Potential Hydrodynamic Cytoplasmic Transfer between Mammalian Cells: Cell-Projection Pumping.

We earlier reported cytoplasmic fluorescence exchange between cultured human fibroblasts (Fibs) and malignant cells (MCs). Others report similar transfer via either tunneling nanotubes (TNTs) or shed membrane vesicles, and this changes the phenotype of recipient cells. Our time-lapse microscopy showed most exchange was from Fibs into MCs, with less in the reverse direction. Although TNTs were seen, we were surprised transfer was not via TNTs but was instead via fine and often branching cell projections that defied direct visual resolution because of their size and rapid movement. Their structure was revealed nonetheless by their organellar cargo and the grooves they formed indenting MCs, which was consistent with holotomography. Discrete, rapid, and highly localized transfer events evidenced against a role for shed vesicles. Transfer coincided with rapid retraction of the cell projections, suggesting a hydrodynamic mechanism. Increased hydrodynamic pressure in retracting cell projections normally returns cytoplasm to the cell body. We hypothesize "cell-projection pumping" (CPP), in which cytoplasm in retracting cell projections partially equilibrates into adjacent recipient cells via microfusions that form temporary intercellular cytoplasmic continuities. We tested plausibility for CPP by combined mathematical modeling, comparison of predictions from the model with experimental results, and then computer simulations based on experimental data. The mathematical model predicted preferential CPP into cells with lower cell stiffness, expected from equilibration of pressure toward least resistance. Predictions from the model were satisfied when Fibs were cocultured with MCs and fluorescence exchange was related to cell stiffness by atomic force microscopy. When transfer into 5000 simulated recipient MCs or Fibs was studied in computer simulations, inputting experimental cell stiffness and donor cell fluorescence values generated transfers to simulated recipient cells similar to those seen by experiment. We propose CPP as a potentially novel mechanism in mammalian intercellular cytoplasmic transfer and communication.

Seasoned adaptive antibody immunity for highly pathogenic pandemic influenza in humans.

Fundamentally new approaches are required for the development of vaccines to pre-empt and protect against emerging and pandemic influenzas. Current strategies involve post-emergent homotypic vaccines that are modelled upon select circulating 'seasonal' influenzas, but cannot induce cross-strain protection against newly evolved or zoonotically introduced highly pathogenic influenza (HPI). Avian H5N1 and the less-lethal 2009 H1N1 and their reassortants loom as candidates to seed a future HPI pandemic. Therefore, more universal 'seasoned' vaccine approaches are urgently needed for heterotypic protection ahead of time. Pivotal to this is the need to understand mechanisms that can deliver broad strain protection. Heterotypic and heterosubtypic humoral immunities have largely been overlooked for influenza cross-protection, with most 'seasoned' vaccine efforts for humans focussed on heterotypic cellular immunity. However, 5 years ago we began to identify direct and indirect indicators of humoral-herd immunity to protein sites preserved among H1N1, H3N2 and H5N1 influenzas. Since then the evidence for cross-protective antibodies in humans has been accumulating. Now proposed is a rationale to stimulate and enhance pre-existing heterotypic humoral responses that, together with cell-mediated initiatives, will deliver pre-emptive and universal human protection against emerging epidemic and pandemic influenzas.

Vascularity during wound maturation correlates with fragmentation of serum albumin but not ceruloplasmin, transferrin, or haptoglobin.

Reduced vascularity during wound maturation is mediated by endothelial apoptosis. Albumin has an anti-apoptotic activity for endothelium, which increases up to 100-fold on albumin fragmentation (AF). We now report that levels of AF correlate with changing vascularity during wound maturation. Both scarring and adipogenic wound-healing models were established in mice. Western blots of granulation tissue revealed AF concurrent with periods of high vascularity as determined by thin-section microscopy, with reduced AF on wound maturation (p<0.02). In profiling AF, the levels of 27.5 and 39 kDa fragments were reduced on maturation of both scarring and adipogenic wounds (p<0.005), as were the levels of an additional 17.5 kDa fragment prominent only in adipogenic wounds (p<0.001). A 49 kDa albumin fragment was found to be reduced during maturation of scarring (p<0.001) but not adipogenic wounds. For comparison, we probed for transferrin, ceruloplasmin, and haptoglobin fragmentation on the basis that like albumin, these are considered acute-phase transport proteins. Minimal fragmentation of transferrin and ceruloplasmin was seen, along with partial dissociation of haptoglobin subunits, but these did not correlate with AF or vascularity. Our findings are consistent with a role for AF in regulating granulation tissue vascularity during healing.

Acquired heterosubtypic antibodies in human immunity for avian H5N1 influenza.

