Garry W. Lynch

Marked structural and functional heterogeneity in CXCR4: Separation of HIV-1 and SDF-1α responses

CXCR4, the chemotactic cell receptor for SDF‐1α, 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 CXCR4s 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‐1α‐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‐1α. 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‐1α responses and may indicate therapeutic possibilities.

Direct evidence for native CD4 oligomers in lymphoid and monocytoid cells

CD4 is expressed by T lymphocytes and monocytes and is generally considered a monomer even though its structure was originally modelled on the REI Bence‐Jones homodimer. However, native CD4 was demonstrated as both monomer and dimers of 55 and 110 kDa in lymphoid and monocytoid cells by immunoprecipitation and immunoblotting after solubilization with alkylating (iodoacetamide) or reducing (dithiothreitol, 2‐mercaptoethanol) reagents. Full reduction yielded only the 55‐kDa monomeric form. Purified CD4 oligomers from CEM‐T4 cells were also resolved as homodimers by MALDI‐Tof mass fingerprinting after tryptic digestion. Cell treatment with the membrane impermeable, free‐thiol reactive, 5,5′‐dithiobis‐2‐nitrobenzoic acid enhanced cell surface CD4 dimers and tetramers. The interaction sites producing dimerization were probably in the D4 domain as OKT4 inhibited self association of recombinant CD4 (rCD4). Oligomerization of rCD4 by glutathione and thioredoxin indicates that thiol exchange interactions were responsible. Enhanced CD4 dimer expression was also observed after PMA (20 ng/ml) activation of THP‐1 cells. These findings demonstrate that different quaternary forms of CD4 such as monomers, homodimers and tetramers are expressed by T lymphocytes and monocytes/macrophages.

HIV infection of macrophages and pathogenesis of AIDS dementia complex: interaction of the host cell and viral genotype.

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. J. Leukoc. Biol. 62: 117–125; 1997.

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 ± 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/).

The Anti-Apoptotic Activity of Albumin for Endothelium Is Mediated by a Partially Cryptic Protein Domain and Reduced by Inhibitors of G-Coupled Protein and PI-3 Kinase, but Is Independent of Radical Scavenging or Bound Lipid

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.

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.

Seminal plasma aids the survival and cervical transit of epididymal ram spermatozoa

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.

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‐β‐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.

The level of HIV infection of macrophages is determined by interaction of viral and host cell genotypes

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.

Variation in seminal plasma alters the ability of ram spermatozoa to survive cryopreservation

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 (0-4h), 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<0.001). Similarly, spermatozoa from low-resilience rams frozen with high-resilience seminal plasma exhibited higher motility than spermatozoa from low-resilience rams frozen with low-resilience seminal plasma immediately after thawing (0h; P<0.001). The present study shows that variation in freezing resilience of ram spermatozoa is related to the source and composition of the seminal plasma.