Gas chromatographic-tandem mass spectrometric determination of anabolic steroids and their esters in hair. Application in doping control and meat quality control

We have developed a powerful and simple sensitive method for testing hair for anabolic steroids and their esters. A 100-mg amount of powdered hair was treated with methanol in an ultrasonic bath for extraction of esters, then alkaline digested with 1 M NaOH for an optimum recovery of other drugs. The two liquid preparations were subsequently extracted with ethyl acetate, pooled, then finally highly purified using a twin solid-phase extraction on amino and silica cartridges. The residue was derivatized with N-methyl-N(trimethylsilyl)-trifluoracetamide (MSTFA) prior to injection. Analysis was conducted by gas chromatography coupled to a triple quadrupole mass spectrometer. The generally chosen parent ion was the molecular ion while two daughter ions were selected for each compound with collision energies ranging from -16 to -21 eV. Internal standards were nandrolone d3 for non-esterified drugs and testosterone phenyl propionate for esters. The limits of detection calculated from an analysis of the blanks (n=30) were 0.08 pg/mg for nandrolone, 6.20 pg/mg for boldenone, 0.07 pg/mg for methyl testosterone, 0.15 pg/mg for ethinyl estradiol, 2.10 pg/mg for metandienone, 0.86 pg/mg for testosterone propionate, 0.95 pg/mg for testosterone cypionate, 1.90 pg/mg for nandrolone decanoate, 3.10 pg/mg for testosterone decanoate and 4.80 pg/mg for testosterone undecanoate. Application to doping control has been demonstrated. In a series of 18 sportsmen, two tested positive for anabolic steroids in hair whereas urinalysis was negative for both of them. The first positive case was nandrolone and the second case concerned the identification of testosterone undecanoate. Measured in 10 white males aged between 22 and 31 years, the testosterone concentration was in the range 1.7-9.2 pg/mg (mean=5.0 pg/mg). The method was also applied in meat quality control. Of the 187 analyses realized based upon hair and urine sampling in slaughter houses, 23 were positive for anabolic steroids in hair: one case for boldenone, one case for metandienone, two cases for testosterone propionate, three cases for nandrolone, five cases for testosterone decanoate and 11 cases for methyl testosterone. In the meantime, urinalysis was always negative for these drugs or their metabolites.     

Effect of cell polarization and differentiation on entry of Listeria monocytogenes into the enterocyte-like Caco-2 cell line.

The entry of Listeria monocytogenes into the enterocyte-like Caco-2 cell line was studied as a function of cell polarization and differentiation. L. monocytogenes entered through the entire surface of nonpolarized cells and, predominantly, through the basolateral surface of polarized cells based on the following observations: (i) sites of L. monocytogenes invasion paralleled the distribution of the transferrin receptor, a well-known basolateral marker of polarization; (ii) numbers of internalized bacteria decreased dramatically when Caco-2 monolayers cultured beyond confluency were used (about 0.1% of the inoculated bacteria versus 1 to 2% with nonconfluent monolayers); and (iii) L. monocytogenes entry into postconfluent monolayers was greatly enhanced by treating cells with Ca(2)+ -free medium, a procedure that disrupts intercellular junctions and thus exposes the basolateral surface to bacteria. Ethylene glycol-bis (beta-aminoethyl ether)-N, N, N',N' -tetraacetic acid (EGTA) had contradictory effects on L. monocytogenes entry as this reagent opened intercellular junctions but inhibited binding and internalization of bacteria. Finally, the role of the inlAB locus in L. monocytogenes entry was confirmed because and inlAB mutant was 50- to 100-fold less invasive than the parental strain regardless of the monolayer's age. However, the inlAB mutant was still able to enter cells and to induce intracellular actin polymerization. Entry of inlAB bacteria into Caco-2 cells was not inhibited by EGTA.     