Well understood are the adaptive and dramatic neutralizing homosubtypic antibody responses to hypervariable, immunodominant sites of the hemagglutinin (HA) and neuraminidase (NA) of individual influenza strains. These define influenza subtypes and vaccines modelled upon their HA and NA antigens provide seasonal neutralizing antibody protection against subsequent exposure to the strain and its close relatives, but give little if any protection against antigenically drifted or shifted strains. Contrasting to this is a different form of acquired antibody response, called heterosubtypic immunity. This provides a more seasoned adaptive antibody response to immune-recessive epitopes that are highly-conserved amongst strains. Although, such responses are of lower individual amplitudes than seasonal mechanisms they are active across influenza subtypes, and may give pre-emptive protection against new strains yet to emerge. Heterosubtypic immunities have been well studied in animals, but surprisingly there is minimal evidence for this type of antibody immunity in humans. Thus championed is the notion that seasoned humoral responses can through repeated exposure to sites widely conserved across different strains, cumulatively provide humans with a level of broad protection against emergent novel strains, such as H5N1, that is not afforded by seasonal humoral responses.

Heterosubtypic anti-avian H5N1 influenza antibodies in intravenous immunoglobulins from globally separate populations protect against H5N1 infection in cell culture.

With antigenically novel epidemic and pandemic influenza strains persistently on the horizon it is of fundamental importance that we understand whether heterosubtypic antibodies gained from exposures to circulating human influenzas exist and can protect against emerging novel strains. Our studies of IVIG obtained from an infection-naive population (Australian) enabled us to reveal heterosubtypic influenza antibodies that cross react with H5N1. We now expand those findings for an Australian donor population to include IVIG formulations from a variety of northern hemisphere populations. Examination of IVIGs from European and South East-Asian (Malaysian) blood donor populations further reveal heterosubtypic antibodies to H5N1 in humans from different global regions. Importantly these protect against highly pathogenic avian H5N1 infection in vitro, albeit at low titres of inhibition. Although there were qualitative and quantitative differences in binding and protection between globally different formulations, the heterosubtypic antibody activities for the respective IVIGs were in general quite similar. Of particular note because of the relative geographic proximity to the epicentre of H5N1 and the majority of human infections, was the similarity in the antibody binding responses between IVIGs from the Malayan peninsula, Europe and Australia. These findings highlight the value of employing IVIGs for the study of herd immunity, and particularly heterosubtypic antibody responses to viral antigens such as those conserved between circulating human influenzas and emerging influenza strains such as H5N1. They also open a window into a somewhat ill defined arena of antibody immunity, namely heterosubtypic immunity.

A commercial ELISA detects high levels of human H5 antibody but cross-reacts with influenza A antibodies.

BACKGROUND AND OBJECTIVES: Commercial serological assays to determine influenza A H5N1 infection are available, although the accuracy and reproducibility of these are not reported in detail. This study aimed to assess the validity of a commercial ELISA H5 hemagglutinin (HA) antibody kit. STUDY DESIGN: A commercial ELISA for detection of antibodies towards influenza A H5 HA was evaluated using human sera from vaccinated individuals. The ELISA was used to screen 304 sera with elevated influenza A complement fixation titres collected between the period 1995-2007. RESULTS AND CONCLUSIONS: The ELISA was found to be accurate for sera with high levels of anti-H5 antibodies, and would be useful in clinical settings where a rapid result is required. Thirteen of the stored sera were positive using the ELISA, but were confirmed as negative for H5N1 exposure using further serological tests. Absorption studies suggested that antibodies towards seasonal H3N2 and H1N1 influenza may cross-react with H5 antigen, giving false positive results with the ELISA.

Comparison of human platelet membrane-cytoskeletal proteins with the plasma proteome: Towards understanding the platelet-plasma nexus.

Platelets are essential for maintaining vascular integrity. Given the anucleate nature of platelets, definition of their proteome is essential for understanding platelet pathophysiology. We describe here a detailed MS-based proteomic analysis of the platelet membrane/cytoskeletal sub-proteome from purified, normal, non-activated human platelets. In contrast to previous platelet proteomic purification strategies, the buffy-coat method was utilized in this study to isolate and purify minimally activated platelets, yielding significantly reduced contaminants for leukocytes (0.02 ±â€…0.007×10(6) /L) and erythrocytes (0.21 ±â€…0.02%). Using a false discovery rate of 1%, 203 proteins were identified and characterized with respect to their subcellular localization, biological function, and cellular processes. Of these, 16 have not been identified in previous human platelet proteome studies. As a first approach towards understanding the dynamic platelet-plasma protein composition nexus, we re-analysed the entire HUPO plasma proteome project dataset (647 plasma proteins identified) and compared these data with our platelet proteome dataset. Co-identified proteins (41) were further analysed with respect to their relative abundances (exponentially modified protein abundance index) and functional enrichment in these two proteomes, as well as their correlation with the platelet transcriptome. Both platelet membrane/cytoskeletal and plasma proteome reference datasets, comprising both processed and unprocessed MS/MS spectra, are publicly accessible (http://www.ludwig.edu.au/archive/).

Marked differences in the structures and protein associations of lymphocyte and monocyte CD4: resolution of a novel CD4 isoform.

