Antigenic properties of phage displayed peptides comprising disulfide-bonded loop of the immunodominant region of HIV-1 gp41

The HIV-1 envelope glycoprotein gp41 contains Cys(X)5Cys motif, which has been shown to elicit a strong antibody response in almost all HIV-1 infected individuals. This disulfide-bonded loop region is conserved in most retroviruses suggesting the existence of an essential function in virus life cycle. In this study, we displayed the peptides comprising 12 amino acids of the immunodominant loop of gp41 on the surface of M13 phage as N-terminal fusions to the minor coat protein pIII and major coat protein pVIII of the phage and demonstrated that cysteine loop containing peptide expressed on phage recognized 62 out of 63 (98.4%) HIV-1 positive samples but not control negative sera while phage bearing linear peptides detected 4-30% of HIV-1-positive sera. The main advantage of phage-based ELISA or other antibody detection-based diagnostic tests of HIV-infection to be used for massive screening in developing countries is the reproducible, simple, rapid and low-cost production of recombinant antigens.     

Immunoenzymatic assay that measures the expression of murine histocompatibility antigens in macrophages and lymphocytes

A convenient—simple, sensitive, rapid and reproducible—enzyme immunoassay to measure H-2 particulated and solubilized cellular antigens is described. Cellular antigens were measured by ELISA through the binding of specific biotinylated antibodies and streptoavidin-peroxidase conjugate to cells in suspension. Endogenous peroxidase activity of activated cells was inhibited by addition of sodium azide and H₂O₂ in acid conditions. The assay proved capable of distinguishing between two cell lines (EL–4/H–2^b and P815/H–2^d) and even between the cells of three congenic mouse strains (BALB/B, H–2^b, BALB/c, H–2^d, and BALB/K, H-2^k) and was sensitive to as few as 2.5 × 10⁴ cells/well. Results were comparable to those obtained with FACS. An inhibition version of this assay was found to be very useful for the detection of H–2 antigens present in whole antigen cells extracts. © 1993 Wiley-Liss, Inc.     

Prolame ameliorates anxiety and spatial learning and memory impairment induced by ovariectomy in rats

N-(3-hydroxy-1, 3, 5 (10) estratrien-17beta-yl)-3-hydroxypropylamine (17β aminoestrogen, prolame) is a steroidal compound with weak estrogen-related trophic-proliferative effects in uterus. Contrasting with 17β-estradiol (E2) pro-coagulant effects, this compound has high anticoagulant and antiplatelet effects. It has been extensively demonstrated that E2 plays important roles in brain function. However, prolame's influence on central nervous system has not been documented. In this study, we evaluated the effects of prolame replacement in young ovariectomized rats on spatial learning and memory and anxiety, correlating pyramidal cell dendritic spine density changes and neuronal nitric oxide synthase (nNOS) expression in the hippocampus. Ovariectomized young rats were treated with prolame for 4 weeks. Three other groups were used as physiological, pathological, and pharmacological references as follow: gonadally intact cycling females, ovariectomized, and ovariectomized with 17β-estradiol treatment respectively, for the same time period. Experiment 1 investigated the behavioral effects of prolame on anxiety and spatial learning using elevated plus maze (EPM) and Morris water maze (MWM) paradigms respectively. Experiment 2 studied the dendritic spine density and neuronal nitric oxide synthase expression in the hippocampus of the 4 experimental groups. Similar to estradiol, prolame reversed the anxiogenic effects of ovariectomy, evaluated by EPM, and enhanced MWM performance to the level of gonadally intact subjects. Hippocampi from prolame-treated rats exhibited enhanced nNOS immunoreactivity and its relocation in dendritic compartments, as well as recovery of dendritic spine density loss in pyramidal neurons. Hence, prolame may provide an alternative option for ameliorating neurological symptoms caused by surgical menopause.     

Immunodiagnosis of human cysticercosis in cerebrospinal fluid. Antigens from murine Taenia crassiceps cysticerci effectively substitute those from porcine Taenia solium

Tapeworm antigens from Taenia crassiceps performed as well as those antigens from Taenia solium in an enzyme-linked immunosorbent assay for the detection of Cysticercus antibodies in 96 cerebrospinal fluid samples from patients with neurocysticercosis and in 96 CSF samples from patients with other varied neurological ailments. Thus, this manageable murine model of experimental cysticercosis solved the problem of antigen supply for clinical and epidemiological applications, and it provided an immediate means of abundant production of antigens for the wide distribution and standardization of immunodiagnostic tests for cysticercosis.     

Phage-displayed T-cell epitope grafted into immunoglobulin heavy-chain complementarity-determining regions: an effective vaccine design tested in murine cysticercosis.

A new type of immunogenic molecule was engineered by replacing all three complementarity-determining-region (CDR) loops of the human immunoglobulin (Ig) heavy-chain variable (V(H)) domain with the Taenia crassiceps epitope PT1 (PPPVDYLYQT) and by displaying this construct on the surfaces of M13 bacteriophage. When BALB/c mice were immunized with such phage particles (PIgphage), a strong protection against challenge infection in very susceptible female hosts was obtained. When specifically stimulated, the in vivo-primed CD4(+) and CD8(+) T cells isolated from mice immunized with PT1, both as a free peptide and as the PIgphage construct, proliferated in vitro, indicating efficient epitope presentation by both major histocompatibility complex class II and class I molecules in the specifically antigen-pulsed macrophages used as antigen-presenting cells. These data demonstrate the immunogenic potential of recombinant phage particles displaying CDR epitope-grafted Ig V(H) domains and establish an alternative approach to the design of an effective subunit vaccine for prevention of cysticercosis. The key advantage of this type of immunogen is that no adjuvant is required for its application. The proposed strategy for immunogen construction is potentially suitable for use in any host-pathogen interaction.     

