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Intravenous Immunoglobulin’s (IVIG’s) are prepared from thousands of donor plasmas and as a result comprise an extreme broad mix and depth of Antibody (Ab) specificities. IVIG formulations available in Australia are from both local and overseas donor sources and extracted using a variety of purification methods and immunoglobulin purities, with the Australia-derived and prepared IVIG listed at >98% and the overseas-derived preparations at ≥ 95%. Because of these differences it was predicted that the formulations might individually vary in composition and that together with obvious genetic and geographic antigenic (Ag) environment differences may result in notable variability between formulations. Hence a focussed comparative proteomic profiling of IVIG formulations was undertaken to identify notable similarities and differences across products. Comparisons between formulations did reveal marked qualitative differences in 2D-gel Antibody profiling that included parameters of isoelectric charge (pI), as well as immunoglobulin (Ig) monomer to dimer ratio variability between products, including high molecular weight (MW) immunoglobulin multimers for some. These notable differences were in part quite likely a product of the respective purification methods used, and capacity to select (or de-select) for antibodies of such different properties. Furthermore, for identification of non-Ig proteins carried over from plasma through purifications Mass spectrometry was performed. This identified a few such ancillary proteins, and their identities, in general, differed between formulations. Proteins detected included the most abundant protein of plasma, albumin, as well as other mostly large and abundant proteins; RAG1 - V(D)J recombination activating protein1, gelsolin, complement Factor-B, serotransferrin, tetranectin, NADH ubiquinone oxidoreductase, caspase 3 and VEGFR1. An alternate strategy used commercial Multiplex xMAP assay to detect cytokines, which are small and present in plasma at trace but highly active quantities. This revealed various different cytokine profiles across the formulations studied. The identification of additional proteins, and especially cytokines in IVIGs, is particularly notable, and the positive, negative or null biological relevance for clinical use, needs resolution. Collectively these findings reveal marked differences between Australian and overseas-derived (non-Australian) IVIGs in immunoglobulin composition and biochemical characteristics, and presence of additional carry-over proteins from plasma. These findings prompt the need for further evaluation of the micro-compositions of individual formulations. Perhaps detailed mining and improved comparative understanding of each IVIG formulation, may enable highly tailored and strategic clinical use of certain formulations that are personalised best-fit treatments for particular conditions. Such as in treatment of a neuropathy, as compared to another formulation, more suited for treating a particular infectious disease. The most salient and overarching study conclusion is need for caution in attributing equivalence across IVIGs.

We earlier reported cytoplasmic fluorescence exchange between cultured human fibroblasts (Fib) and malignant cells (MC). Others report similar transfer via either tunneling nanotubes (TNT) or shed membrane vesicles and this changes the phenotype of recipient cells. Our current time-lapse microscopy showed most exchange was from Fib into MC, with less in the reverse direction. Although TNT were seen, we were surprised transfer was not via TNT, 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 MC, while this 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), where cytoplasm in retracting cell-projections partially equilibrates into adjacent recipient cells via micro-fusions that form temporary inter-cellular cytoplasmic continuities. We tested plausibility for CPP by combined mathematical modelling, 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 towards least resistance. Predictions from the model were satisfied when Fib were co-cultured with MC, and fluorescence exchange related with cell stiffness by atomic force microscopy. When transfer into 5000 simulated recipient MC or Fib 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 inter-cellular cytoplasmic transfer and communication.SIGNIFICANCETime-lapse observations of co-cultured cells led us to hypothesize what we believe to be a novel hydrodynamic mechanism transferring cytoplasm between cells. Similar transfer by other mechanisms markedly affects cell behavior. Combined mathematical modelling, satisfaction of predictions from the mathematical model in cell culture experiments, and separate computer simulations that generate outcomes similar to experimental observations, support our hypothesized mechanism.

Variation in the effect of seminal plasma on sperm function and fertility has been hypothesised to be due to differences between males and their seminal plasma composition. The freezing resilience of individual rams (n = 17) was investigated to characterise inter-male variation. This was determined by measuring the degree of change in motility induced by cryopreservation (Experiment 1). Experiment 2 examined the effect of pooled seminal plasma from rams identified as having high or low resilience to freezing on the cryosurvival of washed spermatozoa from either high (n = 3) or low (n = 3) sperm freezing resilience rams. Immediately after thawing and throughout the incubation period (0–4 h), spermatozoa from high-resilience rams frozen with high-resilience seminal plasma demonstrated superior motility to spermatozoa from high-resilience rams frozen with low-resilience seminal plasma (P

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.

