Antibody-Dependent Cellular Cytotoxicity Directed against Cells Expressing Human Immunodeficiency Virus Type 1 Envelope of Primary or Laboratory-Adapted Strains by Human and Chimpanzee Monoclonal Antibodies of Different Epitope Specificities

The characteristics of antibody-dependent cellular cytotoxicity (ADCC) directed by a panel of human and chimpanzee antienvelope (anti-Env) monoclonal antibodies (MAbs) of different epitope specificities were studied; this was accomplished by using target cells expressing human immunodeficiency virus type 1 (HIV-1) Envs of either primary or laboratory-adapted strains. Human MAbs of similar apparent affinities (1 × 10⁹ to 2 × 10⁹ liters/mol) against either a “cluster II”-overlapping epitope of gp41 or against the CD4 binding site, V3 loop, or C5 domain of gp120 directed substantial and comparable levels of specific lysis against targets infected with laboratory-adapted strains of HIV-1. As expected, those MAbs specific for relatively conserved regions of Env generally exhibited ADCC activity against a broader range of HIV-1 strains than those directed against variable epitopes. Significant ADCC activities of selected MAbs against primary isolate Env-expressing cells were demonstrated. In addition, a new ADCC epitope in the V2 domain of gp120 was defined. CD56⁺ cells were demonstrated to be the effector cells in these studies by fluorescence-activated cell sorting followed by ADCC assays. Notably, all anti-Env MAbs tested in this study, including MAbs directed against each of the known neutralization epitope clusters in gp120, directed significant levels of ADCC against targets expressing Env of one or more HIV-1 strains. These results imply that many, if not most, HIV-1-neutralizing human Abs of high affinity (≥3 × 10⁸ liters/mol in these studies) and of the immunoglobulin G1 (IgG1) subclass (i.e., the predominate IgG subclass) are capable of directing ADCC. Since neutralizing Abs have been associated with long-term survival following HIV-1 infection, this suggests that ADCC activity may be beneficial in vivo.The in vivo role(s) of antibodies (Abs) that can direct antibody-dependent cellular cytotoxicity (ADCC) against human immunodeficiency virus type 1 (HIV-1) Env-expressing cells in vitro remains unclear. In ADCC, anti-Env Abs direct effector cells to kill target cells bearing HIV-1 envelope on their surfaces; this is accomplished via specific binding of the Abs’ antigen-binding sites to Envs and their Fc regions to Fc receptors on the effector cells. Broadly strain reactive, ADCC-directing Abs arise early in the immune response to HIV-1 infection in vivo (14) and may be partially responsible for the initial clearance of viremia.Earlier in the HIV-1 epidemic, concerns were raised that shed soluble gp120 in HIV-1-infected individuals might bind to CD4⁺ cells, including uninfected ones, and could target these cells for “innocent bystander” killing by ADCC (6). However, effector cells armed with serum Abs able to direct ADCC in vitro against either innocent bystanders or HIV-1-infected cells were found at highest frequency in asymptomatic, seropositive individuals; patients with AIDS-related complex and AIDS showed progressively diminished reactivities (20). Furthermore, in a recent study (1), the ability of monoclonal Abs (MAbs) against three distinct gp120 epitopes to direct ADCC against uninfected CD4⁺ cells to which rgp120_SF2 had been adsorbed (i.e., innocent bystanders) was demonstrated to be less efficient by at least an order of magnitude than their ability to direct ADCC against HIV-1-infected cells.The existing data from in vivo studies (reviewed in reference 1) supports the efficacy, rather than the pathogenicity, of ADCC-directing Abs against HIV-1. Consistent with this data is our recent characterization of two MAbs, 42F and 43F, isolated from a long-term survivor of HIV-1 infection (1); these MAbs directed significant levels of ADCC and defined a new, conserved ADCC epitope in the C5 domain of HIV-1 gp120. Preliminary evidence indicated that concentrations of 42F- and 43F-like Abs in the serum of the donor were in the range required to direct high levels of ADCC, and these MAbs were shown to bind both oligomeric primary-isolate and laboratory-adapted Env efficiently (1).Because of the potential importance of ADCC-directing Abs against HIV-1, in this study we have evaluated ADCC directed against cells expressing HIV-1 Envs of primary or laboratory-adapted strains by a panel of human and chimpanzee anti-Env MAbs of different epitope specificities. Significant ADCC activities of selected MAbs against primary-isolate Env-expressing cells were demonstrated, and a new ADCC epitope in the V2 domain of gp120 was defined. Finally, a MAb’s ability to direct ADCC against a specific target cell type was shown to be dependent on additional factors beyond its ability to efficiently bind antigen on the target cell and its possession of an Fc region of the appropriate isotype to engage FcγR on effector cells.     

