A system establishment compatibility profiles for artificial oxygen carriers and other substances

Worldwide, great efforts are being made to develop a clinically useful artificial oxygen carrier. Toxicological and immunological compatibility is generally tested using animal experiments but inflammatory parameters in particular show large species-specific differences. Therefore, we developed an in vitro system using human components to establish a compatibility profile of unknown compounds. The test system comprises induction of hemolysis, activation of complement (C3a), induction/suppression of cytokine production, influence on cell proliferation, direct toxicity on peripheral leukocytes, and phagocytosis of the material under test and of microbes. The test system will be described, along with results of various perfluorocarbon emulsions. When testing lecithin-based perfluorodecalin (PFD) emulsions, and comparing them to Pluronic-based PFD emulsions, we could show that Pluronic-based emulsions were virtually untoxic to peripheral human leukocytes. They neither inhibited cell proliferation nor caused any hemolysis, but caused mild to moderate inhibition of endotoxin-induced cytokine production. At the same time, lecithin-based PFD emulsion caused substantial cytotoxicity in phagocytic cells like monocytes (60-100% after 24 h incubation) and granulocytes (10-20% after 24 h incubation). They also suppressed endotoxin-induced cytokine production in monocytes to more than 98% and inhibited cell proliferation of an endothelial (ECV 304) and a monocytic cell line (MonoMac6) to more than 95%.     

Artificial oxygen carriers--the new doping threat in endurance sport?

Maximal oxygen uptake is the major performance limiting factor in endurance sports. Sophisticated training methods have been developed to increase this variable. On the other hand, attempts have been made to improve maximal oxygen uptake by artificial means: blood doping and the misuse of recombinant human erythropoietin have beneficial effects on aerobic exercise capacity. Both methods have been banned by international sporting federations. A new class of substances might represent the next step of fraudulent improvement of the maximal oxygen uptake: artificial oxygen carriers, such as solutions based on recombinant, bovine or human hemoglobin and perfluorocarbon-emulsions have been shown to improve oxygen delivery to the muscle. Hemoglobin-based solutions improve aerobic exercise capacity in animal and human testing. Both substances have potentially lethal side effects including renal toxicity, increased systemic and pulmonary blood pressure and impairment of the immune system. Hemoglobin-based carriers can be detected in drug testing with routine laboratory tests based on the detection of free hemoglobin. Perfluorocarbon is not metabolized by the body and exhaled through the lung and can be measured with chromatography. No screening for these substances in drug tests has been performed so far. International sporting federations should be aware of this new, emerging doping threat.     

Time-related changes of collected shed blood in autologous retransfusion after total knee arthroplasty

A prospective study was done to determine the changes in blood quality parameters of collected drainage blood in retransfusion systems at 6 and 12 h after surgery to verify whether the blood was still suitable for retransfusion purposes for an additional 6 postoperative hours beyond the so far accepted first 6-h time window after surgery. Eighty-one patients received retransfusion within the first 6 h immediately following total knee arthroplasty. Additionally, drainage blood was collected for another 6 h using the same retransfusion system. Samples for laboratory analysis were taken from both the first and second 6-h blood collection interval. Hemoglobin values increased from 9.6 to 10.4 g dl(-1) (p = 0.021). Platelet counts increased from 65,500 to 80,900 microl(-1) (p < 0.001). Leukocyte counts increased from 5,550 to 8,190 1(-1) (p < 0.001). Lactate dehydrogenase (672 U l(-1) during the first vs 651 U l(-1) during the second collection period) and free hemoglobin (71.7 mg dl(-1) vs 67.0 mg dl(-1)) did not change significantly. The potassium concentration decreased slightly from 4.33 to 4.20 mg dl(-1) (p = 0.002). The lactate concentration increased from 4.44 to 7.21 mg dl(-1) (p < 0.001). The pH decreased from 7.07 to 6.94 (p < 0.001). Interleukin-6 concentration increased from 6,500 to 46,500 ng l(-1) (p < 0.001). In this study, we found no relevant difference in most of the drainage blood quality parameters between the first 6-h collection period and the second 6-h collection with regard to its suitability for autologous retransfusion except higher interleukin-6 levels. Due to the higher interleukin concentration, a possible increase in febrile reactions should be taken into account during retransfusion.     

Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal

One of the main impediments for obtaining DNA sequences from ancient human skeletons is the presence of contaminating modern human DNA molecules in many fossil samples and laboratory reagents. However, DNA fragments isolated from ancient specimens show a characteristic DNA damage pattern caused by miscoding lesions that differs from present day DNA sequences. Here, we develop a framework for evaluating the likelihood of a sequence originating from a model with postmortem degradation—summarized in a postmortem degradation score—which allows the identification of DNA fragments that are unlikely to originate from present day sources. We apply this approach to a contaminated Neandertal specimen from Okladnikov Cave in Siberia to isolate its endogenous DNA from modern human contaminants and show that the reconstructed mitochondrial genome sequence is more closely related to the variation of Western Neandertals than what was discernible from previous analyses. Our method opens up the potential for genomic analysis of contaminated fossil material.Retrieval and sequencing of DNA from fossil material has been revolutionized by the rapid surge of high-throughput sequencing technology (1). One of the advantages of the new generation of sequencing technologies over previous PCR-based approaches is the routine sequencing of molecules across their entire length in contrast to specific priming sites. Although this approach has been extended to ancient material from a number of organisms, there is particular interest in genomic analyses of ancient human populations (2–9). However, despite extensive precautions against contamination during laboratory sample preparation, many fossil samples show evidence of contamination from not only the microenvironment of the fossil, but also present day humans (2, 10–14). Although the former type of contamination can introduce statistical noise in comparisons of ancient DNA with modern populations, the latter type of contamination (from present day humans) can impose major biases on population genetic and phylogenetic analyses (15, 16).Ancient DNA sequences show several features indicative of postmortem degradation (PMD) (17). The main diagnostic feature documented so far is a pattern of Cytosine (C) →Thymine (T) substitutions (18–20) that increases toward the 5′ end of the sequence reads (21), which in most applications, results in a complementary Guanine (G) →Adenine (A) pattern in the 3′ end caused by enzymatic repair (5). This pattern has been attributed to Cytosine deamination at single-stranded ends of the molecules (21) and shows a clear tendency of increase over time in contrast to other potential diagnostic features, such as fragment length and preferential fragmentation at purines (22, 23). However, although the observation of such a pattern suggests the presence of degraded DNA in a sequence dataset, it does not prove that modern contamination is absent or even at low frequency. For high-coverage data from extinct close relatives of modern humans, an efficient approach to estimate the contamination proportion is to leverage SNPs that are known to be fixed or nearly fixed in present populations (2, 24). If it can be assumed that contamination is minor relative to endogenous DNA, this approach can also be extended to high-coverage data from ancient modern humans by investigating whether more alleles are present than expected from the ploidy level of the chromosome (4, 24, 25) or making assumptions about the ancestry of both ancient and contaminating individuals (4). For low-coverage data, stratifying sequences based on whether a mismatch consistent with PMD degradation have occurred, and investigating the consistency of population genetic patterns across categories can be used to investigate whether contamination is present at such levels as to affect the conclusions of the analysis (8). However, these approaches only allow investigation of whether contamination is present. To our knowledge, no approach has been developed to overcome significant contamination levels, except for the case of targeted PCR-based sequencing of haploid regions, for which most of the informative termini of the fragments are lost (26). This situation has limited large-scale ancient DNA analyses of human specimens to those specimens with very low levels of present day DNA contamination, leaving the genetic information in potentially crucial fossil material often unstudied.Here, we propose a likelihood approach that incorporates sequence errors and alignment to the reference genome to provide a score that is informative on whether a given sequence is likely to have arisen from a degraded template molecule. We show that this method allows reduction of high contamination rates down to levels that are negligible for most evolutionary purposes and give examples using both mitochondrial (mt) and genome-wide data from the literature. Finally, we produced a high-coverage mtDNA dataset from a Siberian Neandertal fossil excavated in Okladnikov Cave that is contaminated with a substantial amount of modern human DNA and use our method for separating endogenous sequences from contaminating sequences to reconstruct its near-complete mitochondrial genome sequence.     

Studies on the interaction between interleukin 6 and human malignant nonhematopoietic cell lines