Dick内固定器治疗胸腰段骨折并截瘫66例

0　引言　在脊柱损伤中 ,最常见的是胸腰椎骨折脱位并截瘫 ,在其治疗过程中 ,恢复脊柱正常力线、重建脊柱稳定性、恢复椎管容积和解除脊髓压迫是首要问题 .近年来作者采用Dick内固定器治疗胸腰段骨折脱位并截瘫 6 6例 ,经随访观察 ,取得满意效果 .1　对象和方法1.1　对象　本组 6 6 (男 48,女 18)例 ;年龄 13～ 6 0 (平均 32 )岁 ;职业工人 2 0例 ,农民 36例 ,学生 4例 ,教师 6例 ,家属 2例 .损伤原因 :交通事故 13例 ,直接砸伤 2 2例 ,高处坠落伤31例 .损伤节段 :T1 1 2例 ,T1 2 10例 ,L1 2 2例 ,L2 11例 ,L32例 ,L42例 ,L5 1例 ,T8,91…     

Brain evoked potential topographic mapping based on the diffuse approximation

Evoked potential mapping is a convenient technique to describe brain electrical activity using pictorial representation. A new interpolation method based on the diffuse approximation is applied to represent evoked potential distribution over the skull. The method retains most of the attractive features of the finite-element method but does not require explicit elements. In the simulation examples, the human head is assumed to be a single-layer sphere with homogeneous conductivity, and Ary eccentricity transformation is considered to approximate the more realistic three-shell model. The patterns shown in the computed maps suggest the ability of the proposed method to extract coherent information from the data from different electrodes. In the application protocol, visual evoked potentials are used to test the method with a realistic head shape.     

Afferents to Visually Responsive Grafts of Embryonic Occipital Neocortex Tissue Implanted into V1 (Oc1) Cortical Area of Adult Rats

The aim of this study is to determine, as precisely as possible, the topography and the density of host afferents to visually responsive grafts of occipital embryonic cells implanted in block form into the occipital neocortex of adult rats. The presence of visual activity in the grafts was assessed through field potential and single unit electrophysiological recordings. Field potentials appeared triphasic in shape, had low peak-to-peak amplitude (= 100 micro V), and had normal time latencies (? 30 msec). Polarity reversal was never observed. Single unit recordings showed that graft neurons exhibited normal (desynchronized) spontaneous activity, had discrete receptive fields (? 20 masculine in dia.), and responded to small stationary light flashes. A topic projection of the visual field in the grafts was also observed. Injections of cholera toxin sabunit B (CTB) into these responsive grafts induced retrograde labeling in almost all the brain regions normally projecting to the occipital cortical areas. The visual related cortical (Oc 1, Oc2) and thalamic (LP, LD, LGB) regions of the host provide the largest contingent (70-75%) of afferents to the graft. Finally, one of the major findings of this study is that 93-97% of the labeled cortical cells were found in cortical layers V and VI with a net preference for layer VI. A noticeable proportion of these layer VI labeled neurons (15-20%) was systematically observed in sublayer VIb, very close to, or even within, the white matter. We suggest thus that grafts inserted into the occipital cortex of adult rats receive functional visual inputs through various neuronal circuits. Visual inputs could be conveyed to graft eells by: (i) regenerating axons of geniculate neurons previously innervating the injured cortical site; (ii) formation of collateral branches from thalamic axons ending normally in the host cortex close to the graft boundary; and (iii) development of neuronal processes from cells located in the host cortex, mainly in layers V and VI of the occipital areas. Depending on multiple factors, yet unknown but very likely related to the host-graft integration, the cortical circuits might be either the principal afferent inflow to the graft or only a complement to the thalamic input.     

In vivo ciliogenesis in human fetal tracheal epithelium

Development of ciliated (CC) in the fetal human trachea was studied by light and electron microscopy in specimens obtained from 45 embryos or fetuses aged from 9 to 27 weeks of gestation (menstrual age). Four stages could be recognized during tracheal development. Up to 11 weeks (stage I), the trachea was covered with a columnar undifferentiated epithelium with abundant glycogen, apical microvilli, and primary cilia. From 12 to 18–19 weeks (stage II), centriolo-genesis and secondary ciliogenesis were very active, and the percentage of CC and secretory cells (SC) progressively increased. From 20 to 22–23 weeks, the density of CC was higher but, in parallel, the percentage of SC decreased (stage III). Throughout this period, the different steps of ciliogenesis could be identified in the same field, and the ciliated borders consisted of ciliary shafts with a disorderly arrangement. Megacilia were identified. Some of the preciliated cells had both cilia and secretory granules in their apical cytoplasm. After 24 weeks (stage IV), the ciliated border was apparently mature, the rootlets lengthened, and the cilia were correctly orientated. Whatever the fetal age, the density of CC was significantly higher (P < .01) in the dorsal trachea compared to the ventral trachea. There are many similarities between animal and human ciliogenesis, but in human fetuses, most of the ciliary differentiation occurs early, during the first half of gestation. As demonstrated in experimental models, SC likely play a major role in genesis of CC during the fetal development of the human trachea.     

