Mycobacterium tuberculosis exposure of livestock in a German dairy farm: implications for intra vitam diagnosis of bovine tuberculosis in an officially tuberculosis-free country.

Germany has been an officially bovine tuberculosis (bTB)-free (OTF) country since 1996. Gradually rising numbers of bTB herd incidents due to Mycobacterium bovis and M. caprae in North-Western and Southern Germany during the last few years prompted the competent authorities to conduct a nationwide bTB survey in 2013/2014. This led to the detection of a dairy herd in which as many as 55 cattle reacted positively to consecutive intra vitam testing. Test-positive animals lacked visible lesions indicative of bTB at necropsy. Extensive mycobacterial culturing as well as molecular testing of samples from 11 tissues for members of the M. tuberculosis complex (MTC) yielded negative results throughout. However, caseous lymphadenitis of Ln. mandibularis accessorius was observed during meat inspection of a fattening pig from the same farm at regular slaughter at that time. Respective tissue samples tested MTC positive by polymerase chain reaction, and M. tuberculosis T1 family were identified by spoligotyping. Four human reactors within the farmer's family were also found to be immunoreactive. As exposure of livestock to M. tuberculosis is not generally considered, its impact may result in regulatory and practical difficulties when using protocols designed to detect classical bTB, particularly in OTF countries.

Nutritional regulation of the anabolic fate of amino acids within the liver in mammals: concepts arising from in vivo studies.

At the crossroad between nutrient supply and requirements, the liver plays a central role in partitioning nitrogenous nutrients among tissues. The present review examines the utilisation of amino acids (AA) within the liver in various physiopathological states in mammals and how the fates of AA are regulated. AA uptake by the liver is generally driven by the net portal appearance of AA. This coordination is lost when demands by peripheral tissues is important (rapid growth or lactation), or when certain metabolic pathways within the liver become a priority (synthesis of acute-phase proteins). Data obtained in various species have shown that oxidation of AA and export protein synthesis usually responds to nutrient supply. Gluconeogenesis from AA is less dependent on hepatic delivery and the nature of nutrients supplied, and hormones like insulin are involved in the regulatory processes. Gluconeogenesis is regulated by nutritional factors very differently between mammals (glucose absorbed from the diet is important in single-stomached animals, while in carnivores, glucose from endogenous origin is key). The underlying mechanisms explaining how the liver adapts its AA utilisation to the body requirements are complex. The highly adaptable hepatic metabolism must be capable to deal with the various nutritional/physiological challenges that mammals have to face to maintain homeostasis. Whereas the liver responds generally to nutritional parameters in various physiological states occurring throughout life, other complex signalling pathways at systemic and tissue level (hormones, cytokines, nutrients, etc.) are involved additionally in specific physiological/nutritional states to prioritise certain metabolic pathways (pathological states or when nutritional requirements are uncovered).

A prospective, randomized, controlled trial of autologous haematopoietic stem cell transplantation for aggressive multiple sclerosis: a position paper.

BACKGROUND: Haematopoietic stem cell transplantation (HSCT) has been tried in the last 15 years as a therapeutic option in patients with poor-prognosis autoimmune disease who do not respond to conventional treatments. Worldwide, more than 600 patients with multiple sclerosis (MS) have been treated with HSCT, most of them having been recruited in small, single-centre, phase 1-2 uncontrolled trials. Clinical and magnetic resonance imaging outcomes from case series reports or Registry-based analyses suggest that a major response is achieved in most patients; quality and duration of response are better in patients transplanted during the relapsing-remitting phase than in those in the secondary progressive stage. OBJECTIVES: An interdisciplinary group of neurologists and haematologists has been formed, following two international meetings supported by the European and American Blood and Marrow Transplantation Societies, for the purpose of discussing a controlled clinical trial, to be designed within the new scenarios of evolving MS treatments. CONCLUSIONS: Objectives of the trial, patient selection, transplant technology and outcome assessment were extensively discussed. The outcome of this process is summarized in the present paper, with the goal of establishing the background and advancing the development of a prospective, randomized, controlled multicentre trial to assess the clinical efficacy of HSCT for the treatment of highly active MS.