Network slicing to improve multicasting in HPC clusters

In high performance computing (HPC) resources’ extensive experiments are frequently executed. HPC resources (e.g. computing machines and switches) should be able to handle running several experiments in parallel. Typically HPC utilizes parallelization in programs, processing and data. The underlying network is seen as the only non-parallelized HPC component (i.e. no dynamic virtual slicing based on HPC jobs). In this scope we present an approach in this paper to utilize software defined networking (SDN) to parallelize HPC clusters among the different running experiments. We propose to accomplish this through two major components: A passive module (network mapper/remapper) to select for each experiment as soon as it starts the least busy resources in the network, and an SDN-HPC active load balancer to perform more complex and intelligent operations. Active load balancer can logically divide the network based on experiments’ host files. The goal is to reduce traffic to unnecessary hosts or ports. An HPC experiment should multicast, rather than broadcast to only cluster nodes that are used by the experiment. We use virtual tenant network modules in Opendaylight controller to create VLANs based on HPC experiments. In each HPC host, virtual interfaces are created to isolate traffic from the different experiments. The traffic between the different physical hosts that belong to the same experiment can be distinguished based on the VLAN ID assigned to each experiment. We evaluate the new approach using several HPC public benchmarks. Results show a significant enhancement in experiments’ performance especially when HPC cluster experiences running several heavy load experiments simultaneously. Results show also that this multi-casting approach can significantly reduce casting overhead that is caused by using a single cast for all resources in the HPC cluster. In comparison with InfiniBand networks that offer interconnect services with low latency and high bandwidth, HPC services based on SDN can provide two distinguished objectives that may not be possible with InfiniBand: The first objective is the integration of HPC with Ethernet enterprise networks and hence expanding HPC usage to much wider domains. The second objective is the ability to enable users and their applications to customize HPC services with different QoS requirements that fit the different needs of those applications and optimize the usage of HPC clusters.     

High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

Background

Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods.

Results

A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point.

Conclusions

Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.     

The Nexus between VEGF and NFκB Orchestrates a Hypoxia-Independent Neovasculogenesis

Nuclear Factor-Kappa B [NFκB] activation triggers the elevation of various pro-angiogenic factors that contribute to the development and progression of diabetic vasculopathies. It has been demonstrated that vascular endothelial growth factor [VEGF] activates NFκB signaling pathway. Under the ischemic microenvironments, hypoxia-inducible factor-1 [HIF-1] upregulates the expression of several proangiogenic mediators, which play crucial roles in ocular pathologies. Whereas YC-1, a soluble guanylyl cyclase [sGC] agonist, inhibits HIF-1 and NFκB signaling pathways in various cell and animal models. Throughout this investigation, we examined the molecular link between VEGF and NFκB under a hypoxia-independent microenvironment in human retinal microvascular endothelial cells [hRMVECs]. Our data indicate that VEGF promoted retinal neovasculogenesis via NFκB activation, enhancement of its DNA-binding activity, and upregulating NFκB/p65, SDF-1, CXCR4, FAK, αVβ3, α5β1, EPO, ET-1, and MMP-9 expression. Conversely, YC-1 impaired the activation of NFκB and its downstream signaling pathways, via attenuating IκB kinase phosphorylation, degradation and activation, and thus suppressing p65 phosphorylation, nuclear translocation, and inhibiting NFκB-DNA binding activity. We report for the first time that the nexus between VEGF and NFκB is implicated in coordinating a scheme that upregulates several pro-angiogenic molecules, which promotes retinal neovasculogenesis. Our data may suggest the potential use of YC-1 to attenuate the deleterious effects that are associated with hypoxia/ischemia-independent retinal vasculopathies.     