Quantitative analysis of inflammatory cells infiltrating the cystic fibrosis airway mucosa

Airway inflammation represents a hallmark of the cystic fibrosis (CF) disease. However, the mucosal distribution of immune cells along the CF airways has not been clearly defined, particularly in intermediate bronchi and distal bronchioles. We analysed lung tissues collected at the time of transplantation from homozygous DeltaF508+/+CF patients versus non-CF donors. Using immunohistochemistry, the distribution of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, polymorphonuclear neutrophils (PMN), mast cells, CD3+ T cells, including the CD4+ and CD8+ subsets, CD20+ B cells, CD38+ plasma cells and CD68+ macrophages, was analysed at lobar, segmental and distal levels of the bronchial tree. Using image cytometry, the number of cells per mm2 was assessed in the depth of the bronchial wall. In CF airways, alterations mainly consisted in lesions of the surface epithelium. Numerous immune cells were heterogeneously distributed all along the bronchial tree and mainly located in the mucosa, beneath the surface epithelium. Compared to non-CF donors, the lymphoid aggregates formed by B cells were significantly larger all along the CF airways (P = 0.001). The number of T lymphocytes was higher at the CF distal level (P = 0.035), where we observed an intense tissue damage. PMN preferentially accumulated (P = 0.033) in the CF surface epithelium, which overexpressed ICAM-1 but not VCAM-1 and E-selectin. These results highlight the nature of the inflammatory infiltrate in the CF airway mucosa and emphasize a prominent implication of PMN, B and T lymphocytes in the CF disease.     

Human anti-mouse antibody response to the injection of murine monoclonal antibodies against IL-6.

We analysed human anti-mouse antibodies (HAMA) in 12 patients (six with multiple myeloma (MM) and six with metastatic renal cell carcinoma (MRCC) who were treated with B-E8, an IgG1 MoAb against IL-6. Efficiency of the treatment was evidenced by the drop in the serum levels of C-reactive protein (CRP), the in vivo production of which is under the control of IL-6. Three patients with MM and the six patients with MRCC became immunized to the injected MoAb. HAMA appeared between days 7 and 15 after the beginning of the treatment. The nine patients made IgG antibodies; four also made IgM. All immunized patients made anti-idiotype antibodies specific to B-E8. Two of them also developed HAMA directed to murine IgG1 isotype; in these two patients B-E8 MoAb cleared rapidly from the circulation with loss of treatment efficiency. In the patients who developed only anti-idiotype antibodies, serum levels of B-E8 remained unchanged and CRP production remained inhibited, indicating that treatment remained efficient in the presence of HAMA. Circulating B-E8 MoAbs were still able to bind to IL-6 and to inhibit IL-6-dependent proliferation despite the presence of anti-idiotypic HAMA. Therefore, in contrast to HAMA produced against MoAb directed against cellular targets, HAMA against anti-IL-6 MoAb idiotopes led neither to clearance nor to functional inactivation of the injected MoAb. This was further shown by resuming the B-E8 treatment with success in a patient who still had anti-idiotypic HAMA.     

Left ventricular relaxation and hypertension. Effect of captopril on gamma-angiographic parameters]

In order to evaluate the action of captopril on left ventricular filling in hypertension, 14 hypertension (158 +/- 10/101 +/- 5 mmHg) patients aged 51 +/- 6 years were investigated by Technetium 99m gamma-angiography. The time/activity curve was used to determine the maximum filling rate (MFR) and maximum filling time (MFT) of the ventricle before and after treatment with captopril (mean dose : 44 +/- 26 mg/day for 7 months). Blood pressure was significantly lowered by treatment and there was a decrease in left ventricular mass from 128 +/- 17 to 118 +/- 15 g/m2 (p = 0.07). Maximum filling rate was accelerated by treatment from 2.27 +/- 0.57 to 2.57 +/- 0.43 VTD . s-1, p = 0.005). This variation was due essentially to half of the patients, suggesting an "all or nothing" type response. Maximum filling time did not vary. The basic question raised by this type of study is to know whether the improvement in the available relaxation parameter, MFR, was associated with actual improvement in filling, which is the true aim. Although the explanations offered for the observed findings are hypothetical, taking into account of all the trial data together with the morphological data provided by echocardiography suggests that captopril does have an actual and hemodynamically significant action on filling.