Quantitative testing of pain perception in subjects with PTSD--implications for the mechanism of the coexistence between PTSD and chronic pain

Post-traumatic stress disorder (PTSD) often co-occurs with chronic pain. Neither the underlying mechanism of this comorbidity nor the nature of pain perception among subjects with PTSD is well defined. This study is the first systematic and quantitative evaluation of pain perception and chronic pain in subjects with PTSD. The study group consisted of 32 outpatients with combat- and terror-related PTSD, 29 outpatients with anxiety disorder and 20 healthy controls. Quantitative somatosensory testing included the measurement of warm, cold, light touch and heat-pain thresholds and responses to acute suprathreshold heat and mechanical stimuli. Chronic pain was characterized, and levels of PTSD and anxiety symptomatology were assessed by self-report questionnaires. Subjects with PTSD exhibited higher rates of chronic pain, more intense chronic pain and more painful body regions compared with the other two groups. PTSD severity correlated with chronic pain severity. Thresholds of subjects with PTSD were significantly higher than those of subjects with anxiety and healthy controls, but they perceived suprathreshold stimuli as being much more intense than the other two groups. These results suggest that subjects with PTSD exhibit an intense and widespread chronic pain and a unique sensory profile of hyposensitivity to pain accompanied by hyper-reactivity to suprathreshold noxious stimuli. These features may be attributed to the manner with which PTSD subjects emotionally interpret and respond to painful stimuli. Alternatively, but not mutually exclusive, the findings may reflect altered sensory processing among these subjects.     

Experimental envenoming of mice with venom from the scorpion Centruroides limpidus limpidus: differences in mortality and symptoms with and without antibody therapy relating to differences in age, sex and strain of mouse

C57Bl/6J and BALB/cAnN inbred strains of mice differed significantly in mortality and symptoms when intoxicated subcutaneously with one LD₅₀ of venom from Centruroides limpidus limpidus. Higher mortality was observed in C57Bl/6J than in BALB/cAnN. Also, C57Bl/6J mice more quickly developed muscular and respiratory collapse whilst BALB/cAnN mice were hyperactive before dying. Also, the symptoms in the survivors lasted for 24 h in C57Bl/6J and for 2 h in BALB/cAnN. The age and sex of mice were also related to mortality: younger mice were more resistant than older mice and females were more susceptible than males, especially in the younger groups. Antivenom (horse F(ab′)₂) administration 5–10 min after envenoming of mice with one LD₅₀ rescued 60% of BALB/cAnN and 52% of C57Bl/6J mice, respectively. Results indicate that genetic background, gender and age differences are of consequence in the pathogenesis of C. limpidus scorpion envenomation in mice, and that timely treatment with active antivenom F(ab′)₂ saves a significant fraction of intoxicated mice without statistically significant distinction of strains.     

Human single chain Fv antibodies and a complementarity determining region-derived peptide binding to amyloid-beta 1-42

A library of phage-displayed human single-chain Fv (scFv) antibodies was selected against the human amyloid-beta peptide (Abeta42). Two new anti-Abeta42 phage-displayed scFvs antibodies were obtained, and the sequences of their V(H) and Vkappa genes were analyzed. A synthetic peptide based on the sequence of Ig heavy chain (V(H)) complementarity-determining region (HCDR3) of the clone with the highest recognition signal was generated and determined to bind to Abeta42 in ELISA. Furthermore, we showed for the first time that an HCDR3-based peptide had neuroprotective potential against Abeta42 neurotoxicity in rat cultured hippocampal neurons. Our results suggest that not only scFvs recognizing Abeta42 but also synthetic peptides based on the V(H) CDR3 sequences of these antibodies may be novel potential candidates for small molecule-based Alzheimer's disease (AD) therapy.     

Amyloid-beta peptide-specific single chain Fv antibodies isolated from an immune phage display library

A single-chain fragment variable (scFv) antibody library displayed on phage was constructed using spleen cells from mice immunized with human amyloid-beta peptide (Abeta42). This first anti-Abeta42 scFv immune antibody library was selected against human Abeta42. A number of positive clones were obtained, and sequences of VH and Vkappa genes were analyzed using ExPASy and BLAST computer tools. This analysis revealed that only two unique clones with identical VH and Vkappa complementarity determining region (CDR) (except HCDR2) and identical germline genes were selected, indicating that oligoclonal immune response was occurring in Abeta42-immunized mice. Abeta42-specific scFv antibodies selected from this first immune anti-Abeta42 phage antibody library may be an important tool for the development of therapeutic molecules for Alzheimer's disease (AD).     

Immunodiagnosis of neurocysticercosis. Disappointing performance of serology (enzyme-linked immunosorbent assay) in an unbiased sample of neurological patients.

To ascertain the reliability of serological diagnosis of neurocysticercosis in the everyday a priori situation of neurological consultation, the enzyme-linked immunosorbent assay test was used to predict the eventual diagnosis of neurocysticercosis in an unselected sample of 1064 consecutive neurological cases. Results showed 69% sensitivity and 71% specificity of the enzyme-linked immunosorbent assay test for the diagnosis of neurocysticercosis. In sharp contrast with publications that have proclaimed the excellent diagnostic performance of immunodiagnostic tests, our results suggest that identification of serum antibodies with standard enzyme-linked immunosorbent assay techniques is of little value when applied to a large and heterogeneous group of neurological patients in an endemic area of cysticercosis, and our results urge a reevaluation of currently used immunodiagnostic tests that are practiced in the serum of suspected cases.