3D STRUCTURE OF C3b

Retrieved from reference article:

http://www.ncbi.nlm.nih.gov/pubmed/19136636

PRETTY COOL I THINK

Fig. 1. Electron microscopy and 3D reconstruction of C3b and C3bB(Ni2). (A) Reference-free 2D averages obtained for the data set containing images of single molecules of C3b. These averages reveal a characteristic ‘‘L’’ shape evocative of the C3b crystal structure. (B) Reference-free 2D averages of C3bB(Ni2) display a bulky appearance compatible with fB binding to C3b. (C) Front view of the 3D structure of C3b derived from the EM data at a resolution of 28 Å (gray density). The atomic structure of C3b (PDB file 2i07) has been fitted within the EM map and displayed in purple with the C345C and CUB domains highlighted in orange and blue, respectively. (D) Several views of the 3D structure of C3bB(Ni2) at 27 Å resolution (gray density). Fitting of the atomic structure of C3b (PDB file 2i07) allows the assignment of specific regions of the EM map to specific C3b domains. Some densities of the 3D reconstruction cannot be accounted by C3b (asterisks) and correspond to C3b-bound fB.

BACK TO REALITY

Chemokines as immunotransmitters?

Alain Trautmann

Figure 1. The chemokine receptor CCR5 is recruited at a T cell−B cell synapse (1) and this recruitment probably requires chemokine secretion by the APC¹.
This CCR5 recruitment may help explain why conjugated T cells resist ‘distracting’ chemokines better than free T cells (2).
Additional explanations for the chemokine-dependent stability of T cell−B cell conjugates should be considered. For example, chemokines binding to their synaptic receptors may provide direct costimulation for T cell activation (3),
and chemokines as well as TCRs might contribute to the increase in the avidity of integrins for their ligands (4).
Here, the chemokines and receptors are those found at synapses between EBV−B cells and Jurkat T cells. For dendritic cells and naive T cells, similar effects could be exerted by actions of CCL19 on CCR7. MHC, major histocompatibility complex.

Katie Ris

CHEMOKINES

Chemotactic cytokines
Chemokines are classified into polypeptide groups identified by the location of cysteine residues near their amino termini (for example, C-C, C-X-C, C and CX3C).
Chemokines represent the largest family of cytokines (~41 human members), forming a complex network for the chemotactic activation of all leukocytes.
Chemokine receptors, members of the seven-transmembrane-spanning G-protein-coupled receptors, vary by cell type and degree of cell activation.
There is considerable redundancy in chemokine-receptor interaction, as many ligands bind different receptors, or vice versa.
The composition of chemokines produced at sites of tissue wounding not only recruits downstream effector cells (as discussed above), but also dictates the natural evolution of immune reactivity.
For example, MCP-1/CCL2, a potent chemotactic protein for monocytes and lymphocytes, simultaneously induces expression of lymphocyte-derived IL-4 in response to antigen challenge while decreasing expression of IL-12.
The net effect of this alteration facilitates a switch from a TH1-type to a TH2-type inflammatory response.

The chemokine connection
Chemokines were initially defined functionally as soluble factors regulating directional migration of leukocytes during states of inflammation; however, chemokine biology extends to all cell types, including most human neoplastic cells.
Regulation of tumour growth by chemokines—Some tumour cells not only regulate their chemokine expression to help recruit inflammatory cells, but also use these factors to further the tumour growth and progression.

Regulation of angiogenesis by chemokines—
Although angiogenesis is strictly controlled, it is associated with chronic inflammatory diseases, such as psoriasis, rheumatoid arthritis and fibrosis, as well as with tumour growth and metastasis. It is well established that CXC chemokines with the three amino acids (Glu-Leu-Arg/ELR) immediately amino-terminal to the CXC motif (ELR+) are pro-angiogenic and stimulate endothelial cell chemotaxis, whereas ELR− CXC chemokines (for example, PF-4/CXCL4, MIG/CXCL9 and IP-10/CXCL10) possess angiostatic activities.
ELR+ CXC ligands bind to CXCR2 and to a lesser degree to CXCR1, whereas ELR− CXC ligands bind to CXCR3, CXCR4 and CXCR5.

Chemokines and metastasis—
Malignant cells that possess metastatic capacity have properties endowing them with the ability to invade and survive in ectopic tissue, venous and/ or lymphatic environments, as well as ability to reside and proliferate at a distal site (Fig. 3).
Much debate exists as to whether malignant cells metastasize to environments favouring their specific growth or whether different organs are endowed with the ability to arrest or attract specific types of malignant cells through chemotactic factors (the so-called homing theory) 48.
Studies using a mouse model by Muller and colleagues suggest that the pattern of breast cancer metastases is in part governed by specific interactions between CXCR4 and its ligand SDF-1/CXCL12.
CXCL12 is a rather unique chemokine in that it is the product of resting cells in multiple organs, and is particularly highly expressed in target organs for breast cancer metastasis.
CXCL12 triggers chemotaxis of malignant mammary carcinoma cells in vitro, and the chemotactic activity of extracts of organs targeted by breast cancer cells (bone marrow, liver, lung and lymph nodes) can be neutralized by anti-CXCR4 antibodies.
The involvement of CXCR4 in metastasis is not limited to breast cancer, as CXCR4 is expressed in tumour cell lines (for example, prostate carcinomas, B-cell lymphomas, astrogliomas and chronic lymphocytic leukaemias) that also respond to CXCL12.
The broader implications of these observations are that chemokines may be involved in regulating the spectrum of metastases in diverse cancer types.

