparin group’ compared to the ‘early’ and ‘late heparin groups’ (10.8 vs. 4.8 vs. 1.3%; p = 0.066).

Conclusion

In patients with SOI, heparin was administered early in a high percentage of patients and was not associated with an increased NOM failure rate or higher in-hospital mortality.

     

Spontaneous priapism after radical retropubic prostatectomy

We present the first case of priapism following radical prostatectomy. A 66-year-old man with normal erections underwent radical retropubic prostatectomy with unilateral nerve sparing. Pathology showed a pT2c pN0 Gleason score 3 + 3 = 6 prostate cancer and the postoperative course was uneventful. Ten days after surgery he recognized a spontaneous painful penile erection without sexual stimulation which occurred in a standing position and disappeared in a supine position. These episodes recurred several times during the next 3 weeks and then completely vanished. Pathophysiologically, we postulate intermittent position-depending venous obstruction due to local hematoma or thrombosis.     

A novel technique to follow fast PaO2 variations during experimental CPR

OBJECTIVE: An ultrafast responding fluorescent-quenching PO2 probe allows time-resolved, in vivo measurement of PO2. This study describes several validation experiments of this new device in vitro, and reports its first use during cardiopulmonary resuscitation in an animal model of cardiac arrest. METHODS: The influence of CO2, temperature and motion artefacts on the signal response of the PO2 probe was analysed in vitro by systematic variation of these values. Thereafter, with approval of the Review Board for the care and use of animals, CPR was performed in four pigs. The PaO2 course was recorded continuously at time resolution of <80 ms in the abdominal aorta using an uncoated fluorescence-quenching probe (Foxy AL-300, OceanOptics Inc., USA). RESULTS: In vitro experiments showed that signal intensity is dependent on CO2 concentration (DeltaPfaO2=4 mmHg/vol.% CO2) and temperature (DeltaPfaO2=16 mmHg/ degrees C), but it is robust with regards to probe motion. In the animal experiments, the uncoated fluorescence-quenching probe was calibrated by repeated simultaneous measurements with the Paratrend 7 sensor to correct the PfaO2 for a potential signal drift, PCO2 and temperature variations. In all animal experiments, the individual PaO2 courses were clearly related to therapeutic interventions and their haemodynamic effects during CPR and allowed recording of ultrafast PO2 changes with a time resolution of 80 ms. CONCLUSIONS: The results demonstrate the feasibility of ultrafast PO2 measurement during CPR and low-flow states. They also demonstrate very rapid systemic effects of CPR upon aortic PO2. Among many other useful applications, the information derived from this technique may help to define the optimum conditions for successful defibrillation and restoration of spontaneous circulation.     

Memory for performed actions in dementia of Alzheimer type: further evidence for a global semantic memory deficit

Findings in patients with dementia of the Alzheimer type (AD patients) concerning the enactment effect, i.e. reaching a better memory performance after encoding actions by performing them in contrast to encoding them by reading, are ambiguous. In order to get more insight into memory deterioration in patients suffering from AD, an experimental study was run. Patients with mild-to-moderate AD (n = 23) and healthy elderly control persons (n = 20) took part. They had to encode actions verbally as well as enactively. Memory performance was tested by using recognition judgments instead of the predominating free recall. The AD patients showed no enactment effect and a rate of false alarm judgments that was more than twice as high as found in healthy elderly controls. This enactive encoding failure may be indicative of memory deficits underlying the conceptual system of action representations.     

Antibiotic prophylaxis in day surgery in dermatology

Antibiotic prophylaxis for the prevention of surgical wounds infections is still a matter of debate in dermatology. The authors have performed an open and randomized study on the prophylactic efficacy of azithromycin, 500 mg per os 1 h prior to the procedure in a dermosurgical office. The absence of postsurgical infections when the intervention has been executed in sites without risk of contamination both in the treated and no treated group, has demonstrated that, in the presence of correct prophylactic measures, no antibiotic prophylaxis is necessary. On the other hand, when the procedure has been performed in sites under risk of infection, the antibiotic prophylaxis has instead demonstrated a significant efficacy. The study has documented that azithromycin allows to efficacely prevent bacterical suprainfection with a good compliance of the patient in the cases in which dermosurgical activity is performed in sites with risk of infection (face, scalp, genitals, perineo, feet).