Physiologic action of glucagon on liver glucose metabolism.

Glucagon is a primary regulator of hepatic glucose production (HGP) in vivo during fasting, exercise and hypoglycaemia. Glucagon also plays a role in limiting hepatic glucose uptake and producing the hyperglycaemic phenotype associated with insulin deficiency and insulin resistance. In response to a physiological rise in glucagon, HGP is rapidly stimulated. This increase in HGP is entirely attributable to an enhancement of glycogenolysis, with little to no acute effect on gluconeogenesis. This dramatic rise in glycogenolysis in response to hyperglucagonemia wanes with time. A component of this waning effect is known to be independent of hyperglycemia, though the molecular basis for this tachyphylaxis is not fully understood. In the overnight fasted state, the presence of basal glucagon secretion is essential in countering the suppressive effects of basal insulin, resulting in the maintenance of appropriate levels of glycogenolysis, fasting HGP and blood glucose. The enhancement of glycogenolysis in response to elevated glucagon is critical in the life-preserving counterregulatory response to hypoglycaemia, as well as a key factor in providing adequate circulating glucose for working muscle during exercise. Finally, glucagon has a key role in promoting the catabolic consequences associated with states of deficient insulin action, which supports the therapeutic potential in developing glucagon receptor antagonists or inhibitors of glucagon secretion.

[Proper lighting for color vision testing: The TRUE COLOR LED lamp - an innovation in this field]. / Bien éclairer les tests de vision des couleurs, la lampe TRUE COLOR LED : une innovation dans ce domaine.

Given the latest trend in light sources, and in particular, the widespread use of LED lamps, we began to rethink the old lighting standards for colour vision testing. We (J. Leid) therefore conceived a new, easy to use, calibrated lamp with a specific ergonomic design, emitting light with a spectrum close to that of the standard D65, and perfectly adapted to pigmentary colour vision tests. A prototype conforming to a strict set of specifications in terms of quality and quantity of light, as well as ergonomics, was tested against a Macbeth lamp and fluorescent lighting, on controls and patients with congenital and acquired dyschromatopsia. Tests performed using our new lighting reveal, at a minimum, the same types of dyschromatopsia as the ones found with the old devices, and, in some cases, led to a more precise diagnosis, which we confirmed using an anomaloscope. These results demonstrate that an appropriate LED lamp permits perfect colour vision testing and appears superior to prior technology. The objective of the True Color LED Lamp is to offer practitioners a modern, energy-efficient, maintenance-free, compact, effective technological tool with specific ergonomics and a colour spectrum that provides optimal lighting for pigmentary colour vision testing, coupled with a double level illumination, which constitutes a favourable replacement for older devices which are no longer available or insufficiently calibrated.

Proteolipid protein is necessary in peripheral as well as central myelin.

Alternative products of the proteolipid protein gene (PLP), proteolipid protein (PLP) and DM20, are major components of compact myelin in the central nervous system, but quantitatively minor constituents of Schwann cells. A family with a null allele of PLP has a less severe CNS phenotype than those with other types of PLP mutations. Moreover, individuals with PLP null mutations have a demyelinating peripheral neuropathy, not seen with other PLP mutations of humans or animals. Direct analysis of normal peripheral nerve demonstrates that PLP is localized to compact myelin. This and the clinical and pathologic observations of the PLP null phenotype indicate that PLP/DM20 is necessary for proper myelin function both in the central and peripheral nervous systems.

Agricultural pollutant penetration and steady state in thick aquifers.