Wound healing versus invasive tumour growth.  
a, Normal tissues have a highly organized and segregated architecture. Upon wounding or tissue assault, platelets are activated and form a haemostatic plug where they release vasoactive mediators that regulate vascular permeability, influx of serum fibrinogen, and formation of the fibrin clot. Chemotactic factors such as transforming growth factor-β and platelet-derived growth factor, derived from activated platelets, initiate granulation tissue formation, activation of fibroblasts, and induction and activation of proteolytic enzymes necessary for remodelling of the extracellular matrix (for example, matrix metalloproteinases and urokinase-type plasminogen activator). In combination, granulocytes, monocytes and fibroblasts are recruited, the venous network restored, and re-epithelialization across the wound occurs. Epithelial and stromal cell types engage in a reciprocal signalling dialogue to facilitate healing. Once the wound is healed, the reciprocal signalling subsides. 
 b, Invasive carcinomas are less organized. Neoplasia-associated angiogenesis and lymphangiogenesis produces a chaotic vascular organization of blood vessels and lymphatics where neoplastic cells interact with other cell types (mesenchymal, haematopoietic and lymphoid) and a remodelled extracellular matrix. Although the vascular network is not disrupted in the same way during neoplastic progression as it is during wounding, many reciprocal interactions occur in parallel. Neoplastic cells produce an array of cytokines and chemokines that are mitogenic and/or chemoattractants for granulocytes, mast cells, monocytes/macrophages, fibroblasts and endothelial cells. In addition, activated fibroblasts and infiltrating inflammatory cells secrete proteolytic enzymes, cytokines and chemokines, which are mitogenic for neoplastic cells, as well as endothelial cells involved in neoangiogenesis and lymphangiogenesis. These factors potentiate tumour growth, stimulate angiogenesis, induce fibroblast migration and maturation, and enable metastatic spread via engagement with either the venous or lymphatic 
networks.

 Cytokine and chemokine balances regulate neoplastic outcome. The balance of cytokines in any given tumour is critical for regulating the type and extent of inflammatory infiltrate that forms. Tumours that produce little or no cytokines or an overabundance of anti-inflammatory cytokines induce limited inflammatory and vascular responses, resulting in constrained tumour growth. In contrast, production of an abundance of pro-inflammatory cytokines can lead to a level of inflammation that potentiates angiogenesis, thus favouring neoplastic growth. Alternatively, high levels of monocytes and/or neutrophil infiltration, in response to an altered balance of pro-versus anti-inflammatory cytokines, can be associated with cytotoxicity, angiostasis and tumour regression. In tumours, interleukin-10 is generally a product of tumour cells and tumour-associated macrophages.

This is from an article called: Inflammation and cancer; Lisa M. Coussens^*†§ and Zena Werb^‡§

NEUTRALIZATION

Neutralisation of a virus is defined as the loss of infectivity through reaction of the virus whith specific antibody.

Virus is inoculated into cell culture and the presence of unneutralized virus may be detected by plaque assay.
Loss of infectivity can be achieved by bound antibody interfering with the release of the viral genome into the host cells.

There are two types of neutralization:

Reversible neutralization
- the process can be reversed by diluting teh antibody-antigen mixture within a short time of the formation of the antibody- antigen complexes (30mins).
It is thought that reversible neutralization is due to the interference with attachment of virions to the cellular receptors, f.ex the attachment of the hemagglutinin (HA) protein of influenza viruses to sialic acid.
The process requires that the surface of the virus is saturated with antibodies.

Stable neutralization
-with time antigen-antibody complexes usually become more stable (several hours) and the process cannot be reversed by dilution. The neutralized virus can be reactivated by proteolytic cleavage. Stable neutralization has a different mechanism to that of reversible neutralization.
The number of antibody molecules required for stable neutralization is smaller than that of reversible neutralization.. Even a single antibody can neutralize a virion, and such neutralization is generally produced by antibody molecules that establish contact with 2 antigenic sites on different monomers of a virion, greatly increasing the stability of the complexes.

Viral evolution must select for mutations that change the antigenic determinants inovlved in neutralization. In contrast, other antigenic sites would remain unchanged because mutations affecting them would not be selected for and could be detrimental.
A virus would thus evolve to develop a variety of  types differing in neutralization mechanisms.

Before a neutralization test is carried out, known components must be standardized. To identify a virus isolate, a known pretitred antiserum is used. Conversely, to measure the antibody response of an individual to a virus, a known pretitred virus is used. To titrate a known virus, serial tenfold dilutions of the isolate si prepared and inoculated into a cell culture or an animal.

The virus endpoint titre is the reciprocal of the highest dilution of virus that infects 50% of the host system, e.g. 50% of cell cultures develop cytpathic effect (CPE) or 50% of animals develop disease.
This endpoint dilution contains one 50% tissue culture infecting dose (TCID50) or one 50% lethal dose (LD50) of virus per unit volume. The concentration of virus generally used in the neutralization test is 100TCID50 or 100LD50 per unit volume.

Micrograph showing the viral cytopathic effect of herpes simplex virus (multi-nucleation, ground glass chromatin).