Detection and typing of human papillomavirus by e6 nested multiplex PCR

A nested multiplex PCR (NMPCR) assay that combines degenerate E6/E7 consensus primers and type-specific primers was evaluated for the detection and typing of human papillomavirus (HPV) genotypes 6/11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, 66, and 68 using HPV DNA-containing plasmids and cervical scrapes (n = 1,525). The performance of the NMPCR assay relative to that of conventional PCR with MY09-MY11 and GP5+-GP6+ primers, and nested PCR with these two primer sets (MY/GP) was evaluated in 495 cervical scrapes with corresponding histologic and cytologic findings. HPV prevalence rates determined with the NMPCR assay were 34.7% (102 of 294) in the absence of cervical intraepithelial neoplasia (CIN 0), 94.2% (113 of 120) in the presence of mild or moderate dysplasia (CIN I/II), and 97.8% (44 of 45) in the presence of severe dysplasia (CIN III). The combination of all four HPV detection methods applied in the study was taken as "gold standard": in all three morphological subgroups the NMPCR assay had significantly (P < 0.0001) higher sensitivities than the MY09-MY11 and GP5+-GP6+ assays and sensitivities comparable or equal to those of the MY/GP assay. All 18 HPV genotypes investigated were detected among the clinical samples. The ratio of high- to low-risk HPV genotypes increased from 4:1 (80 of 103) in CIN 0 to 19:1 (149 of 157) in CIN I to III. Multiple infections were detected in 47.9% (124 of 259) of the patients. In conclusion, the novel NMPCR method is a sensitive and useful tool for HPV DNA detection, especially when exact HPV genotyping and the identification of multiple HPV infections are required.     

Characteristic genotypes discriminate between Babesia canis isolates of differing vector specificity and pathogenicity to dogs

The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene were characterized in eight Babesia canis isolates of differing geographic origin, vector specificity, and pathogenicity to dogs. The genotypes determined by sequencing segregated into three clearly separated groups close to or near the species level and correspond to the previously proposed subspecies B. canis canis, B. canis vogeli, and B. canis rossi. The three genotypes can be distinguished by Sau96I digestion of the polymerase chain reaction (PCR)-amplified rDNA target. Received: 12 December 1997 / Accepted: 5 January 1998     

Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase C gamma 2 that specifically increases external Ca2+ entry

The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype.     

The contribution of vestibular input to the stabilization of human posture: a new experimental approach

An experiment was designed to evaluate the vestibular contribution to the stabilization of upright stance in normals and in two patients with loss of vestibular function. A forward or backward displacement of a load (2 kg) by a torque motor attached to the subject induced opposing movements in the head and trunk. The small linear acceleration of the head in space of about 0.1 g was followed, with a latency of 50-65 ms, by EMG responses in the tibialis anterior and rectus femoris (backward acceleration) or gastrocnemius muscles (forward acceleration). These responses were absent in patients with a vestibular deficit. It is suggested that the observed EMG responses are due to fast acting vestibulospinal reflexes involved in the regulation of upright stance. For comparable head accelerations the integrated EMG responses induced by the vestibulospinal mechanism are about ten times smaller than those induced by spinal stretch reflexes during displacement of the feet. Vestibulospinal reflexes would appear, therefore, to play only a minor role in the compensation of stumbling.     

Induced histidine decarboxylase in endotoxic shock: Identification of the enzyme in rat liver and influence of its inhibitors on survival parameters

The hypothesis of a causal relationship between a progressive and unrestrained increase of tissue histamine formation by activation of an inducible histidine decarboxylase (HDC) and lethality in endotoxic shock (Schayer's induced histamine concept) was tested in a standardized rat endotoxic shock model.Initial enzyme identification studies in the rat shock liver (8 hrs after endotoxin challenge) clearly demonstrate that the induced histidine decarboxylase is an acid (specific) HDC. The succeeding randomized, controlled study with appropriate inhibitors of the enzyme, -methyl-histidine (competitive inhibitor) and -fluoromethyl-histidine (irreversible inhibitor) using doses of 2, 20 or 100 mg/kg showedno significant effect on the survival rate of rats in endotoxin shock. The survival rate of the non-treated endotoxin control group (NaCl) was 25%; all methylprednisolone treated rats (50 mg/kg) survived.Thus, the induced histamine isnot a predominant factor (necessary or sufficient determinant) for the lethal outcome in rat endotoxic shock. The protective effect of MP isnot predominantly due to the inhibition of the induced histidine decarboxylase. The use of HDC-inhibitors as the appropriate instruments for evaluation of the significance of this mechanism is discussed.Clinic of General Surgery, City Hospital Sieburg.     

