CCL-2 as a possible early marker for remission after traumatic spinal cord injury.

STUDY DESIGN: Prospective observational study. OBJECTIVES: To describe the correlation between CCL-2, CCL-3, CCL-4 and CXCL-5 serum levels and remission after traumatic spinal cord injury (SCI) in a human protocol compared with animal studies. SETTING: Germany, Rhineland-Palatinate (Rheinland-Pfalz). METHODS: We examined the serum levels of CCL-2, CCL-3, CCL-4 and CXCL-5 over a 12-week period; in particular, at admission and 4, 9 and 12 h, 1 and 3 days and 1, 2, 4, 8 and 12 weeks after trauma. According to our study design, we matched 10 patients with TSCI and neurological remission with 10 patients with an initial ASIA A grade and no neurological remission. In all, 10 patients with vertebral fracture without neurological deficits served as control. Our analysis was performed using a Luminex Cytokine Panel. Multivariate logistic regression models were used to examine the predictive value with respect to neurological remission vs no neurological remission. RESULTS: The results of our study showed differences in the serum expression patterns of CCL-2 in association with the neurological remission (CCL-2 at admission P=0.013). Serum levels of CCL-2 and CCL-4 were significantly different in patients with and without neurological remission. The favored predictive model resulted in an area under the curve (AUC) of 93.1% in the receiver operating characteristic (ROC) analysis. CONCLUSIONS: Our results indicate that peripheral serum analysis is a suitable concept for predicting the patient's potential for neurological remission after TSCI. Furthermore, the initial CCL-2 concentration provides an additional predictive value compared with the NLI (neurological level of injury). Therefore, the present study introduces a promising approach for future monitoring concepts and tracking techniques for current therapies. The results indicate that future investigations with an enlarged sample size are needed in order to develop monitoring, prognostic and scoring systems.

Remote ultrasound diagnostics disrupting traditional military frontline healthcare delivery.

Accurate and reliable diagnostic capability is essential in deployed healthcare to aid decision-making and mitigate risk. This is important for both the patient and the deployed healthcare system, especially when considering the prioritisation of scarce aeromedical evacuation assets and frontline resources. Novel ultrasound tele-guidance technology presents a valuable diagnostic solution for remotely deployed military clinicians. This report discusses the first use of a consultant radiologist guiding a clinician, untrained in ultrasound, to perform an ultrasound scan via a live tele-guidance feed in the deployed environment using the Butterfly iQ+ tele-guidance system. Distance scanning provided a diagnostic quality report when compared with locally performed imaging to improve patient care and maintain operational output. This example demonstrates feasibility of remote point-of-care imaging systems in provision of location-agnostic high-quality diagnostic capability. Future opportunities to develop care pathways using bedside tele-diagnostics will democratise access, drive efficiency and improve patient care experience and outcomes.

[Artificial intelligence in orthopedics and trauma surgery]. / Künstliche Intelligenz in der Orthopädie und Unfallchirurgie.

Artificial intelligence (AI) is a very relevant topic for the medicine of the future. This article focuses on the field of AI in the context of orthopedics and trauma surgery. The main focus is on the potentials of AI in the analysis of symptoms, radiological images, clinical data sets, use in hospitals and operating theaters as well as for training and education. For the orthopedics and trauma surgery of the future AI is much more than pure fiction; however, there is still a long way to go before the potential of an optimized and individualized patient care can be utilized. Interdisciplinary and international approaches, including personnel, economic, legal and ethical aspects will play a decisive role in this respect.

Prevention of cold inactivation of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase by NADPH.

Solubilized 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) from rat liver microsomes has been reported to be reversibly inactivated by temperatures below 19 degrees C. Cold inactivation has now been found to be completely prevented by NADPH and by NADP+ at a concentration of 3 mM. NADPH, however, was more active than NADP+ at lower concentrations and prevented 50% of the cold inactivation at 0.2 mM, whereas a 1.1 mM NADPH+ without effect and the substrate 3-hydroxy-3-methylglutaryl coenzyme A prevented only 30% of the cold inactivation at a concentration 50 times greater than the Km value.

