Dextran sulfate and heparin interact with CD4 molecules to inhibit the binding of coat protein (gp120) of HIV

Dextran sulfate, heparin, and certain other sulfated polysaccharides potently inhibit the adsorption of HIV to CD4+ cells. The mechanism of this inhibition is unclear and, specifically, it is unknown if these agents act at the level of CD4-gp120 binding. For example, previous reports have demonstrated that dextran sulfate does not inhibit the cell surface binding of anti-CD4 mAb known to be directed at the gp120 binding site. In order to confirm and extend these observations, in the present study, it was shown that dextran sulfate does not inhibit the binding of OKT4A, OKT4C, Leu3a, or B66.6 to CD4+ cells as measured by cytofluorography. Next, recombinant forms of CD4 (rT4) and gp120 (rgp120) were utilized to directly study their molecular interaction in the absence of other viral or cellular structures. Reciprocal solid phase ELISA assays were developed to study directly the effects of sulfated polysaccharides on the binding of rT4 to immobilized rgp120 and vice versa. Dextran sulfate, heparin, and fucoidan, but not chondroitin sulfate, inhibited the binding of rgp120 to rT4. Importantly, dextran sulfate and heparin pre-treatment of immobilized rT4, but not immobilized rgp120, inhibited rT4-rgp120 binding. Taken together, these data suggest that while both sulfated polysaccharides and anti-CD4 mAb inhibit gp120 binding, the sulfated polysaccharides interact with sites on CD4 that are distinct from those with which the antibodies bind.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Functional analysis of human T cell subsets defined by monoclonal antibodies. V. Suppressor cells within the activated OKT4+ population belong to a distinct subset

In the present report, we characterize a monoclonal antibody directed at a surface differentiation antigen on human T cells. The monoclonal antibody, OKT17, recognizes a cell surface antigen present on the majority of resting normal peripheral T cells. In contrast, OKT17 is unreactive with normal B cells, B cell lines, T cell lines, or SIg+ CLL. Interestingly, after activation, the antigen recognized by OKT17 is lost from a subset of OKT4+ cells. We took advantage of this finding to explore further the functional heterogeneity within activated OKT4+ cells. Evidence was obtained that the PWM-activated OKT4+ subset remaining after depletion of OKT17-reactive T cells (OKT4+ 17-) contains radiosensitive helperr cells but is devoid of suppressor cells. In contrast, the activated OKT4+ 17+ population contains potent radiosensitive suppressor cells as well as radioresistant helpe cells. Taken together, these studies suggest that the OKT17 monoclonal antibody can differentiate two functionally mature, activated OKT4+ human T cells: OKT4+ OKT17+ radiosensitive suppressor cells and OKT4+ 17- radiosensitive helper cells.     

Isolation and characterization of naturally occurring subclasses of human peripheral blood T cells with regulatory functions.

By utilizing naturally occurring autoimmune antibodies from patients with juvenile rheumatoid arthritis, we have isolated and functionally characterized two unique subpopulations of T cells. JRA+ T cells, i.e., those identified by sera from these patients, react poorly in response to allogeneic cells, respond to Con A but not PHA, and do not help in the synthesis and secretion of Ig by B cells. In contrast, JRA- T cells, i.e., those not identified by sera from these patients, respond very well to allogeneic cells, proliferate well in response to PHA but not Con A, and more interestingly, can greatly enhance the secretion of Ig by B cells.     

Functional analysis of human T cell subsets defined by monoclonal antibodies. III. Regulation of helper factor production by T cell subsets

In the present report we extended our previous studies demonstrating that obligatory T-T interactions are important in regulating human immune responses in vitro. Functionally distinct human T cell subsets were isolated by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Evidence was obtained that during allogeneic interactions, OKT4+, but not OKT8+, responder T cells are required to generate helper factor(s) capable of polyclonally activating human B cells independent of additional T cell help. Importantly, the alloantigen-induced helper factor(s) production and/or release was found to be suppressed by addition of graded numbers of radiosensitive OKT8+ cells. On the other hand, no evidence was obtained that supernatant derived from alloactivated OKT8+ cells could counterbalance the helper activity generated in the presence of supernatant from alloactivated OKT4+ cells. Furthermore, OKT8+ cells, known to suppress PWM-driven B cell differentiation in the presence of OKT4+ cells, do not suppress B cell differentiation induced by preformed helper factor even in the presence of OKT4+ cells. These data further underscore the importance of functional T-T interactions in immunoregulation in vitro and support the idea that the target of suppression of B cell differentiation, induced either by alloantigen-triggered helper factor or PWM, are OKT4+ cells and not B cells themselves.     

