Quantitative immunofluorescent assay of full-length,recombinant CD4 in solution and mapping of its epitopes

A specific, rapid, and sensitive method for the detection of CD4 in solution was developed using pairs of fluorescently stained monoclonal antibodies which do not cross-compete. The assay is quantitated by flow cytometry using Simply Cellular microbeads (SC beads) as the primary support for the first anti-CD4 mAb. This method uses the standard conditions for anti-CD4 monoclonal antibody binding, washing, detection, and quantitation by flow cytometry of the CD4 antigen either bound to the SC beads or expressed on the cell surface. The monoclonal antibody used (Leu 3a PE) is the standard reference used to evaluate the CD4 concentration. This method differs from ELISA techniques, which need an antigen standard curve and thus can be influenced by the quality and source of the antigen. This type of assay is also a procedure which enables determination of the level of oligomerization of the bound antigen. It can be used for any antigen to which monoclonal antibodies recognizing at least two distinct epitopes are available. The use of soluble or full-length CD4 derivatives as potential therapeutic agents against AIDS, would benefit from a precise quantitation of the CD4 molecules which still have their proper tertiary structure.     

New planar substrates for the in situ synthesis of peptide arrays

The performance of new cellulose membranes, aminofunctionalized via a PEG spacer, as a solid support in the synthesis of peptide arrays is described. The new membranes are stable to trifluoroacetic acid (TFA) and strong aqueous base for days. These properties extend the scope of synthesis considerably, e.g., more efficient side chain cleavage protocols can be applied which yielded peptides of improved purity. For the first time, cellulose membranes with a loading as high as 5 micromol/cm2 were accessible. Additionally, newly developed polypropylene membranes with hydroxy- or amino functionalities were successfully employed for the SPOT synthesis of peptides and phosphopeptides. The membranes are compatible with antibody binding as well as enzymatic phosphorylation assays.     

Profiling the immune responses of human patients treated with recombinant streptokinase after myocardial infarct

The SPOT synthesis of peptide arrays on continuous cellulose membranes should be generally applicable in the analysis of sequential antibody binding sites using the enzyme-substrate or other standard detection protocols. The use of total serum is limited by the occurrence of high background levels. This may be overcome if affinity purified antibodies or sera with high antibody titers are used, which allows work at high dilutions and a consequent reduction of background level. Here we demonstrate the mapping of antigenic regions located on recombinant streptokinase SK-2 (Heberkinase) using cellulose-bound peptide scans and human total sera from patients treated with SK-2 (Heberkinase). Streptokinase (SK) is a 47 kDa protein produced by various strains of hemolytic streptococci and is a potent activator of the fibrinolytic enzyme system in humans. SK is in widespread clinical use to treat acute infarction because of its function as an activator of vascular fibrinolysis. Since streptococcal infections are common, normal individuals are immunized with SK and antibodies (Abs) to SK can be detected in most of them. This therapy generates significant T-cell responses to SK and the neutralizing capacity of the Abs rises significantly. Neutralizing Abs reduces the efficiency of thrombolytic therapy and may cause allergic reactions. The widespread use of SK in humans makes its antigenicity an important clinical problem. In this regard the study of the immunodominant regions of SK becomes an important aspect for the improvement of this thrombolytic agent.     

Phantom and in vivo study of the Look-Locher T1 mapping method

This paper describes and tests the LL-EPI method for obtaining quantitative T₁ estimates in a few seconds thereby allowing dynamic T₁ studies. It is shown that the method works even when there is an inflow into the imaged volume, e.g., in a vessel. No calibration is needed. The method has been tested in a phantom study with several different scan parameter set-ups, with and without inflow. The method shows robustness and individual scan parameters and inflow rates do not influence the ability to calculate the Gd-DTPA concentration. Linearity prevail between the measured 1/T₁ and the Gd-DTPA concentration in the range 150 < T₁ < 2500 ms. In a dynamic Gd-DTPA phantom study, it was shown that the dynamic LL-EPI T₁ mapping technique was three times more sensitive than the signal from a T^*₂-weighted EPI sequence. In an in vivo study, dynamic T₁ mapping of the Gd-DTPA uptake in a meningioma was performed. Inspection of the uptake curves indicates that the method is feasible in clinical perfusion studies.     

