Site-directed in vitro immunization leads to a complete human monoclonal IgG4λ that binds specifically to the CDR2 region of CTLA-4 (CD152) without interfering the engagement of natural ligands

<p>Abstract</p> <p>Background</p> <p>The ability to acquire fully human monoclonal antibodies (mAbs) with pre-defined specificities is critical to the development of molecular tags for the analysis of receptor function in addition to promising immunotherapeutics. Yet most of the arriving affinity maturated and complete human immunoglobulin G (IgG) molecules, which are actually derived from single human B cells, have not widely been used to study the conserved self antigens (Ags) such as CD152 (cytotoxic T lymphocyte antigen-4, CTLA-4) because proper hosts are lacking.</p> <p>Results</p> <p>Here we developed an optimized protocol for site-directed <it>in vitro </it>immunizing peripheral blood mononuclear cells (PBMC) by using a selected epitope of human CD152, an essential receptor involved in down-regulation of T cell activation. The resultant stable trioma cell lines constantly produce anti-CD152 mAb (γ4λhuCD152), which contains variable (V) regions of the heavy chain and the light chain derived from the VH3 and Vλ human germline genes, respectively, and yet displays an unusual IgG4 isotype. Interestingly, γ4λhuCD152 has a basic pI not commonly found in myeloid monoclonal IgG4λs as revealed by the isoelectric focusing (IEF) analysis. Furthermore, γ4λhuCD152 binds specifically, with nanomolar affinity, to an extracellular constituency encompassing the putative second complementarity determining region (CDR2) of CD152, whereby it can react to activated CD3⁺cells.</p> <p>Conclusion</p> <p>In a context of specific cell depletion and conditioned medium,<it>in vitro </it>induction of human Abs against a conserved self Ag was successfully acquired and a relatively basic mAb, γ4λhuCD152, with high affinity to CDR2 of CD152 was thus obtained. Application of such a human IgG4λ mAb with designated CDR2 specificity may impact upon and prefer for CD152 labeling both <it>in situ </it>and <it>ex situ</it>, as it does not affect the binding of endogenous B7 ligands and can localize into the confined immunological synapse which may otherwise prevent the access of whole IgG1 molecules.</p

K 1-6: an asymmetric planetary nebula with a binary central star

We present new imaging data and archival multiwavelength observations of the little studied emission nebula K 1-6 and its central star. Narrow-band images in H-alpha (+ [NII]) and [OIII] taken with the Faulkes Telescope North reveal a stratified, asymmetric, elliptical nebula surrounding a central star which has the colours of a late G- or early K-type subgiant or giant. GALEX ultraviolet images reveal a very hot subdwarf or white dwarf coincident in position with this star. The cooler, optically dominant star is strongly variable with a period of 21.312 +/- 0.008 days, and is possibly a high amplitude member of the RS CVn class, although an FK Com classification is also possible. Archival ROSAT data provide good evidence that the cool star has an active corona. We conclude that K 1-6 is most likely an old bona fide planetary nebula at a distance of ~1.0 kpc, interacting with the interstellar medium, and containing a binary or ternary central star. The observations and data analyses reported in this paper were conducted in conjunction with Year 11 high school students as part of an Australian Research Council Linkage Grant science education project, denoted Space To Grow, conducted jointly by professional astronomers, educational researchers, teachers, and high-school students.Comment: 13 pages, 5 figures, accepted by the Publications of the Astronomical Society of Australia (PASA

A novel method of estimating dose responses for polymer gels using texture analysis of scanning electron microscopy images.

Polymer gels are regarded as a potential dosimeter for independent validation of absorbed doses in clinical radiotherapy. Several imaging modalities have been used to convert radiation-induced polymerization to absorbed doses from a macro-scale viewpoint. This study developed a novel dose conversion mechanism by texture analysis of scanning electron microscopy (SEM) images. The modified N-isopropyl-acrylamide (NIPAM) gels were prepared under normoxic conditions, and were administered radiation doses from 5 to 20 Gy. After freeze drying, the gel samples were sliced for SEM scanning with 50×, 500×, and 3500× magnifications. Four texture indices were calculated based on the gray level co-occurrence matrix (GLCM). The results showed that entropy and homogeneity were more suitable than contrast and energy as dose indices for higher linearity and sensitivity of the dose response curves. After parameter optimization, an R (2) value of 0.993 can be achieved for homogeneity using 500× magnified SEM images with 27 pixel offsets and no outlier exclusion. For dose verification, the percentage errors between the prescribed dose and the measured dose for 5, 10, 15, and 20 Gy were -7.60%, 5.80%, 2.53%, and -0.95%, respectively. We conclude that texture analysis can be applied to the SEM images of gel dosimeters to accurately convert micro-scale structural features to absorbed doses. The proposed method may extend the feasibility of applying gel dosimeters in the fields of diagnostic radiology and radiation protection

Effects of cancer, chemotherapy and cytokines on subjective and objective cognitive functioning among patients with breast cancer

