BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

Identification of noninvasive biomarkers for alcohol-induced liver disease using urinary metabolomics and the Ppara-null mouse

Alcohol-induced liver disease (ALD) is a leading cause of nonaccident-related deaths in the United States. Although liver damage caused by ALD is reversible when discovered at the earlier stages, current risk assessment tools are relatively nonspecific. Identification of an early specific signature of ALD would aid in therapeutic intervention and recovery. In this study, the metabolic changes associated with ALD were examined using alcohol-fed male Ppara-null mouse as a model of ALD. Principal components analysis of the mass spectrometry-based urinary metabolic profile showed that alcohol-treated wild-type and Ppara-null mice could be distinguished from control animals without information on history of alcohol consumption. The urinary excretion of ethyl-sulfate, ethyl-beta-d-glucuronide, 4-hydroxyphenylacetic acid, and 4-hydroxyphenylacetic acid sulfate was elevated and that of the 2-hydroxyphenylacetic acid, adipic acid, and pimelic acid was depleted during alcohol treatment in both wild-type and the Ppara-null mice albeit to different extents. However, indole-3-lactic acid was exclusively elevated by alcohol exposure in Ppara-null mice. The elevation of indole-3-lactic acid is mechanistically related to the molecular events associated with development of ALD in alcohol-treated Ppara-null mice. This study demonstrated the ability of a metabolomics approach to identify early, noninvasive biomarkers of ALD pathogenesis in Ppara-null mouse model.

Tenocytes of chronic rotator cuff tendon tears can be stimulated by platelet-released growth factors

BACKGROUND Bone-to-tendon healing after rotator cuff repairs is mainly impaired by poor tissue quality. The tenocytes of chronic rotator cuff tendon tears are not able to synthesize normal fibrocartilaginous extracellular matrix (ECM). We hypothesized that in the presence of platelet-released growth factors (PRGF), tenocytes from chronically retracted rotator cuff tendons proliferate and synthesize the appropriate ECM proteins. MATERIALS AND METHODS Tenocytes from 8 patients with chronic rotator cuff tears were cultured for 4 weeks in 2 different media: standard medium (Iscove's Modified Dulbecco's Media + 10% fetal calf serum + 1% nonessential amino acids + 0.5 μg/mL ascorbic acid) and media with an additional 10% PRGF. Cell proliferation was assessed at 7, 14, 21, and 28 days. Messenger (m)RNA levels of collagens I, II, and X, decorin, biglycan, and aggrecan were analyzed using real time reverse-transcription polymerase chain reaction. Immunocytochemistry was also performed. RESULTS The proliferation rate of tenocytes was significantly higher at all time points when cultured with PRGF. At 21 days, the mRNA levels for collagens I, II, and X, decorin, aggrecan, and biglycan were significantly higher in the PRGF group. The mRNA data were confirmed at protein level by immunocytochemistry. CONCLUSIONS PRGFs enhance tenocyte proliferation in vitro and promote synthesis of ECM to levels similar to those found with insertion of the normal human rotator cuffs. CLINICAL RELEVANCE Biologic augmentation of repaired rotator cuffs with PRGF may enhance the properties of the repair tissue. However, further studies are needed to determine if application of PRGF remains safe and effective in long-term clinical studies. LEVEL OF EVIDENCE Basic Science Study, Cell Biology.

Learning to drive: learners' self-reported cognitive failure level predicts driving instructor's observation rating of driving performance

OBJECTIVES Evidence increases that cognitive failure may be used to screen for drivers at risk. Until now, most studies have relied on driving learners. This exploratory pilot study examines self-report of cognitive failure in driving beginners and error during real driving as observed by driving instructors. METHODS Forty-two driving learners of 14 driving instructors filled out a work-related cognitive failure questionnaire. Driving instructors observed driving errors during the next driving lesson. In multiple linear regression analysis, driving errors were regressed on cognitive failure with the number of driving lessons as an estimator of driving experience controlled. RESULTS Higher cognitive failure predicted more driving errors (p < .01) when age, gender and driving experience were controlled in analysis. CONCLUSIONS Cognitive failure was significantly associated with observed driving errors. Systematic research on cognitive failure in driving beginners is recommended.