It is necessary to understand whether some humans possess natural humoral-immune protection for avian- H5N1 influenza. To broadly assess an exposure naive cohort we have examined intravenous immunoglobulins (IVIGs) isolated from pools of many thousands of normal Australian blood donations. In studies of the anti-H5N1 antibody poten- tial of these highly purified IVIG therapeutics and of individual donor sera we have identified antibodies that bind to both H5N1 surface envelope and internal viral proteins and neutralize in vitro MDCK and Vero cell infections by highly pathogenic avian influenza clade I and II and human-derived H5N1 isolates. As this reactivity is removed by adsorption with purified H3N2 and H1N1 strains, anti-H5N1 cross-reacting hetero-typic antibodies are implicated. These findings support that some individuals do contain low levels of specific and neutralizing anti-H5N1 antibodies. The protective relevance of this in vivo remains yet to be determined.

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 ± 0.007 × 106/ L) and erythrocytes (0.21 ± 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/). © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

International audience; The source and composition of seminal plasma has previously been shown to alter the ability of spermatozoa to survive cryopreservation. In the present study, the ionic and proteomic composition of seminal plasma from rams with high (HSP; n = 3) or low (LSP; n = 3) freezing resilient spermatozoa was assessed. 75 proteins were identified to be more abundant in HSP and 48 proteins were identified to be more abundant in LSP. Individual seminal plasma proteomes were established for each of the six rams examined. For each ram, correlations were conducted between previously recorded freezing resilience [1] and individual spectral counts in order to identify markers of freezing resilience. 26S proteasome complex, acylamino acid releasing enzyme, alpha mannosidase class 2C, heat shock protein 90, tripeptidyl-peptidase 2, TCP-1 complex, sorbitol dehydrogenase and transitional endoplasmic reticulum ATPase were found to be positively correlated (r2 > 0.7) with freezing resilience. Cystatin, zinc-2-alpha glycoprotein, angiogenin-2-like protein, cartilage acidic protein-1, cathepsin B and ribonuclease 4 isoform 1 were found to be negatively correlated (r2 > 0.7) with freezing resilience. Several negative markers were found to originate from the accessory sex glands, whereas many positive markers originated from spermatozoa and were part of or associated with the 26S proteasome or CCT complex.

The Ebola surface glycoprotein, GP, facilitates receptor binding, cell infection and cytopathology, and is a principal target for immune protection. The structure of the GP central core structure and antibody target for the 1976 Ebola Zaire Mayinga strain of Ebola has been resolved by X-ray crystallography (e.g., pdb-ID: 3CSY). But other important GP regions have defied crystal structure determination. These include the apical mucin-like domain (MLD) that contains immuno-protective sites and is most distal to the membrane, and the membrane proximal C terminus region (meD) where GP is tethered to the Ebola surface. A molecular structure-based strategy in vaccine design necessitates detailed fine-structure knowledge of exposed sites for immuno-targeting. To address the lack of MLD and meD structures we have performed computational modeling of those regions using the programs iTasser, Phyre2 and CABS-flex. Candidate MLD model structures for the 1976 Mayinga strain were screened for continuity and fit with the 3CSY core and tested further for 3D compliance for its fit within the spatial boundaries of a lowresolution GP trimer cryoelectron tomograph (EMDB-ID: EMD-6003). Under these constraints only 1 of 6 initial MLD models generated by iTasser or Phyre2 met the required structural criteria of continuity, orientation and spatial fit. That best-fit structure (MLDm1p1) was additionally evaluated using the MolProbity geometric analysis, as well by the generation of highly similar and compatible structures (e.g., MLDm2c2) by the CABS-flex program. Notably, documented protective antibody sites of the MLD mapped to the MLDm1p1 apex surface, as anticipated for domain functionality or exposure to antibody attack. These studies describe best fit proximate in silico models of the MLD and meD regions of GP to for improved molecular understanding and therapy design. Biochemical and immunologic validation and refinements of the models are continuing, to establish to their biostructural compatibilities and value to inform on GP structure-function and immuno-target potential.

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.