A new compact disc format of high density array synthesis applied to peptide nucleic acids and in situ MALDI analysis

A fully automated synthesizer was constructed and designed to perform high speed miniaturized syntheses of compound libraries using the SPOT technique. Utilizing magnetically controlled drop-on-demand ink jet nozzles, an r/phi array format of 2500 spots can be simultaneously dispensed from up to 24 separate reagent valves onto a rotating disc as the solid phase in less than three minutes. In addition, a complete wash station is on board allowing for fully programmable combinatorial syntheses without manual attention. A new carbon black/polypropylene composite solid phase disc was developed and tested for its functionalisation/loading, spot detection, durability and MALDI-TOF target capabilities. The carbon black/polypropylene composite was then successfully employed jointly as the solid phase in the syntheses of short peptide and PNA oligomers and as the target probe holder for MALDI-TOF measurement without transfer of the material. Several protocols for PNA syntheses were also investigated and an optimised PNA methodology for the carbon black/polypropylene composite is reported.     

Assembly and infection process of bacteriophage T4

Bacterophage T4 consists of three parts, namely, a head, a tail, and six tail fibers, each of which is assembled along an independent pathway and then joined. In contrast to simple plant viruses such as tobacco mosaic virus, disassembly and reassembly of the virion is not possible. This is due mainly to the fact that the assembly involves not only irreversible steps such as cleavage of covalent bonds of some constituent proteins, but also that it requires a scaffold and involves the inner membrane of the host cell. Another unique feature of the assembly as a biological nanomachine is the involvement of specific protein devices such as a "ruler molecule," which determines the length of the tail, an ATP-driven DNA packaging protein complex, and phage-encoded molecular chaperones. Recent structural biological studies of the phage started to unveil the molecular mechanics of structural transformation of the tail upon infection.     

Defining the antigenic structure of human GM-CSF and its implications for receptor interaction and therapeutic treatments

Three epitopes of human Granulocyte-Macrophage Colony-Stimulating Factor (hGM-CSF) recognised by neutralising and non-neutralising monoclonal antibodies (mAbs) were characterised using competitive binding assays, dissociation constant measurements with glycosylated and non-glycosylated rhGM-CSF, bioactivity inhibition studies, and synthetic peptide arrays. Based on the first approach, two different binding sites were identified: an area referred to as A, recognised by mAbs M1B8 and CC5B5, and an area referred to as B, recognised by mAbs CC1H7 and CC3C12. These binding sites on hGM-CSF were accurately delineated using cytokine-derived overlapping peptide scans and combinatorial hexapeptide libraries prepared by SPOT synthesis on cellulose membranes. We assigned the identical linear epitope A₁P₂A₃R₄ to both non-neutralising mAbs CC1H7 and CC3C12. The conformational epitopes A₁₈E₂₁R₂₃R₂₄F₁₁₉ and R₂₃E₂₁N₁₇W₁₃ recognised by mAb CC5B5, and P₁₁₈F₁₁₉EW₁₃E₁₄ recognised by mAb M1B8, were delineated by dual-positional scanning and subsequent iterative searches with two interrogating positions. Competitive binding assays with mAbs M1B8 and CC5B5 revealed the overlapping nature of the cytokine recognition. However, peptide library screening confirmed their binding to different epitopes of which the essential amino acids were found very closely located on the native protein surface. Inhibition of hGM-CSF biological activity by these mAbs demonstrated that these epitopes are located within or very near the receptor binding site of hGM-CSF. Finally, this work supports the importance of residues from helix A and residues from the C-terminal region of the cytokine, composing a common area that is indispensable for the cytokine's biological activity.     