The leakage of pollutants from agricultural lands to aquifers has increased greatly, driven by increasing fertilizer and pesticide use. Because this increase is recent, ground water pollutant concentrations, loads, and exports may also be increasing as pollutants penetrate more deeply into aquifers. We established in an aquifer profile a ground water recharge and pollutant leakage chronology in an agricultural landscape where 30 m of till blankets a 57-m thick sandstone aquifer. Pollutant concentrations increased from older ground water (1963) at the aquifer base to younger ground water (1985) at its top, a signal of increasing pollutant leakage. Nitrate-N increased from 0.9 to 13.2 mg/L, implying that leakage increased from 1.9 to 16.5 kg/ha/year. Nitrate load and export could increase from 130% to 230% before reaching a steady state in 20 to 40 years. Chloride increases were similar. Pesticide residues alachlor ethane sulfonic acid (ESA), metolachlor ESA, and atrazine residues partially penetrated the aquifer profile. Their concentration-age-date patterns exhibited an initial increase and then a leveling corresponding to the timing of product adoption and leveling of demand. Unlike NO(3), projecting pesticide residue steady states is complicated by the phasing in and out of pesticide products over time; for example, neither alachlor nor atrazine is currently used in the area, and newer products, which have not had time to transit to the aquifer, have been adopted. The circumstances that resulted in the lack of a pollutant steady state are not rare; thus, the lack of steady states in agricultural region aquifers may not be uncommon.

Experimental and theoretical study of the neutron dose produced by carbon ion therapy beams.

High-energy (12)C ions offer favourable conditions for the treatment of deep-seated local tumours. Several facilities for the heavy ion therapy are planned or under construction, for example the new clinical ion-therapy unit HIT at the Radiological University Clinics in Heidelberg. In order to improve existing treatment planning models, it is essential to evaluate the secondary fragment production and to include these contributions to the therapy dose with higher accuracy. Secondary neutrons are most abundantly produced in the reactions between (12)C beams and tissues. The dose contribution to tissues by a neutron is fairly small compared with the projectile and the other charged fragments due to no ionisation and the small reaction cross-sections; however, it distributes in a considerably wider region beyond the bragg-peak because of the strong penetrability. Systematic data on energy spectra and doses of secondary neutrons produced by (12)C beams using water targets of different thicknesses for various detection angles have therefore been measured in this study at GSI Darmstadt.

MR nerve imaging in a prospective cohort of patients with suspected carpal tunnel syndrome.

OBJECTIVES: To evaluate the reliability and diagnostic accuracy of high-resolution MRI of the median nerve in a prospectively assembled cohort of subjects with clinically suspected carpal tunnel syndrome (CTS). METHODS: The authors prospectively identified 120 subjects with clinically suspected CTS from five Seattle-area clinics. All subjects completed a hand-pain diagram and underwent a standardized nerve conduction study (NCS). The reference standard for determining CTS status was a classic or probable hand pain diagram and NCS with a difference >0.3 ms between the 8-cm median and ulnar peak latencies. Readers graded multiple imaging parameters of the MRI on four-point scales. The authors also performed quantitative measurements of both the median nerve and carpal tunnel cross-sectional areas. NCS and MRI were interpreted without knowledge of the other study or the hand pain diagram. RESULTS: Intrareader reliability was substantial to near perfect (kappa = 0.76 to 0.88). Interreader agreement was lower but still substantial (kappa = 0.60 to 0.67). Sensitivity of MRI was greatest for the overall impression of the images (96%) followed by increased median nerve signal (91%); however, specificities were low (33 to 38%). The length of abnormal signal on T2-weighted images was significantly correlated with nerve conduction latency, and median nerve area was larger at the distal radioulnar joint (15.8 vs 11.8 mm(2)) in patients with CTS. A logistic regression model combining these two MR variables had a receiver operating characteristic area under the curve of 0.85. CONCLUSIONS: The reliability of MRI is high but the diagnostic accuracy is only moderate compared with a research-definition reference standard.

The formation of radiation-induced DNA breaks: the ratio of double-strand breaks to single-strand breaks.