Analysis of clinical forensic examination reports on sexual assault

Medical-forensic examination of sexual assault victims and alleged offenders is a common task of many forensic institutes. In the current study, the results from samples taken at the Institute of Legal Medicine, Hanover Medical School, during a period from 2005 to 2007 were retrospectively evaluated. In total, 292 victims (283 females and nine males) and 88 suspects were examined. At the time of the assault, 41.8% of the victims and 43.2% of the alleged perpetrators were under the influence of alcohol. Injuries were found in 84.9% of the victims and 39.8% of the suspects. Thirty victims (10.3%) reported having been choked or strangled. Cytology was performed in 218 victims. In 81 cases (38.0%), sperm could be detected in vaginal swabs up to 3 days post-assault. In seven (18.9%) out of 37 anal samples, evidence of sperm could be found 24 h post-assault. None of 22 oral samples was positive for sperm. Out of 301 sexual assault cases, 171 could be proved by means of medical-forensic examination. In summary, our evaluation shows that an early medical-forensic examination of both victim and suspect can secure numerous medical findings. Furthermore, persons intoxicated by alcohol, handicapped persons and persons with psychiatric disorders are more vulnerable to become a sexual assault victim.

Critical review of the use of erythropoietin in the treatment of anaemia during therapy for chronic hepatitis C

Combined pegylated interferon (PegIFN) and ribavirin represents the standard therapy for patients with chronic hepatitis C (CHC), which allows for sustained viral response (SVR) in up to 90% of patients depending on certain viral and host factors. Clinical studies have demonstrated the importance of adherence to therapy, that is, the ability of patients to tolerate and sustain a fully dosed therapy regimen. Adherence is markedly impaired by treatment-related adverse effects. In particular, haemolytic anaemia often requires dose reduction or termination of ribavirin treatment, which compromises treatment efficacy. Recent evidence points to a beneficial role of recombinant erythropoietin (EPO) in alleviating ribavirin-induced anaemia thereby improving quality of life, enabling higher ribavirin dosage and consequently improving SVR. However, no general consensus exists regarding the use of EPO for specific indications: its optimal dosing, treatment benefits and potential risks or cost efficiency. The Swiss Association for the Study of the Liver (SASL) has therefore organized an expert meeting to critically review and discuss the current evidence and to phrase recommendations for clinical practice. A consensus was reached recommending the use of EPO for patients infected with viral genotype 1 developing significant anaemia below 100 g/L haemoglobin and a haematocrit of <30% during standard therapy to improve quality of life and sustain optimal ribavirin dose. However, the evidence supporting its use in patients with pre-existing anaemia, non-1 viral genotypes, a former relapse or nonresponse, liver transplant recipients and cardiovascular or pulmonary disease is considered insufficient.

DNase 2 is the main DNA-degrading enzyme of the stratum corneum

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA.

In vivo analysis of uropod function during physiological T cell trafficking

Migrating lymphocytes acquire a polarized phenotype with a leading and a trailing edge, or uropod. Although in vitro experiments in cell lines or activated primary cell cultures have established that Rho-p160 coiled-coil kinase (ROCK)-myosin II-mediated uropod contractility is required for integrin de-adhesion on two-dimensional surfaces and nuclear propulsion through narrow pores in three-dimensional matrices, less is known about the role of these two events during the recirculation of primary, nonactivated lymphocytes. Using pharmacological antagonists of ROCK and myosin II, we report that inhibition of uropod contractility blocked integrin-independent mouse T cell migration through narrow, but not large, pores in vitro. T cell crawling on chemokine-coated endothelial cells under shear was severely impaired by ROCK inhibition, whereas transendothelial migration was only reduced through endothelial cells with high, but not low, barrier properties. Using three-dimensional thick-tissue imaging and dynamic two-photon microscopy of T cell motility in lymphoid tissue, we demonstrated a significant role for uropod contractility in intraluminal crawling and transendothelial migration through lymph node, but not bone marrow, endothelial cells. Finally, we demonstrated that ICAM-1, but not anatomical constraints or integrin-independent interactions, reduced parenchymal motility of inhibitor-treated T cells within the dense lymphoid microenvironment, thus assigning context-dependent roles for uropod contraction during lymphocyte recirculation.

Structure and function of the glucose PTS transporter from Escherichia coli

The glucose transporter IICB of the Escherichia coli phosphotransferase system (PTS) consists of a polytopic membrane domain (IIC) responsible for substrate transport and a hydrophilic C-terminal domain (IIB) responsible for substrate phosphorylation. We have overexpressed and purified a triple mutant of IIC (mut-IIC), which had recently been shown to be suitable for crystallization purposes. Mut-IIC was homodimeric as determined by blue native-PAGE and gel-filtration, and had an eyeglasses-like structure as shown by negative-stain transmission electron microscopy (TEM) and single particle analysis. Glucose binding and transport by mut-IIC, mut-IICB and wildtype-IICB were compared with scintillation proximity and in vivo transport assays. Binding was reduced and transport was impaired by the triple mutation. The scintillation proximity assay allowed determination of substrate binding, affinity and specificity of wildtype-IICB by a direct method. 2D crystallization of mut-IIC yielded highly-ordered tubular crystals and made possible the calculation of a projection structure at 12Å resolution by negative-stain TEM. Immunogold labeling TEM revealed the sidedness of the tubular crystals, and high-resolution atomic force microscopy the surface structure of mut-IIC. This work presents the structure of a glucose PTS transporter at the highest resolution achieved so far and sets the basis for future structural studies.