The use of peptide arrays for the characterization of monospecific antibody repertoires from polyclonal sera of psychiatric patients suspected of infection by Borna Disease Virus

Borna Disease Virus (BDV) is suspected to infect humans and to be associated with psychiatric disorders. To this date, BDV-reactive antibodies provide the only reliable markers to diagnose human BDV infection. Their diagnostic value, however, was recently questioned by the observation that these antibodies recognize BDV antigen with only low avidity, a typical feature of cross-reacting antibodies. This raised the possibility that the human BDV-reactive antibodies were triggered by other pathogens than BDV. The recent establishment of a peptide array-based screening test allowed the further characterization of these antibodies. It revealed the presence of small amounts of BDV-reactive antibodies in crude human sera that specifically recognized various epitopes of three major BDV proteins. Most importantly, the purified epitope-specific antibodies were shown to bind to BDV antigen with high avidity when assayed by conventional immunofluorescence assay (IFA) or by Western blot. These results are compatible with the view that the presence of BDV-reactive antibodies in human sera reflects an infection with BDV, although the poor affinity maturation remains unexplained. Furthermore, it demonstrates that peptide array-based screening tests are a reliable system for identifying monospecific antibodies from human polyclonal sera with high specificity and sensitivity.     

Multiplexed sorting of libraries on libraries: A novel method for empirical protein design by affinity-driven phage enrichment on synthetic peptide arrays

Chemically synthesized peptide arrays on planar cellulose carriers are proposed as libraries of ligands suitable for the multiplexed simultaneous capture of peptide-specific acceptor proteins from a large randomly mutagenized library of acceptor proteins presented on bacteriophage M13 particles. This experimental set-up can be exploited to rapidly screen for individual new, distinct binding partners from two complementary libraries (two-dimensional screening). The technical feasibility of this empirical protein design approach was demonstrated with calmodulin as an aceptor protein using an array of mastoparan variants for multiplexed phage affinity enrichment.     

Practical considerations in T1 mapping of prostate for dynamic contrast enhancement pharmacokinetic analyses

There are many challenges in developing robust imaging biomarkers that can be reliably applied in a clinical trial setting. In the case of dynamic contrast-enhanced (DCE) MRI, one such challenge is to obtain accurate precontrast T₁ maps for subsequent use in two-compartment pharmacokinetic models commonly used to fit the MR enhancement time courses. In the prostate, a convenient and common approach for this task has been to use the same 3D spoiled gradient-echo sequence used to collect the DCE data, but with variable flip angles (VFAs) to collect data suitable for T₁ mapping prior to contrast injection. However, inhomogeneous radiofrequency conditions within the prostate have been found to adversely affect the accuracy of this technique. Herein we demonstrate the sensitivity of DCE pharmacokinetic parameters to precontrast T₁ values and examine methods to improve the accuracy of T₁ mapping with flip angle-corrected VFA SPGR methods, comparing T₁ maps from such methods with “gold standard” reference T₁ maps generated with saturation recovery experiments performed with fast spin-echo (FSE) sequences.     

Irradiation of human lymphocytes with He-Ne laser dose not express the interleukin-2 receptors

The irradiation of human lymphocytes with a He-Ne laser(632.8nm,56J/cm~2)does not express interleukin-2 receptors on membrances of T-lymphocytes     

Decreases in ADC observed in tissue areas during activation in the cat visual cortex at 9.4 T using high diffusion sensitization

Recent studies in the human visual cortex using diffusion-weighted functional magnetic resonance imaging (fMRI) have suggested that the apparent diffusion coefficient (ADC) decreases, in contrast to earlier studies that consistently reported ADC increases during neuronal activation. The changes, in either case, are hypothesized to provide the ability to improve the spatial specificity of fMRI over conventional blood-oxygenation-level-dependent (BOLD) methods. Most recently, the ADC decreases have been suggested as originating from transient cell swelling caused by either shrinkage of the extracellular space or some intracellular neuronal process that precedes the hemodynamic response. All of these studies have been conducted in humans and at lower magnetic fields, which can be limited by the signal-to-noise ratio (SNR). The low SNR can lead to significant partial-volume effects because of the lower spatial resolutions required to attain sufficient SNR in diffusion-weighted images. Human studies also have the potential confound of motion. At high magnetic fields and in animal model studies, these limitations are alleviated. At high fields, SNR increases, tissue signals are enhanced and signal changes inside the blood are significantly reduced compared to lower fields. In this work, we were able to measure a small but significant ADC decrease in tissue areas, in conjunction with brain activation in the cat visual cortex at 9.4 T when using highly diffusion-weighted images (b>1200 s/mm2) where intravascular effects are minimal. When using low b-values, delayed increases in the tissue ADC during activation were observed. No significant changes in ADC were observed in surface vessels for any diffusion weighting. Furthermore, we did not observe any temporal differences in the highly diffusion-weighted data compared to BOLD; however, although the changes may likely be vascular in nature, they are highly localized to the tissue areas.     