Towards building data analytics benchmarks for IoT intrusion detection

Cluster Computing - Data analytics projects span all types of domains and applications. Researchers publish results using certain datasets and classification models. They present results with a...     

Obesity and Dental Decay: Inference on the Role of Dietary Sugar

Objective

To evaluate the relationship of children’s obesity and dental decay.

Methods

We measured parameters related to obesity and dental decay in 8,275 4^th and 5^th grade Kuwaiti children (average age = 11.36 years) in a cross-sectional study. First to determine body weight, height, age for computation of BMI . Second, to determine numbers of teeth, numbers of fillings and numbers of untreated decayed teeth to determine extent and severity of dental disease. From these measurements, we computed measures of dental decay in children from four body weight categories; obese, overweight, normal healthy weight and underweight children.

Results

The percentage of children with decayed or filled teeth varied inversely with the body weight category. The percentage of decayed or filled teeth decreased from 15.61% (n=193) in underweight children, to 13.03% (n=4,094) in normal healthy weight children, to 9.73% (n=1,786) in overweight children to 7.87% (n=2,202) in obese children. Differences between all groups were statistically significant. Male children in this population had more dental decay than female children but the reduction of tooth decay as a function of BMI was greater in male children.

Conclusions

The finding of an inverse obesity-dental decay relationship contradicts the obesity-sugar and the obesity-dental decay relationship hypotheses. Sugar is well recognized as necessary and sufficient for dental decay. Sugar is also hypothesized to be a leading co-factor in obesity. If the later hypothesis is true, one would expect dental decay to increase with obesity. This was not found. The reasons for this inverse relationship are not currently clear.     

Vitamin D Insufficiency in Arabs and South Asians Positively Associates with Polymorphisms in GC and CYP2R1 Genes

Background

A number of genetic studies have reported an association between vitamin D related genes such as group-specific component gene (GC), Cytochrome P450, family 2, subfamily R, polypeptide 1 (CYP2R1) and 7-dehydrocholesterol reductase/nicotinamide-adenine dinucleotide synthetase 1 (DHCR7/NADSYN1) and serum levels of the active form of Vitamin D, 25 (OH) D among African Americans, Caucasians, and Chinese. Little is known about how genetic variations associate with, or contribute to, 25(OH)D levels in Arabs populations.

Methods

Allele frequencies of 18 SNPs derived from CYP2R1, GC, and DHCR7/NADSYN1 genes in 1549 individuals (Arabs, South Asians, and Southeast Asians living in Kuwait) were determined using real time genotyping assays. Serum levels of 25(OH)D were measured using chemiluminescence immunoassay.

Results

GC gene polymorphisms (rs17467825, rs3755967, rs2282679, rs7041 and rs2298850) were found to be associated with 25(OH)D serum levels in Arabs and South Asians. Two of the CYP2R1 SNPs (rs10500804 and rs12794714) and one of GC SNPs (rs1155563) were found to be significantly associated with 25(OH)D serum levels only in people of Arab origin. Across all three ethnicities none of the SNPs of DHCR7/NADSYN1 were associated with serum 25(OH)D levels and none of the 18 SNPs were significantly associated with serum 25(OH)D levels in people from South East Asia.

Conclusion

Our data show for the first time significant association between the GC (rs2282679 and rs7041), CYP2R1 (rs10741657) SNPs and 25(OH)D levels. This supports their roles in vitamin D Insufficiency in Arab and South Asian populations respectively. Interestingly, two of the CYP2R1 SNPs (rs10500804 and rs12794714) and one GC SNP (rs1155563) were found to correlate with vitamin D in Arab population exclusively signifying their importance in this population.     

A novel antibody-dependent cellular cytotoxicity epitope in gp120 is identified by two monoclonal antibodies isolated from a long-term survivor of human immunodeficiency virus type 1 infection.