Ionizing radiation causes the formation of strand breaks in cellular DNA, as well as other types of lesions in the chromatin of cells. Some of the earliest investigations of the molecular basis of radiation-induced damage and the implications of enzymatic repair were done by Dr. H. S. Kaplan. The induction frequency of DNA double-strand breaks is of special importance, and it is of interest to know the relative proportions of single-strand and double-strand breaks. This ratio changes noticeably with the radiation quality (ionization density). Because it is difficult to assay for DNA lesions in the large mammalian genome, we have developed a method of assaying for DNA double-strand breaks in the supercoiled nucleosome-complexed Simian virus 40 (SV40) genome, irradiated intracellularly. In this communication we present our measurements of the DNA double-strand breaks (DSBs) to single-strand breaks (SSBs) ratio obtained from the intracellularly irradiated SV40 genome. After cobalt gamma ray and X ray irradiations, this ratio is about 1/10. Our methods and results are compared with pertinent data in the literature. If the DSBs/SSBs ratio of 1/10 for cellular chromatin is correct, a substantial number of DNA double-strand breaks are formed in a mammalian cell after moderate doses (1 Gy) of radiation. The implications of different types of DNA double-strand breaks are discussed.

Measurement of intracellular dna double-strand break induction and rejoining along the track of carbon and neon particle beams in water.

PURPOSE: The study was aimed at the measurement of effect-depth distributions of intracellularly induced DNA damage in water as tissue equivalent after heavy ion irradiation with therapy particle beams. METHODS AND MATERIALS: An assay involving embedding of Chinese hamster ovary (CHO-K1) cells in large agarose plugs and electrophoretic elution of radiation induced DNA fragments by constant field gel electrophoresis was developed. Double-strand break production was quantified by densitometric analysis of DNA-fluorescence after staining with ethidium-bromide and determination of the fraction of DNA eluted out of the agarose plugs. Intracellular double-strand break induction and the effect of a 3 h rejoining incubation were investigated following irradiation with 250 kV x-rays and 109 MeV/u carbon- and 295 MeV/u neon-ions. RESULTS AND CONCLUSION: While the DNA damage induced by x-irradiation decreased continuously with penetration depth, a steady increase in the yield of double-strand breaks was observed for particle radiation, reaching distinct maxima at the position of the physical Bragg peaks. Beyond this, the extent of radiation damage dropped drastically. From comparison of DNA damage and calculated dose profiles, relative biological efficiencies (RBEs) for both double-strand break induction and unrejoined strand breaks after 3 h were determined. While RBE for the induction of DNA double-strand breaks decreased continuously with penetration depth, RBE maxima greater than unity were found with carbon- and neon-ions for double-strand break rejoining near the maximum range of the particles. The method presented here allows for a fast and accurate determination of depth profiles of relevant radiobiological effects for mixed particle fields in tissue equivalent.

Charcot-Marie-Tooth neuropathy: clinical phenotypes of four novel mutations in the MPZ and Cx 32 genes.

Charcot-Marie-Tooth Hereditary Neuropathy is a heterogeneous syndrome associated with mutations in several different genes including peripheral myelin protein 22, myelin P0, connexin 32, and early growth response 2. There is considerable variability in the phenotypic expression of this syndrome and the relationship of this variability to mutation genotypes requires extensive analysis. Here we describe the phenotypes and genotypes of four new mutations underlying the Charcot-Marie-Tooth syndrome and document segregation with disease. Four families with Charcot-Marie-Tooth were ascertained, examined, and evaluated electrophysiologically. Each family had peripheral blood DNA screened for mutations in myelin protein 22, myelin P0, and connexin 32. Two families were found with new mutations in the myelin P0 gene: S140T in the extracellular domain and K236del in the cytoplasmic domain. All families showed segregation of the mutations with the Charcot-Marie-Tooth phenotype as did a new family with the rare G163R mutation in the membrane domain. A 49-year-old man with the S140T mutation demonstrated conduction block on electrophysiological testing. A family with a novel S49P mutation in the connexin 32 gene had a neuropathy with very slow nerve conduction. These new mutations in the myelin P0 and connexin 32 genes help to clarify the pathophysiology of the clinical Charcot-Marie-Tooth syndrome. The S140T mutation in myelin P0 can be associated with conduction block and Charcot-Marie-Tooth should be part of the differential diagnosis of that phenomenon. Mutations in the cytoplasmic domain of myelin P0 can cause clinical neuropathy. The S49P mutation in the connexin 32 gene can produce aspects of a demyelinating type of X-linked hereditary neuropathy.