Histogram parameters derived from T1 and T2 weighted images correlate with tumor infiltrating lymphocytes and tumor-stroma ratio in head and neck squamous cell cancer

PurposeThe present study used histogram analysis values derived from T1- and T2- weighted (w) images to elucidate possible associations with Tumor-infiltrating lymphocytes (TIL) and Vimentin expression in head and neck squamous cell cancer (HNSCC).Materials and methodsOverall, 28 patients (n = 8 female patient, 28.6%) with primary HNSCC of different localizations were involved in the study. Magnetic resonance imaging (MRI) was obtained on a 3 T MRI. The images were analyzed with a whole lesion measurement using a histogram approach. TIL- and vimentin-expression was calculated on biopsy samples before any form of treatment.ResultsSeveral T1-derived parameters correlated with the expression of TIL within the stroma compartment: mean (r = 0.42, p = 0.025), p10 (r = 0.50, p = 0.007), p25 (r = 0.42, p = 0.025), median (r = 0.39, p = 0.036), and mode (r = 0.39, p = 0.04). No T2-derived parameter correlated with the TIL within the stroma compartment. Several T2-derived parameters correlated with the expression of TIL within the tumor compartment: mean (r = −0.52, p = 0.004), max (r = −0.43, p = 0.02), p10 (r = −0.38, p = 0.04), p25 (r = −0.53, p = 0.004), p75 (r = −0.52, p = 0.004), p90 (r = −0.48, p = 0.009), median (r = −0.52, p = 0.004), mode (r = −0.40, p = 0.03). Kurtosis derived from T2w images had significant higher values in tumor-rich tumors, compared to stroma-rich tumors, (mean 5.5 ± 0.5 versus 4.2 ± 1.2, p = 0.028).ConclusionsHistogram analysis parameters derived from T1w and T2w images might be able to reflect tumor compartments and TIL expression in HNSCC.     

Effective combinatorial strategy to increase affinity of carbohydrate binding by peptides

The Thomsen-Friedenreich antigen, a carcinoma-associated disaccharide involved in carcinoma cell homotypic aggregation and increased metastatic potential, has clinical value as a prognostic indicator and a marker of metastasized cells. Hence, it can reasonably be predicted that antigen-binding macromolecules are valuable clinical in vivo diagnostic/therapeutic targeting agents. Recently, we have selected first-generation antigen-binding peptides from a random peptide bacteriophage display library and have applied combinatorial affinity maturation to select functionally-maturated peptides, which target cultured carcinoma cells and inhibit carcinoma cell aggregation. In the current study we hypothesize that a targeted search of sequence space surrounding the antigen-binding consensus sequence will select unpredictable amino acid sequences in the non-consensus portions of the peptides, leading to increased affinity for the carbohydrate and greater solubility in physiological buffers. This comprehensive in vitro analysis demonstrates that preferential evolution of the amino-terminal sequence of the peptides occurred, which correlated, in structure/function studies, with the acquisition of maturated function. The maturated peptides are more soluble than the earlier peptides. Studies of peptide binding to the disaccharide indicate that two maturated peptides (P-30-1, F03) have higher affinity for the antigen and bind with higher intensity to the surface of cultured human carcinoma cells than the first-generation peptides. The results support our hypothesis that affinity maturation can improve carbohydrate binding by peptides and have theoretical importance as the first report of maturation of carbohydrate-binding affinity in a small, soluble peptide.     

