Intracellular antibodies for proteomics

The intracellular antibody technology has many applications for proteomics studies.

The potential of intracellular antibodies for the systematic study of the proteome has been made possible by the development of new experimental strategies that allow the selection of antibodies under conditions of intracellular expression. The Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid-based method originally developed for the selection of antibodies readily folded for ectopic expression. IACT has been used for the rapid and effective identification of novel antigen–antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by selected intracellular antibodies. IACT opens the way to the use of intracellular antibody technology for large-scale applications in proteomics. In its present format, its use is however somewhat limited by the need of a preselection of the input phage antibody libraries on protein antigens or by the construction of an antibody library from mice immunized against the target protein(s), to provide an enriched input library to compensate for the suboptimal efficiency of transformation of the yeast cells. These enrichment steps require expressing the corresponding proteins, which represents a severe bottleneck for the scaling up of the technology.

We describe here the construction of a single pot library of intracellular antibodies (SPLINT), a naïve library of scFv fragments expressed directly in the yeast cytoplasm in a format such that antigen-specific intrabodies can be isolated directly from gene sequences, with no manipulation whatsoever of the corresponding proteins. We describe also the isolation from SPLINT of a panel of intrabodies against a number of different proteins.

The application of SPLINT on a genome-wide scale should help the systematic study of the functional organization of cell proteome.     

Airway responses to antigen challenge in allergic rhinitis and allergic asthma

The density dependence of the maximum expiratory flow-volume curve, functional residual capacity (FRC), and specific airway conductance (SGaw) were determined before and during bronchial provocation with ragweed extract in 27 subjects with ragweed hypersensitivity and a history of either bronchial asthma (16 subjects) or allergic rhinitis (11 subjects). Mean baseline SGaw was significantly lower while mean volume of isoflow (Visov) and FrC were significantly higher in subjects with bronchial asthma. During antigen challenge, 10 of 16 subjects with bronchial asthma (63%) and five of 11 subjects with allergic rhinitis (45%) showed a greater than 35% decrease in SGaw ("reactors"): mean relative decreases in SGaw from baseline were 46% and 53%, respectively. The remaining subjects showed a less than 35% decrease in SGaw ("nonreactors") with mean relative decreases of 9% (allergic asthma) and 6% (allergic rhinitis). Mean Visov increased in all subjects with bronchial asthma and in eight of 11 subjects with allergic rhinitis. A significant increase in FRC (6%) was seen only in the "reactors" with bronchial asthma. Following antigen challenge, the beta adrenergic agonist, isoetharine, increased SGaw and decreased Visov. We conclude that in asymptomatic subjects with ragweed hypersensitivity, (1) central and peripheral airway function is more abnormal in subjects with bronchial asthma than in subjects with allergic rhinitis, (2) subjects of both groups show quantitatively and qualitatively comparable airway responses during antigen challenge with a decrease in SGaw or an increase in Visov, possibly representing increase in central and/or peripheral airflow resistance, respectively, (3) Visov may be a more sensitive indicator of airway response to antigen challenge than SGaw, and (4) the bronchodilator effects of a beta adrenergic agonist on antigen-induced bronchospasm are similar in both groups.     

Immunity in HIV-1-Infected Adults with a Previous State of Moderate-Severe Immune-Suppression and More Than 500 CD4+ T Cell After Highly Active Antiretroviral Therapy

We evaluated phenotypic and functional parameters of immune restoration of 27 HIV-infected patients on highly active antiretroviral therapy (HAART) (HIV-cases) with HIV-RNA levels below detectable limits at least during 18 months, and CD4+ cell per microliter higher than 500 at the moment of the study and lower than 300 anytime before. These patients were compared with 11 HIV-controls that never had less than 500 CD4+ cell per microliter and 20 healthy-controls (HIV seronegative subjects) in a cross-sectional study. HIV-cases had lower counts of naïve CD4+ than HIV-controls and healthy-controls. HIV-patients (both HIV-cases and HIV-controls) showed higher values of naïve and memory CD8+ counts than healthy-controls. TREC-bearing cell levels were significantly lower in HIV-cases than in healthy-controls. Peripheral blood mononuclear cells (PBMC) cultures, HIV-cases had lower values in proliferation to streptokinase (SK) and tetanus toxin (TT) than in healthy-controls. HIV-cases had lower IFN-γ and higher IL-5 production with pokeweed than healthy-controls (P < 0.01). However, IL-5 production of HIV-cases after TT stimulation was lower than in HIV-controls and healthy-controls. Total IgG and IgG1 levels were significantly higher in HIV-cases than in HIV-controls and healthy-controls. Also, IgM levels were significantly higher in HIV-cases than in healthy-controls. Nevertheless, IgG2 levels were significantly lower in HIV-cases and HIV-controls than in healthy-controls. The levels of specific Igs antipneumococcal capsular polysaccharide and TT were significantly lower in HIV-cases than in healthy-controls. HIV-patients with a previous state of severe-moderate immunosuppression normalizing their CD4+ counts have a incomplete immune reconstitution after HAART. Long-term consequences of this subclinical immune deficiency remain to be determined.     