RBE of carbon ions: experimental data and the strategy of RBE calculation for treatment planning.

The main reason for the application of heavy ions like carbon in radiotherapy is the enhanced relative biological effectiveness RBE. In contrast to neutrons where RBE is widely independent from penetration depth, high energy carbon beams have a low RBE at the entrance and a high RBE in the target-volume. Therefore, the side effects to normal tissue are small, while the tumor response can be maximized. In the paper, experimental RBE values for inactivation are compiled, that demonstrate the RBE dependence from the repair capacity. In a theoretical approach, the local effect model (LEM), this dependence is used to calculate clinical RBE. Examples for clinical RBEs are given that have been applied to patient treatments.

The physical and radiobiological basis of the local effect model: a response to the commentary by R. Katz.

The physical and biological basis of our model to calculate the biological effects of charged particles, termed the local effect model (LEM), has recently been questioned in a commentary by R. Katz. Major objections were related to the definition of the target size and the use of the term cross section. Here we show that the objections raised against our approach are unjustified and are largely based on serious misunderstandings of the conceptual basis of the local effect model. Furthermore, we show that the approach developed by Katz and coworkers itself suffers from exactly those deficiencies for which Katz criticizes our model. The essential conceptual differences between the two models are discussed by means of some illustrative examples, based on a comparison with experimental data. For these examples, the predictions of the local effect model are fully consistent with the experimental data. In contrast, e.g. for very heavy ions, there are significant discrepancies observed for the Katz approach. These discrepancies can be attributed to the inadequate definition of the target size in this model. Experimental data are thus clearly in favor of the definition of the target as used in the local effect model. Agreement with experimental data is achieved for protons within the Katz approach but at the cost of questionable approximations in combination with the violation of the fundamental physical principle of energy conservation.

Influence of radiation quality on the yield of DNA strand breaks in SV40 DNA irradiated in solution.

Using highly energetic particles to irradiate plasmid DNA in aerobic aqueous solution, we have compiled an extensive database on how yields of DNA single- and double-strand breaks (SSBs and DSBs) vary with radiation quality. This study was performed in a low-scavenging buffer system and covers a wide range of ion species (helium to uranium) and LETs (5 to 16,000 keV/microm). For LETs up to around 40 keV/microm for SSBs and 400 keV/microm for DSBs, the total energy deposition determines cross section. At higher LET, cross sections level off and individual plateaus for particles of different atomic numbers are observed. For each ion species this is more pronounced and occurs at lower LET for SSBs than for DSBs, leading to an increase in the DSB:SSB ratio from 1:70 for X rays to 1:6 at 500 keV/microm. At this LET, the influence of track structure becomes evident, with high local concentrations of ionization events favoring the formation of DSBs and also intratrack recombination reactions. For lower-energy ions, a saturation in production of measurable DSBs is apparent, due to correlated lesion induction within densely ionizing particle tracks. For very heavy low-energy ions, both SSB and DSB cross sections decrease with particle velocity at nearly constant LET, forming individual hooked curves when plotted as a function of LET.

Inactivation of Ehrlich ascites tumor cells by heavy ions.

Exponentially growing and plateau-phase cultures of Ehrlich ascites tumor cells were irradiated with heavy ions (Z greater than or equal to 20) and assayed for loss of reproductive capacity either immediately or at delayed times after irradiation. The results indicated no modification of the exponential dose response due to conditions which usually favor the repair of potentially lethal damage at low ionization density. Postirradiation treatment of the cells with beta-arabinofuranosyladenine, a DNA synthesis inhibitor known to act on PLD repair, resulted in effects similar to those observed without this drug and confirmed the hypothesis that at such high values of ionization density only lethal, unmodifiable damage can be expressed. The inactivation cross-section values calculated from the slope of the measured survival curves showed no significant correlations with commonly used parameters of radiation quality such as LET or z2/beta 2. Instead, a functional dependence on the primary ion energy was indicated, being smaller by a factor of two at low energies (less than or equal to 2 MeV/amu) compared with values at energies above 4 MeV/amu, where agreement with the morphological nuclear cross section of the culture was found. This suggests that at higher specific ion energies energetic secondary electrons contribute to the induction of lethal damage, and that interaction of damaged sites between the primary track and the track ends of delta electrons may occur. The data are therefore also discussed in terms of the "penumbra model" which emphasizes the role of delta electrons in cell killing when radiations with very high ionization density are applied.