The structures, molecular interactions and functions of CD4 in a subset of T lymphocytes have been well characterized. The CD4 receptors of other cell types have, however, been poorly documented. We have previously shown that lymphocytes and monocytes/macrophages differ in their expression of CD4 monomers and dimers. In the present study, we have shown further significant differences. Variability in the blocking of CD4 mAb binding by sulfated polyanions indicated differences in exofacial CD4 structures. In contrast to the well-documented 55 kDa monomers in lymphocytic cells, monocytic cells were found to coexpress two monomer isoforms: the 55 kDa form and a novel 59 kDa species. Experimental uncoupling of CD4 disulfides indicated that the oxidized 55 kDa monomer could be converted to the 59 kDa form. This was achieved by chemical reduction of purified native or recombinant CD4, or in cell transfection experiments by mutation of cysteine to alanine in domain 1 (D1) (Cys16 or Cys84) and in domain 4 (D4) (Cys303 or Cys345). All of these modifications promote CD4 distension on SDS-PAGE analysis and indicate that, when CD4 inter-beta-sheet disulfides in the D1 and D4 Ig folds are disrupted, there is an unravelling of the oxidized form to an extended 59 kDa unfolded state. We hypothesize that this may be a transition-state, structural-intermediate in the formation of disulfide-linked homodimers. Also identified were CD4-tyrosine kinase dissimilarities in which lymphocyte CD4 associated with Lck, but monocyte CD4 associated with HcK. These findings show that there is complex heterogeneity in structures and interactions in the CD4 of T lymphocytes and monocytes.

Marked structural and functional heterogeneity in CXCR4: separation of HIV-1 and SDF-1alpha responses.

CXCR4, the chemotactic cell receptor for SDF-1alpha, is essential for immune trafficking and HIV infection. CXCR4 is remarkably heterogeneous and the purpose of this study was to better identify the isoforms expressed by cells and compare their structure and function. We found that cells express either a predominant isoform or multiple isoforms. These were best resolved on SDS-PAGE using sucrose-gradient-fractionated, triton-insoluble, membrane extracts. We hypothesized that glycosyl modification may underpin some of this heterogeneity and that cell isoform(s) differences may underscore CXCR4's multiple cell functions. A comparison of wild-type (WT) and dual N-linked glycosylation site, N11A/N176A, mutant CXCR4 expressed in 3T3 and HEK-293 cells served to implicate variabilities in glycosylation and oligomerization in almost half of the isoforms. Immunoprecipitation of CXCR4 revealed monomer and dimer non-glycosylated forms of 34 kDa and 68 kDa from the N11A/N176A mutant, compared with glycosylated 40 kDa and 47 kDa and 73 kDa and 80 kDa forms from WT. The functional specificity of isoform action was also implicated because, despite CEMT4 cells expressing high levels of CXCR4 and 11 different isoforms, a single 83 kDa form was found to bind gp120 for HIV-1 IIIB infection. Furthermore, comparative studies found that in contrast to SDF-1alpha-responsive Nalm-6 cells that expressed similar levels of a single isoform, CEMT4 cells did not show a Ca(++) flux or a chemotactic response to SDF-1alpha. Thus, CXCR4 can differ both structurally and functionally between cells, with HIV-1 infection and chemotaxis apparently mediated by different isoforms. This separation of structure and function has implications for understanding HIV-1 entry and SDF-1alpha responses and may indicate therapeutic possibilities.

Detergent-resistant membrane fractions contribute to the total 1H NMR-visible lipid signal in cells.

Leukocytes and other cells show an enhanced intensity of mobile lipid in their 1H NMR spectra under a variety of conditions. Such conditions include stimulation, which has recently been shown to involve detergent-resistant, plasma membrane domains (DRMs) often called lipid rafts. As there is much speculation surrounding the origin of cellular NMR-visible lipid, we analysed subcellular fractions, including DRMs, by NMR spectroscopy. We demonstrated that DRMs isolated by density gradient centrifugation from lymphoid (CEM-T4, stimulated Jurkat cells), and monocytoid (THP-1) cells produced NMR-visible, lipid signals. Large scale subfractionation of THP-1 cells determined that while cytoplasmic lipid droplets constituted much of the total NMR-visible lipid, the contribution of DRMs was significant. Qualitative and quantitative lipid analyses revealed that DRMs and lipid droplets differed in their lipid composition. DRMs were enriched in cholesterol and ganglioside GM1, and contained relatively unsaturated fatty acids compared with the lipid droplets. Both lipid droplets and DRMs contained neutral lipids (triacylgycerols, cholesterol ester, fatty acids in THP-1 cells) that could, in addition to phospholipids, contribute to the NMR-visible lipid. The lipid droplets also exhibited different protein profiles and contained 500-fold less protein than DRMs, confirming that DRMs and droplets were fractionated as separate entities. The NMR-visible lipid in DRMs is therefore unlikely to be a contaminant from lipid droplets. We propose a micropartitioning of the NMR-visible mobile lipid of whole cells between intracellular lipid droplets, where most of this lipid resides, and detergent-resistant plasma membrane domains.