Vascular remodeling and antitumoral effects of mTOR inhibition in a rat model of hepatocellular carcinoma

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is amenable to only few treatments. Inhibitors of the kinase mTOR are a new class of immunosuppressors already in use after liver transplantation. Their antiproliferative and antiangiogenic properties suggest that these drugs could be considered to treat HCC. We investigated the antitumoral effects of mTOR inhibition in a HCC model. METHODS: Hepatoma cells were implanted into livers of syngeneic rats. Animals were treated with the mTOR inhibitor sirolimus for 4 weeks. Tumor growth was monitored by MR imaging. Antiangiogenic effects were assessed in vivo by microvessel density and corrosion casts and in vitro by cell proliferation, tube formation and aortic ring assays. RESULTS: Treated rats had significantly longer survival and developed smaller tumors, fewer extrahepatic metastases and less ascites than controls. Sirolimus decreased intratumoral microvessel density resulting in extensive necrosis. Endothelial cell proliferation was inhibited at lower drug concentrations than hepatoma cells. Tube formation and vascular sprouting of aortic rings were significantly impaired by mTOR inhibition. Casts revealed that in tumors treated with sirolimus vascular sprouting was absent, whereas intussusception was observed. CONCLUSIONS: mTOR inhibition significantly reduces HCC growth and improves survival primarily via antiangiogenic effects. Inhibitors of mTOR may have a role in HCC treatment.

Hemodynamic effects of thoracic epidural analgesia in ovine hyperdynamic endotoxemia

BACKGROUND AND OBJECTIVES: Thoracic epidural analgesia (TEA) is increasingly used for perioperative analgesia. If patients with TEA develop sepsis or systemic inflammatory response subsequent to extended surgery the question arises if it would be safe to continue TEA with its beneficial effects of improving gastrointestinal perfusion and augmenting tissue oxygenation. A major concern in this regard is hemodynamic instability that might ensue from TEA-induced vasodilation. The objective of the present study was to assess the effects of TEA on systemic and pulmonary hemodynamics in a sepsis model of hyperdynamic endotoxemia. METHODS: After a baseline measurement in healthy sheep (n = 14), Salmonella thyphosa endotoxin was continuously infused at a rate of 10 ngxkg(-1)xmin(-1) over 16 hours. The surviving animals (n = 12) were then randomly assigned to 1 of 2 study groups. In the treatment group (n = 6), continuous TEA was initiated with 0.1 mLxkg(-1) bupivacaine 0.125% and maintained with 0.1 mLxkg(-1)xh(-1). In the control group (n = 6) the same amount of isotonic sodium saline solution was injected at the same rate through the epidural catheter. RESULTS: In both experimental groups cardiac index increased and systemic vascular resistance decreased concurrently (each P < .05). Functional epidural blockade in the TEA group was confirmed by sustained suppression of the cutaneous (or panniculus) reflex. During the observational period of 6 hours neither systemic nor pulmonary circulatory variables were impaired by TEA. CONCLUSIONS: From a hemodynamic point of view, TEA presents as a safe treatment option in sepsis or systemic inflammatory response syndrome.

[On quality of life after surgical therapy of oral cancer - a retrospective multi-center study: the connection between dedentition, denture, quality of life, and dysphagia, and the resulting rehabilitation schemes]