Langerhans' cells (LC) of skin are CD4 expressing, dendritic, antigen-presenting cells, that are essential for activation of primary immune responses and are productively infected by HIV. We have shown previously that lymphocytes and monocytes express CD4 both as monomers and covalently linked homodimers. In those cells the 55-kDa monomer structure predominates. LC in un-fractionated human epidermal cell (EC) suspension also expresses both forms of CD4, but in EC the dimer form is predominant. Because isolation of LC into single cell suspension by trypsin, as is routinely used for LC isolation, degrades CD4, a systematic study for an alternate procedure for LC isolation was performed. Thus it was found that collagenase blend F treatment can efficiently release LC into suspension, under conditions of only minimal degradation of control soluble recombinant CD4 or CEM-T4 or THP-1 cell CD4, or importantly of LC surface CD4. SDS-PAGE immunoblotting of purified LC extracted from EC by collagenase confirmed CD4 structure as predominantly 110-kDa dimers, with only minimal 55-kDa monomers. The suitability of LC prepared thus for functional studies was demonstrated with binding of functional ligand HIV gp120. It remains to be determined, however, why tissue embedded LC express mainly CD4 dimers, but single-celled blood lymphocytes and monocytes mainly monomers.

Seminal plasma purportedly plays a critical role in reproduction, but epididymal spermatozoa are capable of fertilisation following deposition in the uterus, calling into question the biological requirement of this substance. Through a combination of direct observation of spermatozoa in utero using probe-based Confocal Laser Endomicroscopy, in vivo assessment of sperm fertility and in vitro analysis of various sperm functional parameters, this study investigated the role of seminal plasma in spermatozoa transit through the cervix of the ewe. Following deposition in the cervical os, epididymal spermatozoa previously exposed to seminal plasma displayed an enhanced ability to traverse the cervix as evidenced by both significantly higher pregnancy rates and numbers of spermatozoa observed at the utero-tubal junction when compared with epididymal spermatozoa not previously exposed to seminal plasma. The beneficial effect of seminal plasma on sperm transport was clearly localised to transit through the cervix as pregnancy rates of spermatozoa deposited directly into the uterus were unaffected by exposure to seminal plasma. This phenomenon was not explained by changes to sperm motion characteristics, as seminal plasma had no effect on the motility, kinematic parameters or mitochondrial membrane potential of spermatozoa. Rather, in vitro testing revealed that seminal plasma improved the ability of epididymal spermatozoa to penetrate cervical mucus recovered from ewes in oestrus. These results demonstrate that the survival and transport of ram spermatozoa through the cervix of the ewe is not linked to their motility or velocity but rather the presence of some cervical penetration trait conferred by exposure to seminal plasma.

Depicted is a composite model of the Ebola surface glycoprotein (GP) in its trimeric configuration (monomers comprise GP1GP2 subunits) and represents a single GP spike projecting from the surface of the Ebola Virus. This figure combines the published X-ray crystallographic (core) [1] and cryoelectron tomography (global) [2] structures, together now with in silico modeled sections of the mucinlike domain and separately the GP stalk [3]. The latter are missing from earlier published crystal structure determinations. The core Ebola Virus glycoprotein GP 3CSY structure (of homo-trimers of truncated GP1-GP2 monomers) was determined by x-ray crystallography at 3.4 Angstroms and is shown both at the bottom left (space filled depiction) and in the centre of the image within a composite model (white – in protein cartoon structure depiction) [1]. In silico modeling with Phyre was performed to obtain a structural model of the GP mucin-like domain (space filled top right monomer) [3], and also incorporated into the central composite image (yellow – top). An in silico model for the GP membrane proximal stalk domain was additionally composed and is represented in trimeric configuration (space filled bottom right) and in the centre composite (yellow strands – at the bottom) [3]. These in silico modelled structures are mapped into the composite shown in the centre with 3 separate mucin-like domains represented in yellow at the top of the trimer model, and at the base of that structure a homotrimeric stem (also in yellow) [4,5]. Together these components: the mucin-like (in silico modeled), core binding and fusion (x-ray crystal defined) and GP-membrane stalk (in situ modeled) regions cover the whole GP (i.e. GP1+GP2) monomer sequence and represent the pre-fusion conformation. These have been mapped in 3D space as trimers, and dispayed within the GP structure framework provided by the cryoelectron tomograph of the global GP trimer configuration. The tomograph is shown in surface view in the top left of the figure and in the central composite image as a blue mesh background. Note: the structures of the central composite are enlarged relative to their peripherally displayed individual elements. Figure 1: Compilation of a full trimeric prefusion model of the Ebola virus (EBOV) surface envelope glycoprotein GP.

