Assessment of the viability and pregnancy potential of mouse embryos biopsied at different preimplantation stages of development

The developmental potential in vitro and in vivo of preimplantation mouse embryos biopsied at the 4-cell, 8-cell and morula stages were investigated. Biopsy had the least impact when performed at the 8-cell stage. There was no effect of biopsy on the development of 8-cells of blastocysts in vitro (95% compared with 99% of controls) or the implantation rate after transfers (82 versus 87%, P greater than 0.05); however, fewer embryos (52 versus 71%, P less than 0.05) resulted in viable fetuses. There was no effect of biopsy at the 8-cell stage on fetal weight on day 17. Blastocyst formation in vitro was significantly less for 4-cell biopsies compared with their controls (76 versus 90%, P less than 0.001) and biopsy also affected the implantation rate (44 versus 59%, P less than 0.01). Biopsy was most detrimental when performed on morulae, reducing the implantation rate from 65% for controls to 21% for biopsies (P less than 0.001). Fetal viability was also markedly affected with a reduction on day 17 from 42 to 26% accompanied by a significant reduction (24%, P = 0.02) of the mean fetal weight. Handling of embryos for biopsy at the morula stage, which involved removal of the zona pellucida, was a significant but not complete cause of the reduced implantation potential observed (sham-controls and intact-controls: 34 and 65%, P less than 0.001), while puncture of the zona during the biopsy of 4-cell and 8-cell embryos had no effect. Therefore, the 8-cell mouse embryo is the most suitable state for embryo biopsy.     

Isolation of the equine granulocytic ehrlichiosis agent, Ehrlichia equi, in tick cell culture.

The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) agent. Both are suspected of being transmitted by ticks. We have successfully isolated E. equi in a cell line, IDE8, derived from a putative vector, the tick Ixodes scapularis. Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers. Cultures were incubated in a candle jar at 34 degrees C in tick cell culture medium with NaHCO3 and an organic buffer [3-(N-morpholino)-propanesulfonic acid] (MOPS). Within 2 weeks, infected cells were detected in Giemsa-stained culture samples, and the organisms subsequently spread to uninfected cells in the cultures. E. equi was passaged serially by transferring a portion of an infected culture to new cell layers every 2 to 3 weeks. The identity of the organisms was confirmed by PCR using oligonucleotide primers specific for E. equi and the HGE agent and by immunocytology. Homologous equine antibodies and human anti-HGE convalescent serum recognized E. equi grown in tick cell culture. Electron microscopy revealed electron-lucent and -dense ehrlichia-like forms developing within host cell endosomes. E. equi passaged twice in tick cell culture retained infectivity and pathogenicity for the equine host, as demonstrated by intravenous inoculation of a suspension of infected tick cells and subsequent reisolation from peripheral blood, in fulfillment of Koch's postulates. The horse developed severe clinical signs, i.e., fever, inappetence, thrombocytopenia, icterus, and limb edema, typical of granulocytic equine ehrlichiosis, within 1 week.     

Nested PCR for specific detection and rapid identification of human picornaviruses.

A nested PCR for the detection and rapid identification of human picornaviruses is described. Enteroviruses and rhinoviruses were amplified with the same set of four primers from the 5'-noncoding region. The nested primers allowed the detection of far less than 1 PFU in diluted virus stocks without Southern blot hybridization. In patients with neurological disorders (mainly aseptic meningitis), 43% of 37 specimens (11 of 21 cerebrospinal fluid specimens, 2 of 10 serum specimens, and 3 of 6 stool specimens) were positive by PCR. A total of 21% (10 of 47 specimens) of heart biopsy specimens from patients with dilative cardiomyopathy were PCR positive, whereas 3% (2 of 70 specimens) of control biopsy specimens from patients with coronary artery disease were PCR positive. PCR-amplified fragments from 27 of 29 clinical isolates and 14 of 28 patient samples were successfully serotyped by restriction enzyme digestion. Two specimens were further investigated by direct sequencing of PCR products, leading to the identification of a poliovirus type 3 isolate with a sequence that was highly divergent from previously published sequences.     

Action of single dynamic fusimotor neurones on cat soleus Ia afferents during muscle shortening

Summary 1. The ability of single dynamic fusimotor (d) fibres to sustain the firing of muscle spindle primary (Ia) afferents during shortening was investigated in soleus muscles of anaesthetised cats. 2. Of 11 d fibres, 10 could maintain Ia firing during 10 mm/s shortening. Of the 7 tested at greater velocities, 5 could maintain Ia firing during shortening at velocities greater than 50 mm/s. 3. This ability was, however, critically dependent upon the timing of the stimulation. In particular, it rapidly reduced with increasing duration of stimulation before the onset of shortening. Furthermore, if appreciable stretch occurred between the onset of _d stimulation and the onset of shortening, this could greatly reduce the ability of _d fibres to sustain Ia discharge. 4. If _d neurones are on occasion phasically activated during voluntary shortening movements, their action could be an important determinant of Ia firing, even in the presence of weak _s action. Therefore in chronic recordings, observation of Ia firing during muscle shortening is not an adequate criterion for inferring _d activity.     