Activation of purified 3-hydroxy-3-methylglutaryl-CoA reductase by phospholipids.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), the enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis, has been purified by two previously reported procedures. Enzyme purified by the method of Heller, R. and Shrewsbury, M. (1976) J. Biol. Chem. 251, 3815-3822) shows up to 3-fold enhancement of activity by various types of lipid dispersions while the enzyme purified by the procedure of Tormanen et al. ((1976) Biochem. Biophys. Res. Commun. 68, 754-762) shows no activation. These results suggest that interaction with microsomal membrane lipids may be important in determining the activity of this enzyme. Analysis of bound lipid showed that enzyme prepared by the procedure of Tormanen contained at last 50 times as much phospholipid on a weight basis as enzyme prepared by Heller and Shrewsbury. Analysis of both preparations by gel-electrophoresis indicates that enzyme activities of the two comigrate, but in neither case does activity coincide with the major protein species.

CCAAT/enhancer binding protein expression is rapidly extinguished in TA1 adipocyte cells treated with tumor necrosis factor.

Tumor necrosis factor (TNF) has been shown to have diverse effects on a wide variety of cell types. In the mouse adipogenic TA1 cell line, TNF completely abolishes differentiation and reverts fully differentiated fat cells into fibroblasts. This block in differentiation and its reversal is due to the rapid reduction in the expression of adipose-specific genes. This study reports that the transcription factor, CCAAT/enhancer binding protein (C/EBP), previously reported to promote the differentiation of 3T3-L1 adipocytes, is expressed in TA1 cells. During their growth in culture, the levels of C/EBP, as evidenced by its cellular levels of specific mRNA, protein, and DNA binding activity, increase dramatically when cells reach confluence and proceed to differentiate. Addition of TNF to cultured preadipocytes or fully differentiated adipocytes rapidly reduces C/EBP levels and is accompanied by the decrease in expression of adipose-specific genes. C/EBP binding sites occur in several adipose-specific genes, and here it is demonstrated that its presence in a novel adipose-specific gene, Clone 47, also referred to as FSP27, may be responsible for the strong down-regulation of the expression of the Clone 47 (FSP27) promoter-linked chloramphenicol acetyl transferase gene by TNF. This study proposes that the loss of C/EBP in response to TNF treatment may in part explain the loss of the adipocyte differentiated state.

Microarrays: biotechnology's discovery platform for functional genomics.

Advances in microarray technology enable massive parallel mining of biological data, with biological chips providing hybridization-based expression monitoring, polymorphism detection and genotyping on a genomic scale. Microarrays containing sequences representative of all human genes may soon permit the expression analysis of the entire human genome in a single reaction. These 'genome chips' will provide unprecedented access to key areas of human health, including disease prognosis and diagnosis, drug discovery, toxicology, aging, and mental illness. Microarray technology is rapidly becoming a central platform for functional genomics.

Transcriptional control of matrix metalloproteinases and the tissue inhibitors of matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are enzymes with important roles in a variety of normal physiological processes; these same enzymes are also operative in a range of pathologies. The proteins known as the tissue inhibitors of matrix metalloproteinases (TIMPs) act to limit the enzymatic function of the MMPs. MMPs and TIMPs can be divided into two groups with respect to gene expression: the majority exhibit inducible expression and a small number are produced constitutively or are expressed at very low levels and are not inducible. Among the agents that induce MMP and TIMP production are the inflammatory cytokines TNF alpha and IL1 beta. A marked cell type specificity is a hallmark of both MMP and TIMP gene expression (i.e., a limited number of cell types can be induced to make these proteins). An analysis of the control elements in the promoter regions of these proteins reveals a correlation between the presence of both AP-1 and Ets binding sites and inducible expression. The chromosomal locations of most of the MMPs and TIMPs have been verified; these data will provide the basis for investigations into possible correlations between mutations in these genes and disease states.

The human stromelysin promoter contains a previously unreported 1.0-kb sequence.

Cloning and characterization of the promoter region controlling the gene encoding human stromelysin (Str) has been previously reported [Quinones et al., J. Biol. Chem. 264 (1989) 8339-8344]. We have characterized independently isolated genomic clones of the STR promoter, designated pSKStrB and 682, that are considerably different from the published sequence. Although the sequences up to an XbaI site at -480 of the 5' regions are identical, a novel 1.0-kb segment exists upstream from -480. This sequence is absent from the published clone, but its presence in the genomic DNA from twelve individuals has been confirmed by both PCR analysis and restriction mapping. Upstream of the novel 1-kb segment, the sequence of the published clone reappears, but in pSKStrB exists in inverse orientation.