Enhanced adsorptive removal of Safranine T from aqueous solutions by waste sea buckthorn branch powder modified with dopamine: Kinetics,equilibrium, and thermodynamics

Polydopamine coated sea buckthorn branch powder (PDA@SBP) was facilely synthesized via a one-pot bio-inspired dip-coating approach. The as-synthesized PDA@SBP was characterized using Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). The adsorption progresses of Safranine T on the surface of PDA@SBP adsorbent were systematically investigated. More specifically, the effects of solution pH, contact time, initial concentration and temperature were evaluated, respectively. The experimental results showed the adsorption capacity of PDA@SBP at 293.15 K could reach up to 54.0 mg/g; the adsorption increased by 201.7% compared to that of native SBP (17.9 mg/g). Besides, kinetics studies showed that pseudo-second-order kinetic model adequately described the adsorption behavior. The adsorption experimental data could be fitted well a Freundlich isotherm model. Thermodynamic analyses showed that the ST adsorption was a physisorption endothermic process. Regeneration of the spent PDA@SBP adsorbent was conducted with 0.1 M HCl without significant reduction in adsorption capacity. On the basis of these investigations, it is believed that the PDA@SBP adsorbent could have potential applications in sewage disposal areas because of their considerable adsorption capacities, brilliant regeneration capability, and cost-effective and eco-friendly preparation and use.     

Phosphate and amide III mapping in sialoliths with Raman microspectroscopy

Sialoliths, a cause of the salivary gland infection, are reported to be composed of inorganic and organic substances. However, the precise mechanism of sialolith formation remains unclear. The purpose of this report is to elucidate this mechanism by analyzing the precise distribution of phosphate (an inorganic substance) and amide III (an organic substance) in sialoliths by using Raman microspectroscopy. Sialoliths from the submandibular gland duct were analyzed by this form of observation and by a scanning electron microscope (SEM) equipped with an energy‐dispersive X‐ray spectroscope (EDX). In Raman microspectroscopy we analyzed the spectral peak of the phosphate (PO₄³⁻) symmetric stretching vibrational mode (υ₁) at 960 cm⁻¹ and that of amide III at 1265 cm⁻¹ to demonstrate the mapping of an image of these elements showing a semiquantitative distribution of phosphate and amide III in the sialoliths. It was found that phosphate and amide III were concentrated at the center of the sialoliths, and the phosphate distribution in the sialoliths showed concentric laminations. These results indicated the possibility that the sialoliths originated from a nidus of organic materials and progressively grew by the deposition of layers of organic and inorganic materials. Copyright © 2008 John Wiley & Sons, Ltd.     

High density peptide microarrays. In situ synthesis and applications

The technologies enabling the creation of large scale, miniaturized peptide or protein microarrays are emerging. The focuses of this review are the synthesis and applications of peptide and peptidomimetic microarrays, especially the light directed parallel synthesis of individually addressable high density peptide microarrays using a novel photogenerated reagent chemistry and digital photolithography (Gao et al., 1998, J. Am. Chem. Soc. 120, 12698; Pellois et al. 2002, Nat. Biotechnol. 20, 922). Concepts related to the synthesis are discussed, such as the reactions of photogenerated acids in the deprotection step of peptide synthesis or oligonucleotide synthesis, and the applications of high density peptide chips in antibody binding assays are discussed. Peptide chips provide versatile tools for probing antigen-antibody, protein-protein, peptide-ligand interactions and are basic components for miniaturization, automation, and system integration in research and clinical diagnosis applications.     

The use of ultrasound in the South Cone region. Advances in organic and inorganic synthesis and in analytical methods

In organic and inorganic synthesis and in analytical methods, an external conventional heat source is usually applied to carry out a chemical reaction at a high temperature, or an extraction procedure. In the last decades, the use of ultrasound as an alternative energy source has become an interesting field of research in these topics in the South Cone region (Argentina, Chile, Uruguay, Southern Brazil and Paraguay). For this reason, the present review, covering the period 2009 to mid-2021, is a compilation of ultrasound-assisted synthetic and analytical methodologies.