NADPH oxidase-mediated oxidative stress: genetic studies of the p22(phox) gene in hypertension

Increased vascular production of reactive oxygen species, especially superoxide anion, significantly contributes to the oxidative stress associated with hypertension. An enhanced superoxide production causes an increased inactivation of nitric oxide that diminishes nitric oxide bioavailability, thus contributing to endothelial dysfunction and hypertrophy of vascular cells. It has been shown that NADPH oxidases play a major role as the most important sources of superoxide anion in phagocytic and vascular cells. Several experimental observations have described an enhanced superoxide generation as a result of NADPH oxidase activation in hypertension. Although these enzymes respond to stimuli such as vasoactive factors, growth factors, and cytokines, recent data suggest a significant role of the genetic background in the modulation of the expression of its different components. Several polymorphisms have been identified in the promoter and in the coding region of CYBA, the gene that encodes the essential subunit of the NADPH oxidase p22phox, some of which seem to influence significantly the activity of these enzymes in the context of cardiovascular diseases. Among CYBA polymorphisms, genetic investigations have provided a novel marker, the -930(A/G) polymorphism, which determines the genetic susceptibility of hypertensive patients to oxidative stress.     

Calcium homeostasis and glucose uptake of murine myotubes exposed to insulin,caffeine and 4‐chloro‐m‐cresol

The modulation of glucose uptake by cytosolic calcium and the role of insulin on calcium homeostasis in insulin‐target cells are incompletely understood and results are contradictory. To address this issue, we used the C2C12 murine skeletal muscle cell line model and examined the influence of caffeine and 4‐chloro‐m‐cresol, two ryanodine receptor agonists known to mobilize intracellular calcium stores and increase cytosolic free calcium concentration. We followed ⁴⁵calcium efflux, a validated indicator of cytosolic calcium concentration, and 3‐O‐methyl‐[1–³H]‐d ‐glucose uptake in parallel. We also determined if insulin incubation affected ⁴⁵calcium influx rate. A 30‐min treatment by 1 μm insulin highly significantly increased ⁴⁵calcium efflux by 8.5% (P = 0.0014), despite a significant reduction of ⁴⁵Ca²⁺ influx already measurable after 20 and 30 min of insulin stimulation (?16.6%, P = 0.0119 and ?21.3%, P = 0.0047, respectively). Caffeine (1–20 mm ) and 4‐chloro‐m‐cresol (0.05–10 mm ) concentration‐dependently increased ⁴⁵calcium efflux, the latter being more potent and efficacious. These agents, in a concentration‐dependent manner, inhibited both basal and, more potently, insulin‐stimulated glucose uptake. This resulted in a negative correlation of glucose uptake and ⁴⁵calcium efflux (r > 0.95, P < 0.001). This effect was ～5 times greater for caffeine than for 4‐chloro‐m‐cresol, suggesting a calcium‐independent part of the glucose uptake inhibition by caffeine. In our in vitro model of cultured muscle cells, insulin appears to prevent calcium overload by both stimulating efflux and inhibiting cell storage. This effect, taken together with the observed inhibitory, inverse relationship between ⁴⁵calcium efflux and glucose uptake, contributes to describing the complex insulin–calcium interplay involved in target cells.     

Sensitization and lung function in workers occupationally exposed to natural thickening products

BACKGROUND: Three workers engaged in the manufacture of natural thickener products (Cassia spp., guar, and tamarind flour) were occupationally sensitized to Cassia spp. Therefore, a cross-sectional study on the prevalence of sensitization among and respiratory health of the employees of this plant was conducted. METHODS AND RESULTS: Sixty-two workers (36 with high, 26 with low exposure) participated in the survey. Skin prick tests and specific IgE tests with extracts of these components revealed that 11.3% were sensitized to Cassia spp. and 9.7% to at least one species of storage mites, with a significantly higher portion of atopic subjects in the sensitized group. Overall, 55% of the subjects reported work-related symptoms (upper and lower airways, eyes, or skin). FVC % pred. was significantly lower in highly exposed workers, while RV % pred. and RV%TLC % pred. were significantly higher in this group. In the multivariate model, sensitization was not a risk factor for impairment of lung function. CONCLUSIONS: Dust exposure to flours may not only cause allergic sensitization but also induce chronic changes in lung function.     