SIMPLE SUMMARY: Although cognitive impairments have been complained about in patients with breast cancer who underwent chemotherapy, recent research has described possible neurocognitive decline prior to the start of chemotherapy and suggested that inflammatory cytokines may also have been involved. However, inconsistencies have been found in correlations of cognitive impairments with cancer, chemotherapy, and peridiagnostic cytokine levels. This cross-sectional study aimed to examine associations of cognitive functions and levels of cytokines in patients with newly- diagnosed breast cancer before chemotherapy, those that were 3 to 9 months after completing chemotherapy, and non-cancer controls, adjusting for baseline intelligence quotient, mood, and fatigue. We found that the performance in semantic association of verbal fluency in patients post chemotherapy might be affected by the status of cancer, IL-13, and anxiety. Our results indicated that verbal fluency and anxiety may be important when considering relevant psychosocial managements or prophylactic interventions for cognitive preservation associated with regulations in cytokines. ABSTRACT: Background: We aimed to investigate the associations of breast cancer (BC) and cancer-related chemotherapies with cytokine levels, and cognitive function. Methods: We evaluated subjective and objective cognitive function in BC patients before chemotherapy and 3~9 months after the completion of chemotherapy. Healthy volunteers without cancer were also compared as control group. Interleukins (IL) 2, 4, 5, 6, 10, 12p70, 13, 17A, 1β, IFNγ, and TNFα were measured. Associations of cancer status, chemotherapy and cytokine levels with subjective and objective cognitive impairments were analyzed using a regression model, adjusting for covariates, including IQ and psychological distress. Results: After adjustment, poorer performance in semantic verbal fluency was found in the post-chemotherapy subgroup compared to controls (p = 0.011, η(2) = 0.070); whereas pre-chemotherapy patients scored higher in subjective cognitive perception. Higher IL-13 was associated with lower semantic verbal fluency in the post-chemotherapy subgroup. Higher IL-10 was associated with better perceived cognitive abilities in the pre-chemotherapy and control groups; while IL-5 and IL-13 were associated with lower perceived cognitive abilities in pre-chemotherapy and control groups. Our findings from mediation analysis further suggest that verbal fluency might be affected by cancer status, although mediated by anxiety. Conclusions: Our findings suggest that verbal fluency might be affected by cancer status, although mediated by anxiety. Different cytokines and their interactions may have different roles of neuroinflammation or neuroprotection that need further research

Site-directed immunization leads to a complete human monoclonal IgG4λ that binds specifically to the CDR2 region of CTLA-4 (CD152) without interfering the engagement of natural ligands-5

<p><b>Copyright information:</b></p><p>Taken from "Site-directed immunization leads to a complete human monoclonal IgG4λ that binds specifically to the CDR2 region of CTLA-4 (CD152) without interfering the engagement of natural ligands"</p><p>http://www.biomedcentral.com/1472-6750/7/51</p><p>BMC Biotechnology 2007;7():51-51.</p><p>Published online 23 Aug 2007</p><p>PMCID:PMC2025598.</p><p></p>n value of all subjects analyzed. Filled triangles represent specific frequencies of individual PBMC's. Original data can be retrieved from Additional File . Panel B illustrates a representative ELISA reactivity profile of culture supernatant. Diluted supernatants were tested in duplicate with 100 μL added to each well. Deviation between duplicate was less than 10% for any reported value. Panel C represents immunofluorescent staining of mitogen-activated CD3T cells (right thick peaks). PBMCs were isolated from normal blood donors and stimulated with indicated mitogens for 72 h to induce CD152 expression. Expression of mAb-recognized epitope was verified in gated CD3 population by flow cytometry. Data represent one experiment from three different normal subjects. Appropriate isotype controls were used for all Abs. The basal fluorescence obtained with the use of isotype control (IgG4λ myeloma protein) is indicated by the left thin peaks

Entropy versus dose response curves for the n-NIPAM gels estimated from 50×, 500×, and 3500× magnified SEM images.

<p>A major downward trend was observed as the dose increased due to the increasing smoothness of the SEM micrographs.</p

Sixteen sub-micrographs divided from the original 500× magnified SEM image irradiated with 20 Gy.

<p>The homogeneity is attached in the lower-left corner of each sub-image. Impurities in the gel g007matrix caused variations in the texture analysis results.</p

SEM images of the n-NIPAM gels with absorbed doses of 0, 5, 10, 15, and 20 Gy acquired at 50×, 500×, and 3500× magnifications.

<p>As the dose increased, the fibrous structures gradually disappeared at the 500× and 3500× micrographs.</p

Sensitivity maps of entropy and homogeneity at 500× and 3500× magnifications.

<p>The <i>x</i> and <i>y</i> axes are the distance (<i>d</i>) and exclusion number (<i>e</i>), respectively, and the pixel value in the matrix represents the normalized absolute value of the slope of the corresponding DRC. Entropy had relatively stable results than homogeneity at both magnifications.</p

Linearity maps of entropy and homogeneity at 500× and 3500× magnifications.

<p>The <i>x</i> and <i>y</i> axes are the distance (<i>d</i>) and the exclusion number (<i>e</i>), respectively, and the pixel value in the matrix represents the <i>R</i>² value of the corresponding DRC. Homogeneity was more sensitive to <i>d</i> and <i>e</i> than entropy at both magnification levels.</p