Induction of TCR Vbeta-specific CD8+ CTLs by TCR Vbeta-derived peptides bound to HLA-E

Previous studies have identified murine and human regulatory CD8+ T cells specific for TCR-Vbeta families expressed on autologous activated CD4+ T cells. In the mouse, these regulatory CD8+ T cells were shown to be restricted by the MHC class Ib molecule, Qa-1. In the present study, we asked whether HLA-E, the human functional equivalent of Qa-1, binds Vbeta peptides and whether the HLA-E/Vbeta-peptide complex induces and restricts human CD8+ CTLs. We first created stable HLA-E gene transfectants of the C1R cell line (C1R-E). Two putative HLA-E binding nonapeptides identified in human TCR Vbeta1 and Vbeta2 chains (SLELGDSAL and LLLGPGSGL, respectively) were shown to bind to HLA-E. CD8+ T cells could be primed in vitro by C1R-E cells loaded with the Vbeta1 (C1R-E/V1) or Vbeta2 (C1R-E/V2) peptide to preferentially kill C1R-E cells loaded with the respective inducing Vbeta peptide, compared with targets loaded with the other peptides. Priming CD8+ T cells with untreated C1R-E cells did not induce Vbeta-specific CTLs. Of perhaps more physiological relevance was the finding that the CD8+ CTLs primed by C1R-E/V1 also preferentially killed activated autologous TCR Vbeta1+. Similar results were observed in reciprocal experiments using C1R-E/V2 for priming. Furthermore, anti-CD8 and anti-MHC class I mAbs inhibited this Vbeta-specific killing of C1R-E and CD4+ T cell targets. Taken together, the data provide evidence that certain TCR-Vbeta peptides can be presented by HLA-E to further induce Vbeta-specific CD8+ CTLs.     

Serosurvey of Brucella spp. infection in the Kafue lechwe (Kobus leche kafuensis) of the Kafue flats in Zambia

One of the diseases of veterinary and public health importance affecting the Kafue lechwe (Kobus leche kafuensis) on the Kafue flats is brucellosis, for which only scant information is available. During the 2003 (October), 2004 (December), and 2008 (July-December) hunting seasons in the Kafue flats, we conducted a study to determine the seroprevalence of Brucella spp. in the Kafue lechwe and to evaluate serologic tests for detection of Brucella spp. antibodies in lechwe. The Rose Bengal Test (RBT), competitive enzyme-linked immunosorbent assay (cELISA), and fluorescence polarization assay (FPA) were used. A total of 121 Kafue lechwe were hunted for disease investigations in 2003, 2004, and 2008 in the Kafue Flat Game Management Area. Of these, 21.6%, (95% confidence interval [CI]: 14.2-29.1%) had detectable antibodies to Brucella spp. The Kafue lechwe in Lochnivar National Park had higher antibody results than those in Blue Lagoon National Park (odds ratio=3.0; 95% CI: 0.94-9.4). Infection levels were similar in females (21.6%) and males (21.7%). Results were similar among RBT, FPA, cELISA tests, suggesting that these could effectively be used in diagnosing brucellosis in the Kafue lechwe. Our study demonstrates the presence of Brucella infections in the Kafue lechwe in two national parks located in the Kafue flats and further highlights the suitability of serologic assays for testing the Kafue lechwe. Because the Kafue lechwe is the most hunted wildlife species in Zambia, hunters need to be informed of the public health risk of Brucella spp. infection.     

Human neutrophil clearance of bacterial pathogens triggers anti-microbial γδ T cell responses in early infection

Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis--characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity--show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in early infection and suggest novel diagnostic and therapeutic approaches.     

A deletion at the mouse Xist gene exposes trans-effects that alter the heterochromatin of the inactive X chromosome and the replication time and DNA stability of both X chromosomes

The inactive X chromosome of female mammals displays several properties of heterochromatin including late replication, histone H4 hypoacetylation, histone H3 hypomethylation at lysine-4, and methylated CpG islands. We show that cre-Lox-mediated excision of 21 kb from both Xist alleles in female mouse fibroblasts led to the appearance of two histone modifications throughout the inactive X chromosome usually associated with euchromatin: histone H4 acetylation and histone H3 lysine-4 methylation. Despite these euchromatic properties, the inactive X chromosome was replicated even later in S phase than in wild-type female cells. Homozygosity for the deletion also caused regions of the active X chromosome that are associated with very high concentrations of LINE-1 elements to be replicated very late in S phase. Extreme late replication is a property of fragile sites and the 21-kb deletions destabilized the DNA of both X chromosomes, leading to deletions and translocations. This was accompanied by the phosphorylation of p53 at serine-15, an event that occurs in response to DNA damage, and the accumulation of gamma-H2AX, a histone involved in DNA repair, on the X chromosome. The Xist locus therefore maintains the DNA stability of both X chromosomes.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.