Dendritic cells (DCs) are important targets for human immunodeficiency virus (HIV) because of their roles during transmission and also maintenance of immune competence. Furthermore, DCs are a key cell in the development of HIV vaccines. In both these settings the mechanism of binding of the HIV envelope protein gp120 to DCs is of importance. Recently a single C-type lectin receptor (CLR), DC-SIGN, has been reported to be the predominant receptor on monocyte-derived DCs (MD-DCs) rather than CD4. In this study a novel biotinylated gp120 assay was used to determine whether CLR or CD4 were predominant receptors on MDDCs and ex vivo blood DCs. CLR bound more than 80% of gp120 on MDDCs, with residual binding attributable to CD4, reconfirming that CLRs were the major receptors for gp120 on MDDCs. However, in contrast to recent reports, gp120 binding to at least 3 CLRs was observed: DC-SIGN, mannose receptor, and unidentified trypsin resistant CLR(s). In marked contrast, freshly isolated and cultured CD11c(+ve) and CD11c(-ve) blood DCs only bound gp120 via CD4. In view of these marked differences between MDDCs and blood DCs, HIV capture by DCs and transfer mechanisms to T cells as well as potential antigenic processing pathways will need to be determined for each DC phenotype. (Blood. 2001;98:2482-2488) (C) 2001 by The American Society of Hematology.

Albumin is the major transport protein in blood and intramolecular movement contributes to this function. Nonenzymatic glycosylation (NEG) of albumin occurs in diabetes and, in this study, fluorometric methods were used to determine the effect of increasing levels of NEG upon intramolecular movement in human serum albumin. Low levels of NEG significantly reduced and left-shifted Trp fluorescence, reduced quenching by acrylamide and inhibited penetration of bis-ANS, while these changes became only modestly more pronounced at higher levels of NEG. Mass spectrometry of tryptic and CNBr NEG-HSA fragments identified potential glycosylation sites and demonstrated only late glycosylation of the C- and N-terminal regions of the protein. Similar changes in diabetes may contribute to altered transport function in these patients.

The outcome of HIV infection in vivo and in vitro depends on the interaction of viral and cellular genotypes. Analysis of infection of blood monocyte-derived macrophages by primary HIV strains shows that approximately one-third of 32 isolates was consistently high-replicating, one-third was consistently low-replicating, and one-third was dependent on the donor of the macrophages (i.e., variable). HIV isolates from patients with AIDS showed enhanced replication within macrophages and predominant use of CCR5 for entry, although 13% did use CXCR4. Tissue isolates from brain and CSF showed an enhanced ability to infect 1-day-old monocytes compared with blood isolates from patients with AIDS. The ability of primary isolates to infect neonatal or adult monocytes maturing into macrophages or placental macrophages correlated directly with the extent of CCR5 expression. Studies of macrophages from pairs of identical twins and unrelated donors showed genetic control over CCR5 expression, which was independent of the CCR5▵32 genotype. Furthermore, these studies showed a marked host-cell genetic effect on the variable primary HIV strains. Although CCR5 was essential for the entry of most primary isolates, it was not the essential “bottleneck” determining productivity of infection. The location of this bottleneck in the HIV replication cycle differs according to viral strain and host-cell donor, but it was exerted before the stage of reverse transcription in 80–90% of cases. Such host-cell genetic factors may affect viral load in vivo where macrophages are the predominant target cells.

Boston Biomedical Research Institute, Boston, USACD4 is expressed by T lymphocytes and monocytes and is generally considered a monomereven though its structure was originally modelled on the REI Bence-Jones homodimer. How-ever, native CD4 was demonstrated as both monomer and dimers of 55 and 110 kDa in lym-phoid and monocytoid cells by immunoprecipitation and immunoblotting after solubilizationwith alkylating (iodoacetamide) or reducing (dithiothreitol, 2-mercaptoethanol) reagents. Fullreduction yielded only the 55-kDa monomeric form. Purified CD4 oligomers from CEM-T4cells were also resolved as homodimers by MALDI-Tof mass fingerprinting after trypticdigestion. Cell treatment with the membrane impermeable, free-thiol reactive, 5,5’-dithiobis-2-nitrobenzoic acid enhanced cell surface CD4 dimers and tetramers. The interaction sitesproducing dimerization were probably in the D4 domain as OKT4 inhibited self association ofrecombinant CD4 (rCD4). Oligomerization of rCD4 by glutathione and thioredoxin indicatesthat thiol exchange interactions were responsible. Enhanced CD4 dimer expression was alsoobserved after PMA (20ng/ml) activation of THP-1 cells. These findings demonstrate thatdifferent quaternary forms of CD4 such as monomers, homodimers and tetramers areexpressed by T lymphocytes and monocytes/macrophages.