Heavy-ion effects on bacteria spores: the impact parameter dependence of the inactivation.

The impact parameter dependence of the inactivation of Bacillus subtilis-spores has been measured using a heavy-ion minibeam facility, which permits single-ion exposure of individual spores with a spatial accuracy of about 1 micron. The apparatus consists basically of a collimator and a microscope used to position the biological objects directly behind the collimator. Measurements were obtained for nickel, tin, and uranium ions at 1.4 MeV/u. for central hits the results show an inactivation probability of less than one with a continuous decrease in inactivation with increasing distance. Long-ranging effects which extend beyond the range of the delta electrons could not be confirmed.

Heavy ion effects on yeast: inhibition of ribosomal RNA synthesis.

Diploid wild-type yeast cells were exposed to beams of heavy ions covering a wide range of linear energy transfer (LET) (43-13,700 keV/microns). Synthesis of ribosomal RNA (rRNA) was assessed as a functional measure of damage produced by particle radiation. An exponential decrease of relative rRNA synthesis with particle fluence was demonstrated in all cases. The inactivation cross sections derived were found to increase with LET over the entire range of LET studied. The corresponding values for relative biological effectiveness were slightly less than unity. Maximum cross sections measured were close to 1 micron 2, implying that some larger structure within the yeast nucleus (e.g., the nucleolus) might represent the target for an impairment of synthetic activity by very heavy ions rather than the genes coding for rRNA. Where tested, an oxygen effect for rRNA synthesis could not be demonstrated.

DNA strand break induction and rejoining and cellular recovery in mammalian cells after heavy-ion irradiation.

The induction of intracellular DNA strand breaks by X rays and various heavy charged particles was measured by the alkaline unwinding and alkaline and neutral filter elution techniques. No variations in strand break induction were found between the different cell lines under investigation. For a given particle, both the LET and the particle energy determined the efficiency to induce DNA lesions. RBE values for the total amount of induced strand breaks were always less than 1. For DNA double-strand breaks (DSBs), RBE values only slightly greater than 1 were determined for particle radiation with an LET around 300 keV/microns. Intracellular DSB/SSB ratios were found to be equivalent to data reported for in vitro systems using radioprotective conditions [Christensen et al., Int. J. Radiat. Biol. 22, 457-477, 1972; Taucher-Scholz et al., Adv. Space Res. 12(2-3), (2)73-(2)80, 1992]. Strand break rejoining as an indicator of cellular repair processes was detected even after high-LET irradiation (LET < or = 10,000 keV/microns). However, both the half-times of rejoining and the fraction of residual DNA breaks increased with the atomic number of the particle. After particle irradiation with LET values beyond 10,000 keV/microns, no rejoining of DNA strand breaks was found.

Peripheral neuropathy caused by proteolipid protein gene mutations.

Pelizaeus-Merzbacher disease (PMD) is a dysmyelinating disorder of the central nervous system typically caused by duplications or missense mutations of the proteolipid protein (PLP) gene. Most investigators have found that peripheral nerve function and structure is normal in PMD patients. We have found that null mutations of the PLP gene cause demyelinating peripheral neuropathy, whereas duplications and a proline 14 to leucine mutation do not affect nerve function. A family with a nonsense mutation at position 144, which affects only PLP but not the alternatively spliced gene product DM20, has a very mild syndrome, including normal peripheral nerve function. Our findings suggest that DM20 alone is sufficient to maintain normal nerve function and that there may be domains of PLP/DM20 that have a relatively more active role in the peripheral nervous system compared with that in the central nervous system.