Two monoclonal antibodies (MAbs), 42F and 43F, were isolated some 14 months apart from a single long-term survivor of human immunodeficiency virus type 1 (HIV-1) infection. These MAbs were found to be indistinguishable in terms of their isotypes, specificities, affinities, and biological activities. Both 42F and 43F directed substantial antibody-dependent cellular cytotoxicity (ADCC) against cells infected with four divergent lab-adapted strains of HIV-1, but no neutralizing activity against these strains was detectable. The ability of MAbs 42F and 43F, as well as that of MAbs against two other gp120 epitopes, to direct ADCC against uninfected CD4+ cells to which recombinant gp120SF2 had been adsorbed (i.e., "innocent bystanders") was demonstrated to be less efficient by at least an order of magnitude than their ability to direct ADCC against HIV-1-infected cells. Flow cytometry analyses showed that 42F and 43F also bind to native primary isolate Envs from clades B and E expressed on cell surfaces. By direct binding and competition assays, it was demonstrated that the 42F/43F epitope lies in a domain of gp120 outside the previously described CD4-binding site and V3 loop ADCC epitope clusters. Immunoblot analysis revealed that the 42F/43F epitope is not dependent on disulfide bonds or N-linked glycans in gp120. Epitope mapping of 42F and 43F by binding to linear peptides demonstrated specificity of these MAbs for a sequence of 10 amino acids in the C5 domain comprising residues 491 to 500 (Los Alamos National Laboratory numbering for the HXB2 strain). Thus, 42F and 43F define a new ADCC epitope in gp120. Because of the relative conservation of this epitope and the fact that it appears to have been significantly immunogenic in the individual from which these MAbs were derived, it may prove to be a useful component of HIV vaccines. Furthermore, these MAbs may be used as tools to probe the potential importance of ADCC as an antiviral activity in HIV-1 infection.     

Sequence and analysis of a whole genome from Kuwaiti population subgroup of Persian ancestry

Background

The 1000 Genome project paved the way for sequencing diverse human populations. New genome projects are being established to sequence underrepresented populations helping in understanding human genetic diversity. The Kuwait Genome Project an initiative to sequence individual genomes from the three subgroups of Kuwaiti population namely, Saudi Arabian tribe; “tent-dwelling” Bedouin; and Persian, attributing their ancestry to different regions in Arabian Peninsula and to modern-day Iran (West Asia). These subgroups were in line with settlement history and are confirmed by genetic studies. In this work, we report whole genome sequence of a Kuwaiti native from Persian subgroup at >37X coverage.

Results

We document 3,573,824 SNPs, 404,090 insertions/deletions, and 11,138 structural variations. Out of the reported SNPs and indels, 85,939 are novel. We identify 295 ‘loss-of-function’ and 2,314 ’deleterious’ coding variants, some of which carry homozygous genotypes in the sequenced genome; the associated phenotypes include pharmacogenomic traits such as greater triglyceride lowering ability with fenofibrate treatment, and requirement of high warfarin dosage to elicit anticoagulation response. 6,328 non-coding SNPs associate with 811 phenotype traits: in congruence with medical history of the participant for Type 2 diabetes and β-Thalassemia, and of participant’s family for migraine, 72 (of 159 known) Type 2 diabetes, 3 (of 4) β-Thalassemia, and 76 (of 169) migraine variants are seen in the genome. Intergenome comparisons based on shared disease-causing variants, positions the sequenced genome between Asian and European genomes in congruence with geographical location of the region. On comparison, bead arrays perform better than sequencing platforms in correctly calling genotypes in low-coverage sequenced genome regions however in the event of novel SNP or indel near genotype calling position can lead to false calls using bead arrays.

Conclusions

We report, for the first time, reference genome resource for the population of Persian ancestry. The resource provides a starting point for designing large-scale genetic studies in Peninsula including Kuwait, and Persian population. Such efforts on populations under-represented in global genome variation surveys help augment current knowledge on human genome diversity.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1233-x) contains supplementary material, which is available to authorized users.