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.

Mononuclear phagocytes play a major role during human immunodeficiency virus (HIV) transmission and throughout all stages of HIV infection and disease in most tissues. Cells of monocytic lineage are thought to be the first infected by the virus following sexual and vertical transmission (67, 79), and dendritic cells have been implicated as the first cellular targets for simian immunodeficiency virus in a simian model of mucosal transmission (70). Human monocyte-derived macrophages (MDMs) are susceptible to HIV type 1 (HIV-1) infection in vitro (27, 54, 58, 64). However, some groups have reported that viral inoculation must occur within a temporal window of the monocyte differentiation process for a productive infection to be established. For example, Schuitemaker et al. (64) and Potts et al. (54) suggest that terminally differentiated in vitro MDMs are completely resistant to HIV-1 infection, inferring similar resistance by tissue macrophages in vivo. Differentiated cells of monocytic lineage have been shown to harbor and produce virus in the brains (39), lungs (6), and lymphoid systems (18) of HIV-1-infected individuals. Strong evidence now exists for the infection of circulating blood monocytes with HIV-1 (34, 46, 49, 63, 77), with most studies reporting that the infected cells are a minor subpopulation of the total monocyte pool. The chemokine receptors CXCR-4 and CCR-5 have been found to act predominantly as fusion cofactors for T-cell-line-tropic and macrophage/dual tropic (M-tropic) HIV-1 infection of CD4-positive cells, respectively (1, 15, 17, 23). However, the roles of these receptors, particularly CCR-5, in mediating HIV-1 infection of cultured monocytes, MDMs, and tissue macrophages are ill-defined. Published data comparing relative infectibilities of monocytes, MDMs, and tissue macrophages from the same person have been compiled by using laboratory-adapted (LA) HIV-1 isolates (44, 58), but to date no studies using clinically relevant primary (PR) HIV-1 strains have been reported. Such data need to be correlated with chemokine receptor expression. Differences between adult and neonatal monocytes/macrophages in HIV-1 infectability and chemokine receptor expression require further study. Such differences in susceptibility to HIV-1 infection between neonatal and adult cells may contribute to the different symptoms, severity of neuropathology, and rate of disease progression observed in pediatric AIDS cases (65, 73). To date, relevant reports have shown only that cord blood monocytes are more susceptible to infection with LA M-tropic HIV-1 isolates in vitro than their adult counterparts (30, 68). Therefore, we have analyzed in vitro infection of neonatal cord blood-derived monocytes, MDMs, and placental macrophages (PMs) with a panel of LA and PR isolates of HIV-1. To avoid discrepancies associated with host cell genetic variation in HIV-1 infection studies (5, 69), all three cell types were syngeneic. These infection data were then correlated with CD4, CXCR-4, and CCR-5 expression by neonatal cells and compared with HIV-1 susceptibility in adult monocytes/MDMs. Our results suggest correlations between CCR-5 expression and susceptibility to infection by PR but not LA M-tropic strains of HIV-1 in neonatal cells and differences in susceptibility to PR HIV-1 isolates between neonatal and adult cells. They also provide an explanation for the relative refractoriness of PMs to infection with PR HIV-1 isolates.

AIDS dementia complex (ADC) develops in only a third of HIV-infected patients who progress to AIDS. Macrophages and microglial cells are the major cellular sites of productive HIV replication in brain. Using 11 blood isolates of HIV from asymptomatic patients there was marked variation in tropism and the level of productive infection in recently adherent monocytes and monocyte-derived macrophages cultured in vitro. However, less variation was seen with 19 blood isolates from advanced HIV infection and 11 postmortem tissue isolates from brain, cerebrospinal fluid, spleen, and lung. Newly adherent monocytes expressed CCR5 in all seven patients tested, consistent with their susceptibility to infection but not explaining the above variability. There is also marked regional variability in neuropathology in the brain of patients with ADC. We have demonstrated that there was marked variation in the V3 sequences of HIV clones from different regions of the cortex of a patient with ADC, suggesting independent evolution of HIV replication in brain. Furthermore, production of the neurotoxin quinolinic acid from HIV-infected macrophages varied, depending on the host and source of HIV isolate. Hence variations in viral genotype, production by infected macrophages, and subsequent toxin production may contribute to the variability in neuropathology between individuals and between different regions of the brain in the same individual.