Rapid screening of libraries with radiolabeled DNA sequences generated by PCR using highly degenerate oligonucleotide mixtures.

The PCR technique can use protein-derived oligonucleotide sequences as primers to develop probes for screening recombinant libraries. Here we report a method with highly degenerate mixtures of oligonucleotides as primers for the PCR that eliminates the need to identify or isolate the DNA sequences derived by PCR. The method uses the pool of PCR-generated DNA sequences radiolabeled during the extension reaction as a probe, combined with highly stringent hybridization and wash conditions that permit only homologous sequences to hybridize and therefore target desired clones. This technique was used successfully to clone the receptor for tumor necrosis factor.

Use of oligonucleotide arrays to analyze drug toxicity.

The advent of oligonucleotide arrays allows the simultaneous analysis of the expression of thousands of genes. This powerful technology, highly dependent on advanced analysis tools, can transform the level of information currently available on the mechanisms underlying drug-related toxicity. It is now possible to analyze the global transcriptional response to a drug and determine the global pathways associated with the effects of this agent. This analysis can be performed on samples from patients that developed a toxic effect, on cells exposed to the toxic agent, and in animal models of toxicity. Especially useful is the comparison of transcriptional responses in animals susceptible to drug-induced disease with those of genetically modified animals that are resistant to this effect. This analytic strategy allows the delineation of specific mechanisms relevant and specific to drug-induced toxicity and thus might lead to novel therapeutic interventions in these toxic reactions.

Quantitative B-hCG levels less than 1000 mIU/mL in patients with ectopic pregnancy: pelvic ultrasound still useful.

The purpose of this study was to determine if pelvic ultrasound was useful in suggesting the diagnosis of ectopic pregnancy in patients with a quantitative B-hCG level less than 1000 mIU/mL. We performed a retrospective review of all patients evaluated and diagnosed with ectopic pregnancy in the emergency departments of seven area hospitals during a ten month period. Sixty-four patients with a confirmed diagnosis of ectopic pregnancy, a pelvic ultrasound, and a quantitative B-hCG level were included in the study. Eighteen (28%) of these patients had a quantitative B-hCG less than 1000 mIU/mL. Sixteen of the eighteen patients (89%) with a B-hCG level less than 1000 mIU/mL had sonographic findings suggestive of ectopic pregnancy, such as fluid in the cul-de-sac, or a complex adnexal or cystic mass. Overall, 25% of all patients diagnosed with an ectopic pregnancy during this time period had a quantitative B-hCG level less than 1000 mIU/mL and an ultrasound suggestive for ectopic pregnancy. Pelvic ultrasound is useful as a screening tool in the initial evaluation of suspected ectopic pregnancy, even when the quantitative B-hCG level is below 1000 mIU/mL.

3-Hydroxy-3-methylglutaryl coenzyme A reductase in isolated villous and crypt cells of the rat ileum.

The localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, E.C. 1.1.1.34) in the villous and crypt cells of the small intestine was accomplished after separating these cells from the mucosal layer by sequential dissociation in a "dual-buffer" system. Consistent separation was demonstrated by using the marker enzymes alkaline phosphatase, specific to the villous cell, and thymidine kinase, specific to the crypt cell. Cells obtained were 95-100% viable, and no relative difference in lability was observed, as evidenced by the equal distribution of acid phosphatase. This method of cell separation was an improvement over the "scraping" technique which damaged cells severely and produced villous preparations that contained little or no reductase activity. The HMG-CoA reductase specific activity in whole cell homogenates of the ileal villi was 0.47 and of the crypts was 0.27 nmol/min per mg of protein, considerably higher values than have been reported earlier. Also in comparison to the crypts, the villi incorporated 1.5-fold more [(14)C]-acetate into sterols, a ratio similar to that describing the distribution of HMG-CoA reductase in the two cell populations. These results unequivocally establish that the villi have higher HMG-CoA reductase activity than the crypts and confirm an earlier report from this laboratory that the villi are a major site of sterol synthesis. The sterol bio-synthetic capacity of the small intestine was highest in the ileum and decreased towards the jejunum. The HMG-CoA reductase specific activity of the ileum averaged 0.30 and that of the jejunum 0.10 nmol/min per mg of protein; however, the cholesterol content of the ileum was slightly lower than the jejunum. These results are discussed to suggest the possibility that the sterol content of the ileum may largely be due to in situ synthesis.