Anti-anti-idiotypic (Ab3) antibodies that bind progesterone-11alpha-bovine serum albumin differ in their combining sites from antibodies raised directly against the antigen

Polyclonal rabbit anti-idiotypic (Ab2) antibodies raised against the antiprogesterone mAb DB3 (Ab1) were used to induce an Ab3 antiprogesterone response in BALB/c mice. While the affinity of Ab3 sera for progesterone was 10-50-times lower than that of DB3, their steroid-binding specificity showed considerable similarity to DB3. Two immunoglobulin M (IgM) Ab3 monoclonal antibodies (mAbs), 1A4 and 3B11, were obtained, both of which bound progesterone conjugated to bovine serum albumin (progesterone-BSA). 1A4 also bound free progesterone, although with low affinity and very broad cross-reactivity. Like DB3, 1A4 is encoded by a heavy-chain variable region (VH) gene segment from the small VGAM3.8 family, a restriction that is characteristic of antibodies raised against progesterone-11alpha-BSA. In contrast, 3B11 binds progesterone-11alpha-BSA but not free progesterone and is encoded by an unrelated VH gene from the J558 family. The light chain variable region (VL) of 1A4 lacks the intradomain disulphide bridge owing to replacement of CysL23 by Tyr. Both the 1A4 and 3B11 heavy chains have extremely short complementarity determining region (CDR) H3 loops, comprising three and four amino acids, respectively. Modelling of the combining site of 1A4 from the X-ray crystallographic structure of DB3 indicates that the short H3 loop is a major factor in the loss of affinity and specificity for steroid.     

High-dose inhaled steroids in the management of asthma A comparison of the effects of budesonide and beclomethasone dipropionate on pulmonary function, symptoms, bronchial responsiveness and the adrenal function

Svendsen UG, Frølund L, Heinig JH, Madsen F, Nielsen NH, Weeke B. High-dose inhaled steroids in the management of asthma. A comparison of the effects of budesonide and beclomethasone dipropionate on pulmonary function, symptoms, bronchial responsiveness and the adrenal function.
The efficacy of budesonide (800 μg b.d.) and beclomethasone dipropionate (750 μg b.d.) in controlling the symptoms of asthma, pulmonary function, bronchial responsiveness to histamine, and adrenal function, was assessed in a double-blind, double-dummy cross-over study of 36 adult chronic asthmatic patients. The patients, the majority of whom were assessed to be affected to a severe degree, were insufficiently controlled in their current regimen of inhaled steroids and/or inhaled and oral bronchodilators. A 2 weeks baseline period preceded 6 weeks of treatment with each of the study drugs. Both treatment groups showed improvements from baseline in clinical assessment of lung function carried out after the first 6 weeks of treatment. No significant differences were seen throughout the entire 12 weeks study, when comparing the effects of the treatments on FEV₁ FVC, PEF or the histamine PC₂₀. Asthma severity, symptom score and inhaled bronchodilator use showed the same results after both treatments. It is concluded that inhalations of budesonide and beclomethasone dipropionate in high doses are equally potent in the treatment of severe asthma. There is no significant influence on the adrenal function and no significant side effects during a period equal to that of the present study.     

Antigenic and genetic analyses of human rotavirus with dual subgroup specificity.

In our previous study (S. Urasawa, T. Urasawa, K. Taniguchi, F. Wakasugi, N. Kobayashi, S. Chiba, N. Sakurada, S. Morita, O. Morita, M. Tokieda, T. Kawamoto, K. Minekawa, and M. Oseto, J. Infect. Dis. 160:44-51, 1989) of antigenic characterization of about 300 human rotavirus (HRV) isolates collected at different localities in Japan, we found 4 HRV isolates having unique antigenic and genetic constructions. The four strains possessed both subgroup I and subgroup II antigens, serotype 3 antigen, and a long RNA electropherotype. The reactivity pattern of these four HRV isolates with three monoclonal antibodies (MAbs) directed to an outer capsid protein, VP4, and with one MAb directed to an inner capsid protein, VP2, was clearly different from those of usual subgroup II HRVs having serotype 1, serotype 3, or serotype 4 specificity and a long RNA pattern, whereas their reactivity pattern was similar to that of strain K8 (subgroup II, serotype 1), which possessed unique VP4 and VP2 proteins. RNA-RNA cross-hybridization analysis indicated that while the four isolates were genetically distinct from the two genetic groups of HRV reported previously, i.e., the Wa family (strains KU, S3, and YO) and the DS-1 family (strain S2), they were closely related to strain K8, a strain having unique antigenic and genetic properties (K. Taniguchi, K. Nishikawa, T. Urasawa, S. Urasawa, K. Midthun, A. Z. Kapikian, and M. Gorziglia, J. Virol. 63:4101-4106, 1989).