Knowledge of CD4 conformation within the membranes of human lymphoid and monocytoid cells is essential for a clear understanding of its function as a ligand for major histocompatibility complex II (MHC) molecules in T cell activation and for gp120 in human immunodeficiency virus (HIV) infection. The charge and structure of native (nCD4) and soluble recombinant CD4 (rCD4) were examined by one- and two-dimensional (2-DE) electrophoresis antigen mapping and silver staining. Recombinant CD4 was partitioned by nonequilibrium pH gradient electrophoresis (NEPHGE) and revealed a number of differentially charged 44 kDa species (pI > 9.5). Biotinylation (4 h, room temperature) of rCD4 yielded a single labelled species on sodium dodedyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an increased apparent molecular mass to 50 kDa, consistent with a maximal incorporation of approximately 18 molecules of biotin per rCD4 molecule. The milder biotinylation (15 min, 4 degrees C) of cell-(CEM-T4, THP-1) expressed CD4 was not accompanied by any apparent alteration in molecular weight, nor abrogation of CD4 antigenicity. This was determined by isolation of nCD4 by immunoprecipitation and SDS-PAGE immunoblotting, using anti-CD4 mAbs (leu3a, OKT4A, Q4120, T4, OKT4, Q425) and by flow cytometry (leu4a, T4). The immunoprecipitation of full-length native CD4 from lymphoid MT2 and CEM-T4 cell extracts, however, revealed both monomeric and higher-order CD4 antigen complexes by immunoblotting. These studies describe the biotinylation, 1-DE and 2-DE of CD4 preparations, and indicate the capacity of CD4 of lymphocytes to form complexes which may influence CD4 conformation and epitope availability.

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

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.

Biochemical and immuno-microscopic techniques were used to study temporal involvement of thrombospondin in relation to fibrinogen in muscle regeneration using a rat skeletal muscle-wound model. In undamaged control muscle, no fibrinogen and minimal thrombospondin antigen was found. Following crushing injury, fibrin networks appear immediately, followed by a gradual ordered accumulation of thrombospondin (within a few hours) in the vicinity of the vascular bed and adjacent endomysial connective tissue. Later, thrombospondin becomes associated with connective tissue and basal laminae around muscle fibers throughout the damaged muscle, maximal labelling occurring 3-6 days post-injury. Thrombospondin immunoreactivity decreased thereafter to near normal levels after 7 days post-injury, coincident with the appearance of regenerating muscle fibers. In contrast, little fibrin material remained by five days after injury. Quantitative radioimmunoassay of soluble thrombospondin antigen and radioimmune labelling of thick frozen sections reinforced the qualitative immuno-microscopic observations, with levels peaking at 3-4 days post-trauma, 10-fold over control levels. SDS-PAGE immunoblotting of non-reduced muscle extracts three days after a crush assault shows that the bulk of the thrombospondin incorporated into the injury site exists in a polymerized state (less than or equal to 1000 kD). These results demonstrate that the temporal appearance and disappearance of thrombospondin in the healing of a crushing lesion in muscle is related more closely to the regeneration phase of muscle than to the coagulation phase.

Increased vascular disease occurs with low albumin (human serum albumin, HSA), possibly reflecting specific inhibition of endothelial apoptosis reported for tissue culture. Despite the reported specificity for endothelial protection by HSA, the high but physiological concentrations needed appear more consistent with non-specific low-affinity interactions. We reconcile this contradiction by demonstrating protection is mediated by a partially cryptic HSA protein domain, which becomes more exposed and active following cyanogen bromide fragmentation (p < 0.001). Also, although others reported HSA radical scavenging and bound lipids as important for inhibiting apoptosis in non-endothelial cell types, we demonstrate the protective effect for endothelium is unaffected when HSA radical scavenging is blocked by alkylation, or following delipidation. Further probing the mechanism responsible, we found that the G-coupled protein inhibitors pertussis toxin and suramin reduced protection of endothelium by HSA (p < 0.005), while the tyrosine kinase inhibitor genistein had no effect. Consistent with a role for phosphoinositide 3 kinase (PI3K) was inhibition by both wortmannin and LY294002 (p < 0.05), as well as phosphorylation of Akt, while MAP kinase inhibitors had no effect. We conclude the active site in HSA inhibiting endothelial apoptosis is partially cryptic, and acts via a G-coupled protein PI3K-dependent mechanism.