Comparison of three methods to measure absolute cerebral hemoglobin concentration in neonates by near-infrared spectrophotometry

Three methods by which to determine absolute total cerebral hemoglobin concentration (tHb in micromol/L) by near-infrared spectrophotometry (NIRS) have evolved: (1) tHbo, requiring oxygenation changes and arterial oxygen saturation measurements as a reference using a relative NIRS algorithm, (2) tHbg, using a geometrical multidistance principle and (3) tHbgo, a combination of both. The aim of this study was to compare the three methods quantitatively. Sixteen clinically stable preterm infants with a mean gestational age of 29.6 (range of 25.1-36.4) weeks, birthweight of 1386 (680-2820) g and a postnatal age of 2.5 (0.5-6) days, who needed supplemental oxygen, were enrolled. The mean+/-standard deviation tHbg was 150.2+/-41.8 micromol/L (range of 61.6-228.9 micromol/L), the tHbo was 62.1+/-27.2 micromol/L (26.0-110.8 micromol/L) and the tHbgo was 89.3+/-45.6 micromol/L (26.5-195.9 micromol/L). The correlation coefficient among the three methods were tHbg and tHbgo r=0.736; tHbo and tHbgo r=0.938; tHbg and tHbo r=0.598. A multiple regression with variable selection by Mellow's C(p) showed, that tHbg was correlated to the birthweight, the postnatal age, the heart rate and the pCO2 (r(2)=0.588), tHbo and tHbgo were associated with the hemoglobin concentration in the blood, the mean arterial blood pressure and the pCO2 (r(2)=0.493 and 0.406, respectively). The three methods (tHbg, tHbo, and tHbgo) give systematically different tHb readings and large intersubject variability.     

Properties and regulation of chloride channels in cystic fibrosis and normal airway cells

The present study examines the properties of Cl^–channels in cultured respiratory cells of cystic fibrosis (CF) patients and normal (N) individuals. In excised membrane patches the conductances for CF and N Cl^– channels were larger at positive as compared to negative clamp voltages (V _c): 74±2.6 (V _c > 0) and 47±2.0 pS (V _c < 0) for CF (n= 57) and 69±3.6 (V _c > 0) and 45±2.3 pS (V _c < 0) for N (n=35). The open probability (P _o) of the channel increased markedly with depolarization. Both the voltage dependence of the conductance and of P _o contribute to the outward rectification of the channel. The time histogram analysis reveals two open and two closed time constants. The selectivity of the channel was Cl^–=Br^– =I^– > NO ₃ ^– gluconate. The channel was inhibited reversibly by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) at 10^–7 mol/l to 10^–5 mol/l. While Cl^– channels were present in cell attached patches of N cells, they were absent in those of CF cells. The mean conductance for cell attached (N) Cl^– channels was 76±3.2 pS for positive clamp voltages (V _c) and 46±3.9 pS for negative V _c (n=8). When the membrane patches were excised from CF cells Cl^– currents appeared spontaneously (n=19). The immediate appearance (within 1 s) of Cl^– channels after excision was observed at positive (n=6) as well as at negative clamp voltage (n=13). Excision activation of CF Cl^– channels was observed at low (< 10^–9 mol/l) or high (10^–3 mol/l) calcium activities on the cytosolic side of the excised patch. Variation of the Ca⁺ activity (< 10^–9–10^–3 mol/l) or pH (6.5–8.5) on the cytosolic side exerted no effects on these Cl^– channels. These results suggest that Cl^– channels are present in the apical membrane of CF and N respiratory cells but they seem to be inhibited in intact CF cells. Excision of the patch and hence removal of the cytosolic inhibitor leads to an activation of Cl^– channels. The Cl^– channels in excised patches of N and CF cells have identical properties.     

Involvement of different receptors in the regulation of human posture

Compensatory electromyographic (EMG) responses and several biomechanical parameters were studied following impulsive disturbance of the lower limbs during stance on a treadmill. Treadmill acceleration impulses were backwards or forwards directed, or their direction was inverted after 30 ms. Backwards directed impulses were followed by gastrocnemius and forwards directed ones by tibialis anterior EMG responses (latency 65-75 ms) whose duration depended on impulse duration. When the direction of the impulse was inverted, the respective antagonistic leg muscles were activated, with a delay of 68 to 75 ms after onset of stretch of these muscles. The behaviour of the EMG responses could best be correlated to the displacement at the ankle joint and may be described in terms of a stretch reflex response. The function of this stretch reflex mechanism is suggested to be connected with the control of the body's centre of gravity in order to prevent falling. Head movements induced by the impulses showed little correlation with the appearance of the EMG responses, suggesting that the vestibular system is unlikely to be significantly involved in the generation of these responses.