PURPOSE: The surgical treatment of oral cancer results in functional and aesthetical impairments. Patients' quality of life is considerably impaired by oral symptoms resulting from therapy of oral cancer. In many cases the inevitable resection of the tumor, as well as the adjuvant radiochemotherapy will cause the destruction of physiologically and anatomically important structures. One focus of research was the specific rehabilitation of dental loss by functional dentures. Another was the course of 19 impairments (comprehension of speech for unknown others, comprehension of speech for familiar others, eating/swallowing, mobility of the tongue, opening range of the mouth, mobility of lower jaw, mobility of neck, mobility of arms and shoulders, sense of taste, sense of smell, appearance, strength, appetite, respiration, pain, swelling, xerostomia, halitosis). METHODS: Commissioned by the German, Austrian and Swiss cooperative group on tumors of the maxillofacial region (DOSAK), data were collected in 3.894 questionnaires at 43 hospitals in Germany, Austria and Switzerland. The catalogue comprised 147 items in 9 chapters. At the end of the enquiry, 1.761 anonymous questionnaires were returned by 38 hospitals. 1.652 of these could be evaluated regarding the question. RESULTS: The sum score of the 19 impairments was highly increased immediately after the operation and recovered over the next 6 months, without, however, reaching the pre-surgery level. Of 1.652 patients, only 35% did not lose any teeth during therapy. 23% lost up to 5, 17% up to 10 teeth. A quarter of the patients lost more than 10 teeth. The more teeth were lost, the greater the decline of quality of life (p < or = 0.001), although this could be allayed by the functionality of the dentures (p < or = 0.001). There is a reciprocal dependence between the functionality of dental prosthetics and impairment by eating/swallowing (p < or = 0.001). CONCLUSIONS: Patients' quality of life after radical surgery of a carcinoma of the oral cavity depends not only on the functionality of dentures and the specificity of rehabilitation, but also from the initial findings, the extent and location of the resection, the chosen therapy, the general circumstances of the patient's life as well as their strategies of coping. These factors, however, unlike those of functionality of dental prosthesis and rehabilitation, are not modifiable.

General principles of therapy of pyogenic meningitis

In bacterial meningitis, several pharmacodynamic factors determine therapeutic success-when defined as sterilization of the CSF: (1) Local host defense deficits in the CNS require the use of bactericidal antibiotics to sterilize the CSF. (2) CSF antibiotic concentrations that are at least 10-fold above the MBC are necessary for maximal bactericidal activity. Protein binding, low pH, and slow bacterial growth rates are among the factors that may explain the high antibiotic concentrations necessary in vivo. (3) High CSF peak concentrations that lead to rapid bacterial killing appear more important than prolonged suprainhibitory concentrations, probably because very low residual levels in the CSF prevent bacterial regrowth, even during relatively long dosing intervals. (4) Penetration of antibiotics into the CSF is significantly impaired by the blood-brain barrier and thus, very high serum levels are necessary to achieve the CSF concentrations required for optimal bactericidal activity. Beyond these principles, recent data suggests that rapid lytic killing of bacteria in the CSF may have harmful effects on the brain because of the release of biologically active products from the lysed bacteria. Since rapid CSF sterilization remains a key therapeutic goal, the harmful consequences of bacterial lysis present a major challenge in the therapy of bacterial meningitis. Currently, dexamethasone represents that only clinically beneficial approach to reduce the harmful effects of bacterial lysis, and novel approaches are required to improve the outcome of this serious infection.

Pharmacodynamics of antibiotics in experimental bacterial meningitis--two sides to rapid bacterial killing in the cerebrospinal fluid

In bacterial meningitis, several pharmacodynamic factors determine therapeutic success--when defined as sterilization of the cerebrospinal fluid (CSF); (i) local host defense deficits require the use of bactericidal antibiotics; (ii) CSF antibiotic concentrations that are at least 10-fold above the MBC are necessary for maximal bactericidal activity; (iii) high CSF peak concentrations that lead to rapid bacterial killing appear more important than prolonged suprainhibitory concentrations, probably because very low residual levels in the CSF prevent bacterial regrowth even during relatively long dosing intervals; (iv) penetration of antibiotics into the CSF is significantly impaired by the blood-brain barrier, thus requiring high serum levels to achieve the CSF concentrations necessary for rapid bacterial killing. Beyond these principles, recent data suggest that rapid lytic killing of bacteria in the CSF may have harmful effects on the brain because of the release of biologically active bacterial products. The conflict between the need for rapid CSF sterilization and the harmful consequences of bacterial lysis must be addressed in the therapy of meningitis.

Direct combination of nanoparticle fabrication and exposure to lung cell cultures in a closed setup as a method to simulate accidental nanoparticle exposure of humans

The tremendous application potential of nanosized materials stays in sharp contrast to a growing number of critical reports of their potential toxicity. Applications of in vitro methods to assess nanoparticles are severely limited through difficulties in exposing cells of the respiratory tract directly to airborne engineered nanoparticles. We present a completely new approach to expose lung cells to particles generated in situ by flame spray synthesis. Cerium oxide nanoparticles from a single run were produced and simultaneously exposed to the surface of cultured lung cells inside a glovebox. Separately collected samples were used to measure hydrodynamic particle size distribution, shape, and agglomerate morphology. Cell viability was not impaired by the conditions of the glovebox exposure. The tightness of the lung cell monolayer, the mean total lamellar body volume, and the generation of oxidative DNA damage revealed a dose-dependent cellular response to the airborne engineered nanoparticles. The direct combination of production and exposure allows studying particle toxicity in a simple and reproducible way under environmental conditions.