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.

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.

The chemokine receptor CCR5 and to a lesser extent CCR3 and CCR2b have been shown to serve as coreceptors for human immunodeficiency virus type 1 (HIV-1) entry into blood- or tissue-derived macrophages. Therefore, we examined the expression of the chemokine receptors CCR1, CCR2b, CCR3, CCR5, and CXCR4 as RNAs or as membrane-expressed antigens in monocytes maturing into macrophages and correlated these results with the susceptibility of macrophages to HIV-1 infection, as measured by their concentrations of extracellular p24 antigen and levels of intracellular HIV DNA by quantitative PCR. There was little change in levels of CCR1, CCR2b, and CCR5 RNAs. CCR3 RNA and surface antigen were undetectable throughout maturation of adherent monocytes over 10 days. CXCR4 RNA and membrane antigen were strongly expressed in newly adherent monocytes, but their levels declined at day 7. The amounts of CCR5 RNA remained stable, but the amounts of CCR5 antigen increased from undetectable to peak levels at day 7 and then declined slightly at day 10. Levels of susceptibility to laboratory (HIV-1 BaL ) and clinical strains of HIV-1 showed parallel kinetics, peaking at day 7 and then decreasing at days 10 to 14. The concordance of levels of HIV DNA and p24 antigen suggested that the changes in susceptibility with monocyte maturation were at or immediately after entry and correlated well with CCR5 expression and inversely with CXCR4 expression.

A study was designed to assess the impact of water-soluble proteins and peptides extracted from meat and bone meal (MBM) on broiler chick performance, following their oral delivery during the early post-hatch period. Proteinaceous material was fractionated by size exclusion filtration into weight ranges of 100 kDa (Fraction 3; 0.8 mg protein/mL), which formed the three protein fraction treatments. A total of 1 mL of each of the respective preparations was delivered orally via gavage over 4 days (0.25 µL each day) to Cobb broiler hatchlings. Three control groups: control–unhandled, control–phosphate-buffered saline and control–handled were also included. Chicks were grown to 30 days of age. Feed intake, chick weight gain and feed conversion ratio were determined from day old through to 29 days of age. On Days 10, 16, 23 and 30, the weight of the breast and the small intestine was determined from 10 birds/treatment. For all parameters measured there was no interaction between experimental week and protein fraction treatment. Chicks receiving Fraction 2 had a statistically significant increase in feed intake and weight gain (P = 0.012) compared with the control–unhandled chicks. Chicks receiving Fraction 2 also demonstrated a numerically higher final bodyweight. Mass spectrometric analysis of all three fractions revealed that they each contained a wide array of proteinacious material. The results of this study suggests the likelihood that protein or protein-derived fragment components within the 3–100 kDa molecular weight range of MBM can generate improvements in broiler chick production, and thus promote the need for further research to identify the specific protein(s) responsible for the observed positive growth effects.

Sulfated polyanions (SPs) bind variably to lymphocyte-expressed CD4 and inhibit binding of monoclonal antibodies to the first two domains of CD4. To further define this interaction, soluble recombinant CD4 (sCD4; four extracellular domains), its truncated amino-terminal two-domain derivative, and three linear peptide analogues spanning residues 6–60 (6–24, 20–40, 41–60) in the first domain were investigated for SP binding. Dextran sulfate (DXS) (500 kDa), polyvinyl sulfate, fucoidan, and carrageenan-kappa, each immobilized on carboxymethyl cellulose fibers, bound strongly to both the two-domain and four-domain recombinant CD4 molecules (similar to that observed with native CD4), whereas dextran sulfate (5 kDa), chondroitin 6-sulfate, and pentosan sulfate bound relatively poorly. No peptide binding to SPs was observed. Recombinant gp120 bound poorly (

Factor XIII A subunit was detected in U937 cells and human alveolar macrophages by immunohistology and Western blotting. U937 cells synthesize factor XIII A subunit de novo under serum-free, platelet- free conditions, as indicated by 35S-methionine labeling and immunoprecipitation. Thrombin-dependent activity was demonstrated to account for 98% of the total transglutaminase activity in U937 cells (1.5 micrograms per 0.5 X 10(6) cells/mL). Intact U937 cells and alveolar macrophages and homogenates from these cells cross-linked fibrin to form gamma-gamma and alpha-polymers. Factor XIII A was detected on the surface of intact U937 cells and macrophages by flow cytometry and 125I-labeling and immunoprecipitation. Cell surface expression of factor XIII A was augmented in the presence of several soluble macrophage activators; however, no concurrent increase in its biosynthesis was observed. The presence and cell surface expression of factor XIII A subunit within macrophages suggest new pathways by which these cells may function in clotting and in the remodeling of the extracellular matrix during inflammation and wound healing.