Measurement of functional ability and health status in the arthritic patient

Chronic arthritis may have great impact on the patient but also on his or her family, relatives and friends. The assessment of the consequences of chronic arthritis and the effect of therapy not only in terms of physical, but also psychological and social dimensions deserves more attention. Functional ability and health status can be measured using a questionnaire or ‘instrument’, high-lighting important aspects not quantified with more traditional measurements. In this paper, arguments to apply such instruments more frequently are given. Health status instruments can be used not only to assess beneficial but also deleterious (side-)effects of therapeutic interventions. The properties are summarized of the most frequently used instruments assessing functional ability and health status. Many of these instruments have been evaluated sufficiently for validity and reliability; their sensitivity to detect change seems to be satisfactory. Therefore it is advisable to choose an internationally accepted, frequently used instrument, reflecting the area of interest.     

Epitope specificity is critical for high and moderate avidity cytotoxic T lymphocytes associated with control of viral load and clinical disease in horses with equine infectious anemia virus

Equine infectious anemia virus (EIAV) is a lentivirus that causes persistent infections in horses. We hypothesized that high-avidity CTL specific for nonvariable epitopes might be associated with low viral load and minimal disease in EIAV-infected horses. To test this hypothesis, memory CTL (CTLm) responses were analyzed in two infected horses with high plasma viral loads and recurrent disease (progressors), and in two infected horses with low-to-undetectable viral loads and mild disease (nonprogressors). High-avidity CTLm in one progressor recognized an envelope gp90 epitope, and the data documented for the first time in EIAV that viral variation led to CTL escape. Each of the nonprogressors had high-to-moderate avidity CTLm directed against epitopes within Rev, including the nuclear export and nuclear localization domains. These results suggested that the epitope specificity of high- and moderate-avidity CTLm was an important determinant for disease outcome in the EIAV-infected horses examined.     

尼古丁依赖的功能影像学研究进展

在过去10年中,随着功能脑影像学技术在物质滥用领域的发展,越来越多的研究者运用这些技术以探讨尼古丁成瘾的神经生物学机制。本文主要总结了尼古丁急、慢性暴露下对脑功能影响的神经脑影像学研究进展,证实了尼古丁依赖与中脑边缘犒赏系统、调节注意、记忆、运动以及联想相关脑环路之间的相关性。这些研究发现将为揭示尼古丁依赖的神经生物学机制提供更多的证据支持,进一步促进今后戒烟治疗的发展。     

Determining appropriate models for joint control using surface electrical stimulation of soleus in spinal cord injury

The mechanical impedance of the ankle joint during electrical stimulation of the soleus is studied by applying constant-velocity 10° angular perturbations to the ankle and measuring the resultant torque. Both neurologically intact subjects and spinal cord injured subjects are tested. Lumped, piecewise linear models are developed to predict the torque from the measured displacement and acceleration signals. The commonly used second-order mass-spring-dashpot model fails to predict the changes in torque that occur following imposed movements. A fiveelement, directionally-dependent piecewise linear model is much better at predicting the measured responses for velocities up to 50° s⁻¹. Numerical least squared error indentification techniques are used to estimate the model parameters for three neurologically intact and three spinal cord injured subjects. The average error between the model’s response and the measured response across all subjects is 10·9%. There is some evidence that a velocity-dependent non-linear model could produce better results than the directionally-dependent piecewise linear model.     

Responses of sacral visceral afferents from the lower urinary tract,colon and anus to mechanical stimulation

The discharge characteristics of sacral visceral afferents supplying the urinary bladder, urethra, colon and anus to mechanical stimuli were analyzed in the anaesthetized cat. The stimuli used were passive distension (urinary bladder, colon), isovolumetric contraction (urinary bladder), movements of the urethral catheter and mechanical shearing stimuli (mucosal skin of the anal canal). (1) In total 245 afferent units which projected in the pelvic nerve were isolated from the sacral dorsal roots. From one of the following organs, urinary bladder, colon, urethra and anus 117 afferent units were activated. By these stimuli from the bladder, urethra and anus 122 afferent units could not be activated, and as far as tested also not from the colon; in 6 afferent units the classification was unclear. (2) Afferent units from the urinary bladder and the colon responded consistently to passive distension of the respective organ. The units from the urinary bladder showed graded responses at intraluminal pressures of about 10–70 mm Hg and responded also to isovolumetric contractions of the organ. The thresholds of the units from the bladder to passive distension and contraction varied from about 5 to 20 mm Hg intravesical pressure. (3) The afferent units from the urethra and the anus did not react or showed some weak phasic and irregular responses to distension and contraction applied to the urinary bladder or to distension of the colon. They were consistently excited by low threshold mechanical stimulation of the urethra and anus, respectively. (4) The axons from the bladder, urethra and anus were presumably myelinated (conduction velocity above 2 m/s) and conducted at 10.3±6.1 m/s (n=34, mean±SD), 26.3±9.3 m/s (n=13) and 9.5±5.1 m/s (n=37), respectively. The axons from the colon conducted at about 0.5 to 16 m/s (n=20), 13 of them conducting at less than 2 m/s. About 75% of the axons which could not be activated by mechanical stimulation of the visceral organs were presumably unmyelinated (conduction velocity below 2 m/s). (5) Some ongoing activity was found in 9 out of 26 afferent units from the anus but, with one exception, the afferent units from the bladder, urethra and colon were silent. (6) It is concluded that the pelvic afferent units from the urinary bladder, urethra, colon and anus consist of distinct populations with characteristic response patterns. There is no indication from this investigation that the urinary bladder is supplied by sacral afferents which are only recruited at high intravesical pressures during passive distension and isovolumetric contractions and which are possibly associated with pain.Supported by the Deutsche Forschungsgemeinschaft