We have previously reported on stimulation of clonal growth of cell lines from human solid tumors by recombinant human interleukin 3, recombinant human granulocyte-macrophage colony-stimulating factor, and recombinant human granulocyte colony-stimulating factor (W. E. Berdel et al., Blood, 73: 80-83, 1989; Exp. Hematol., 16: 510, 1988). Within an extensive screening program of hematopoietic growth factor activity on malignant cells, the effects of recombinant human interleukin 6 (rhIL-6) were tested on the growth (tritiated thymidine uptake and human tumor cloning assay) of 26 different human cell lines derived from a wide range of solid tumors (head and neck, 4; lung, 1; pancreatic, 1; gastric, 1; colorectal, 3; renal, 3; bladder, 1; prostate, 1; breast, 2; ovary, 2; choriocarcinoma, 1; sarcoma, 2; glioblastoma, 2; neuroblastoma, 2). rhIL-6 (dose range up to 10(4) IU/ml) caused no reproducible enhancement or inhibition of tritiated thymidine uptake by tumor cell lines from nonhematopoietic origin. Furthermore, 19 of the tumor cell lines were clonogenic in a capillary modification of the human tumor cloning assay. No reproducible stimulation of clonal growth by rhIL-6 was observed in any of the cells tested. Particularly, there was no sensitivity of those cell lines for rhIL-6, which were previously shown to be sensitive for recombinant human interleukin 3 and recombinant human granulocyte-macrophage colony-stimulating factor in this assay. On the other hand, there were no significant growth-inhibitory effects of rhIL-6 on the cell lines tested in this study. Further experiments showed no influence of neutralizing monoclonal anti-hIL-6 antibody on the growth of 3 kidney carcinoma cell lines, making autocrine growth-modulating loops for IL-6 in these lines unlikely. In conclusion, no major interactions between hIL-6 and the growth of the human malignant cell lines from nonhematopoietic origin tested were detected in this study.     

Altered Brain Long-Range Functional Interactions Underlying the Link Between Aberrant Self-experience and Self-other Relationship in First-Episode Schizophrenia

Self-experience anomalies are elementary features of schizophrenic pathology. Such deficits can have a profound impact on self-other relationship, but how they are related through aberrant brain function remains poorly understood. In this functional magnetic resonance imaging (fMRI) study, we provide new evidence for a cortical link between aberrant self-experience and social cognition in first-episode schizophrenia (FES). As identified in previous studies, ventral premotor cortex (vPMC) and posterior insula (pIC) are candidate brain regions underlying disturbances in both self-experience and self-other relationship due to their processing of predominantly externally guided (vPMC; goal-oriented behavior) and internally guided (pIC; interoception) stimuli. Results from functional interaction analysis in a sample of 24 FES patients and 22 healthy controls show aberrant functional interactions (background/intrinsic connectivity) of right vPMC and bilateral pIC with posterior cingulate cortex (PCC), a midline region that has been shown central in mediating self-experience. More specifically, our results show increased functional coupling between vPMC and PCC, which positively correlated with basic symptoms (subjective self-experience disturbances). pIC showed reduced functional coupling with PCC and postcentral gyrus and increased functional interactions with anterior insula. Taken together, our results suggest an imbalance in the processing between internally and externally guided information and its abnormal integration with self-referential processing as mediated by PCC. Due to our correlation findings, we suggest this imbalance to be closely related to basic symptoms in FES and thus anomalous self-experience. The findings further disentangle the cortical basis of how self-experience anomalies may pervade the social domain.Key words: psychosis, posterior insula, ventral premotor cortex, posterior cingulate cortex, functional magnetic resonance imaging, connectivity     

The 'resting-state hypothesis' of major depressive disorder-a translational subcortical-cortical framework for a system disorder

Major depressive disorder (MDD) has traditionally been characterized by various psychological symptoms, involvement of diverse functional systems (e.g., somatic, affect, cognition, reward, etc.), and with progress in neuroscience, an increasing number of brain regions. This has led to the general assumption that MDD is a stress–responsive brain ‘system disorder’ where either one or several alterations infiltrate a large number of functional systems in the brain that control the organism's somatic, affective, and cognitive life. However, while the effects or consequences of the abnormal changes in the functional systems of, for instance affect, cognition or reward have been investigated extensively, the underlying core mechanism(s) underlying MDD remain unknown. Hypotheses are proliferating rapidly, though. Based on recent findings, we will entertain an abnormality in the resting-state activity in MDD to be a core feature. Based on both animal and human data, we hypothesize that abnormal resting-state activity levels may impact stimulus-induced neural activity in medially situated core systems for self-representation as well as external stimulus (especially stress, specifically separation distress) interactions. Moreover, due to nested hierarchy between subcortical and cortical regions, we assume ‘highjacking’ of higher cortical affective and cognitive functions by lower subcortical primary-process emotional systems. This may account for the predominance of negative affect in somatic and cognitive functional system operations with the consecutive generation of the diverse symptoms in MDD. We will here focus on the neuroanatomical and biochemical basis of resting-state abnormalities in MDD including their linkage to the diverse psychopathological symptoms in depression. However, our ‘resting-state hypothesis’ may go well beyond that by being sufficiently precise to be linked to genetic, social, immunological, and endocrine dimensions and hypotheses as well as to clinical dimensions like endophenotypes and various diagnostic-prognostic biomarkers. Taken together, our ‘resting-state hypothesis’ may be considered a first tentative framework for MDD that integrates translational data, the various dimensions, and subcortical–cortical systems while at the same time providing the link to the clinical level of symptoms, endophenotypes and biomarkers.