SummaryPlatelets contain factor XIII, an A subunit zymogen form of transglutaminase (TGase), that is activated by thrombin. In addition a thrombin-independent TGase (A#) was observed. A# was formed in platelet preparations lysed at acid pH, and its generation inhibited by protease inhibitors and alkaline pH. When maximal A# activity was generated in acidified lysates no further TGase activity could be induced by subsequent treatment with thrombin. Both FXIII zymogen and A# copurified as for F XIII, from either alkaline or from acidified platelet lysates respectively, by the conventional procedure. The pH optima, Km’s for NN dimethyl casein, molecular weights, heat lability of active forms, requirements for calcium and reducing agents, and immunological characteristics of both TGases were the same. Studies with inhibitor substrates suggested that a thrombin-like cathepsin C or carboxypeptidase was responsible for A# formation. Purified F XIII zymogen could be activated directly by cathepsin C. Thus, the predominant, and probably only, TGase of platelets is factor XIII, which may be activated either by thrombin or by endogenous platelet acid protease(s).

Thrombin activation of platelets induces the release of a high molecular weight glycoprotein, thrombospondin. On treatment with factor XIII transglutaminase and [3H]putrescine, thrombospondin undergoes specific incorporation of this labeled amine, with 2-3 mol of putrescine being incorporated per mol of thrombospondin. Analysis of plasmin digests of [3H]putrescine-thrombospondin showed that the Mr 53,000-core peptide contains the glutamine site for amine incorporation. In the absence of amine substrate, thrombospondin was found to provide both donor (glutamine) and acceptor (lysine) sites for intermolecular cross-links by factors XIIIa, and high molecular weight protein complexes were formed. Homopolymers of thrombospondin were also observed by electron microscopy. Thrombin-cleaved thrombospondin has more cross-linking sites accessible for [3H]putrescine incorporation or for cross-linkage to itself than does the uncleaved native protein. Examination of thrombospondin cross-linkage in the presence of other protein substrates (fibronectin, collagen, fibrinogen, and von Willebrand factor) for factor XIIIa, resulted in reduced thrombospondin polymer formation. Electron microscopy and autoradiography of fibrin clots formed in the presence of 125I-thrombospondin showed an association of thrombospondin with fibrin fibrils. However, confirmation that this association involves covalent epsilon-(gamma-glutamyl)lysyl cross-links between thrombospondin and fibrin was not obtained.

Plasma factor XIII is a complex of A and B proteins noncovalently linked in a tetramer, A2B2, Enzyme-linked immunosorbent assays (ELISA) were developed to measure the separate factor XIII proteins and the complex. All of the A protein in plasma is in the zymogen complex. The B assay measures the total amount of B protein in plasma (both free B and complexed B). This was confirmed by nondenaturing gel electrophoresis and immunoblotting, which showed two bands for B in plasma with this antibody. Two assays were developed to measure A2B2 complex specifically. One assay used a monoclonal antibody to B to bind antigen and measured B protein in the zymogen complex only and hence the concentration of the complex. The specificity of this antibody was also shown by immunoblotting. In the second assay, the capture antibody was to B and the tag antibody was to A. These two assays gave identical results for the concentration of A2B2 (0.07 mumol/L, 21.6 micrograms/mL in normal plasma). Thus, for the first time, differentiation and quantitation of free B and complexed B in plasma was possible. The assays were used to measure factor XIII proteins in plasma from normal controls, homozygous-deficient factor XIII patients, and their heterozygous relatives. The normal concentration of A in plasma is 0.13 to 0.16 mumol/L (approximately 11 micrograms/mL), all of which is in A2B2. The total B concentration is 0.26 to 0.28 mumol/L (approximately 21 micrograms/mL), half of which is complexed. The free B concentration is 0.13 mumol/L (approximately 10 micrograms/mL). Homozygous-deficient patients have essentially no A protein, but their free B concentration is 0.11 mumol/L. Heterozygotes have decreased A2B2, but their free B is 0.11 mumol/L. These results indicate that the concentration of free B is remarkably constant and does not depend on the concentration of A2 or A2B2.

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.