3-Hydroxy-3-methylglutaryl coenzyme A reductase from rat liver. Its purification, properties, and immunochemical studies.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) was purified from rat liver microsomes after solubilization by a slow freezing and thawing method. The purification was accomplished by a five-step procedure involving incubation at 37 degrees, ammonium sulfate fractionation, ultrafiltration, and column chromatography on Bio-Gel A-0.5m and Sephadex G-200. The specific activities of the purified enzyme preparations were up to 480 nmol of mevalonate formed/min/mg of protein, which represented an increase of 350-fold above that of the microsomes. The purified enzyme was found to be essentially homogeneous as evidences by the usual criteria. A subunit molecular weight of 120,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On gel filtration a number of different molecular weight forms were observed which seem to be influenced by temperature, method of purification, and possibly an enzyme-Bio-Gel A-0.5m interaction. The solubilized enzyme consisted predominantly of a species with a molecular weight slightly greater than 200,000 by gel filtration and may be a dimer of two 120,000 subunits. The purified preparation contained lipids; the total cholesterol content was 18 mug/mg of enzyme protein and corresponds to a ratio of 5 mol of cholesterol/enzyme subunit of 120,000 daltons. The specificity of rabbit antiserum prepared against the purified enzyme was demonstrated by double diffusion analysis and quantitative precipitin reactions with solubilized enzyme. The antiserum, in addition to inhibiting the activity of solubilized enzyme also blocked the activity of intact microsomes. The microsomal HMG-CoA reductase is accessible to the antibody, indicating a localization of the enzyme on the outer cytoplasmic surface of the membranes. Intestinal microsomal HMG-CoA reductase was shown to cross-react with antibody to the liver enzyme.

Mechanisms of tumor necrosis factor cytotoxicity and the cytotoxic signals transduced by the p75-tumor necrosis factor receptor.

We have here provided evidence that TNF mediated cytotoxicity is genetically, pharmacologically, and temporally distinct from the cytotoxicity mediated by TNF/CHX. Most studies on TNF cytotoxicity have been done by the combined use of TNF/CHX. The relevance of this approach to the physiological mechanisms underlying cytotoxicity by TNF alone is at present unclear. We have described a system in which overexpression of the p75-TNFR causes TNF-resistant cells to become TNF-sensitive. These cells are killed by TNF alone in a very short period of time and they are a useful system to study the mechanism of TNF-cytotoxicity.

Tumor necrosis factor receptor genes, TNFR1 and TNFR2, on human chromosomes 12 and 1.

Tumor necrosis factor, TNF, is a 17-kDa protein secreted by macrophages and classified as a cytokine. TNF binds to high-affinity receptors on the cell surface and is involved in a wide variety of biological responses. There are at least two types of receptors, tumor necrosis factor receptors 1 and 2 (TNFR1 and TNFR2). The genes for TNFR1 a 55-kDa protein, and TNFR2, a 70-kDa protein, have been mapped to human chromosomes 1 12 (12pter-cen) and (1pter-p32), respectively, by Southern blot analysis of human x Chinese hamster somatic cell hybrid panels. Recently, the corresponding genes in the mouse have been mapped to chromosomes 4 and 6 in regions that are conserved on human chromosomes 1 and 12.

Cell killing and induction of manganous superoxide dismutase by tumor necrosis factor-alpha is mediated by lipoxygenase metabolites of arachidonic acid.

The signalling pathways utilized by tumor necrosis factor-a (TNF) to elicit its actions have been examined in TA1 adipogenic cells. A role for lipoxygenase metabolites of arachidonic acid as mediators of TNF action in the induction of c-fos has been described. In this paper we report that acute cytotoxicity elicited by TNF, in the presence of cycloheximide (CHX), also utilizes this pathway since inhibitors of lipoxygenase action fully prevent TNF/CHX killing of several cell lines. Our data reveal that TNF induction of manganous superoxide dismutase (MnSOD) is also dependent upon lipoxygenase activity. Radical scavengers such as NAC and PDTC prevent TNF/CHX-induced cell killing and reduce MnSOD induction by TNF. Therefore, cell death by TNF/CHX treatment occurs via a pathway in which lipoxygenase products directly or indirectly operate via the generation of superoxide anions.