Chemokine CCR5 Research Articles

Proteomic and Transcriptomic Profiling Reveals Insights into Mechanisms of Leukemic Immune Escape

Despite the high rates of complete remissions achievable by modern frontline therapies, relapse of acute lymphoblastic leukemia (ALL) remains common and is associated with poor long-term survival. Relapse occurs due to the outgrowth of cells from the residual leukemia niche. These are able to evade populations of leukemia-specific memory/effector CD8+ and CD4+ T-cells that are robustly detectable in most patients. This implies a need for better understanding of basic mechanisms underlying relapse and leukemic immune evasion. The presence of phenotypically exhausted PD1+/- TIM3+ CD4+ T-cells in diagnostic bone marrow biopsy samples has been associated with inferior clinical outcomes in clinical studies of ALL patients. This suggests that CD4+ T-cell dysfunction facilitates immune escape by residual leukemic cells. To examine this, we used single-cell RNAseq/CITESEq to study CD4+ T-cells in diagnostic bone marrow biopsy samples and a murine model of BCR-ABL-rearranged ALL. In both settings, we discovered that FOXP3-PD1+ TIM3+ CD4+ T-cells are primarily composed of a unique population capable of helper and cytotoxic functions ("T-helper cytotoxic" or THCTX cells). THCTX cells expressed the effector cytokines TNF and IFNγ, but also the suppressive cytokine IL-10. In advanced leukemia, THCTX cells maintained IFNγ expression but lost the capacity for TNF synthesis, suggestive of a unique state of exhaustion. Administration of an anti-PDL1 antibody in combination with the BCR-ABL inhibitor nilotinib markedly improved long-term survival from 10% to 70% in preclinical models. Treatment benefit from dual therapy was dependent on the presence of CD4+ T-cells and was associated with the clonal expansion of leukemia-specific THCTX cells. Expanded THCTX clones had relatively preserved expression of the helper molecule CD40L as well as of ligands for the CCR5 chemokine receptor, which plays a key role in recruitment of memory/effector T-cells. Congruent with these observations, anti-PDL1 checkpoint blockade was also associated with higher frequencies of Granzyme+ CD8+ T-cells. T-cell exhaustion occurs due to chronic TCR stimulation occurring in the presence of concerted microenvironmental cues from surrounding cell types. To understand how the topography of the leukemia niche contributes to the observed exhaustion of CD4+ T-cells, we performed spatial proteomic profiling of mouse splenic tissue during leukemia development. Leukemic cells express GFP and were recognized with an anti-GFP antibody. Early leukemic infiltration led to pronounced disruption of the immune structures of the spleen and the adoption of novel cell neighborhoods (Figure 1). Infiltration was marked by loss of boundaries between the red and white pulp, and dissolution of the marginal and mantle zones. CD4+ and CD8+ T-cells were diminished in frequency, infrequently observed in proximity to leukemic cells, and expressed exhaustion markers, suggestive of an immune desert phenotype. Overall, these findings support a model in which ALL progression occurs due to the accrued dysfunction of CD4+ THCTX cells marked by a loss of helper and chemotactic functions. This is coincident with profound disruption and reorganization of the lymphoid environment into T-cell poor zones. These findings provide mechanistic insights into the high relapse risk observed among patients with CD4+ T-cell phenotypic exhaustion. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effect of azithromycin on migration of peripheral blood NK cells from patients with chronic obstructive pulmonary disease

Currently, no drugs have been identified that could slow progression of chronic obstructive pulmonary disease (COPD), or have a significant impact on patient mortality. Therefore, research continues aimed at studying the mechanisms of COPD development and searching for drugs that affect its molecular pathogenesis. The aim of our work was to determine the ability of azithromycin combined with corticosteroids to affect the migration of peripheral blood NK cells from the COPD patients. In the present study, we have measured expression of chemokine receptors CCR5, CCR6, CCR7, CXCR3, CXCR4, CXCR6 on the surface of peripheral blood NK cells (CD3- CD56+) by means of flow cytometry in 54 smoking patients with COPD, 21 healthy smokers, and 20 healthy non-smokers. Moreover, the effect of azithromycin (10 µg/mL) and budesonide (10 nM) on the migration of NK cells from COPD patients (n = 8) towards CCL5 (10 nM) and CXCL10 (10 nM) was determined. We found that the percentage of NK cells expressing CXCR3 and CCR5 chemokine receptors was increased in smoking patients with COPD compared with healthy smokers and healthy non-smokers. However, the proportion of these NK cell subsets did not differ between healthy smokers and healthy non-smokers. There were no significant differences in the percentage of NK cells expressing CXCR4, CXCR6, CCR6, CCR7 chemokine receptors between the three groups of subjects. Addition of budesonide to the cell suspensions decreased the migration of blood NK cells towards CCL5 and CXCL10. Azithromycin was also shown to suppress the migration of blood NK cells towards these chemokines. The combination of azithromycin and budesonide was more potent at inhibiting NK cell chemotaxis towards CCL5 and CXCL10 than any of these drugs added alone. Our results demonstrate a change in the chemokine receptor profile of NK cells in COPD patients and indicate the advantages of the combined use of corticosteroids and azithromycin for COPD treatment.

The primary macrophage chemokine, CCL2, is not necessary after a peripheral nerve injury for macrophage recruitment and activation or for conditioning lesion enhanced peripheral regeneration

BackgroundPeripheral nerve injuries stimulate the regenerative capacity of injured neurons through a neuroimmune phenomenon termed the conditioning lesion (CL) response. This response depends on macrophage accumulation in affected dorsal root ganglia (DRGs) and peripheral nerves. The macrophage chemokine CCL2 is upregulated after injury and is allegedly required for stimulating macrophage recruitment and pro-regenerative signaling through its receptor, CCR2. In these tissues, CCL2 is putatively produced by neurons in the DRG and Schwann cells in the distal nerve.MethodsCcl2fl/fl mice were crossed with Advillin-Cre, P0-Cre, or both to create conditional Ccl2 knockouts (CKOs) in sensory neurons, Schwann cells, or both to hypothetically remove CCL2 and macrophages from DRGs, nerves or both. CCL2 was localized using Ccl2–RFPfl/fl mice. CCL2–CCR2 signaling was further examined using global Ccl2 KOs and Ccr2gfp knock-in/knock-outs. Unilateral sciatic nerve transection was used as the injury model, and at various timepoints, chemokine expression, macrophage accumulation and function, and in vivo regeneration were examined using qPCR, immunohistochemistry, and luxol fast blue staining.ResultsSurprisingly, in all CKOs, DRG Ccl2 gene expression was decreased, while nerve Ccl2 was not. CCL2–RFP reporter mice revealed CCL2 expression in several cell types beyond the expected neurons and Schwann cells. Furthermore, macrophage accumulation, myelin clearance, and in vivo regeneration were unaffected in all CKOs, suggesting CCL2 may not be necessary for the CL response. Indeed, Ccl2 global knockout mice showed normal macrophage accumulation, myelin clearance, and in vivo regeneration, indicating these responses do not require CCL2. CCR2 ligands, Ccl7 and Ccl12, were upregulated after nerve injury and perhaps could compensate for the absence of Ccl2. Finally, Ccr2gfp knock-in/knock-out animals were used to differentiate resident and recruited macrophages in the injured tissues. Ccr2gfp/gfp KOs showed a 50% decrease in macrophages in the distal nerve compared to controls with a relative increase in resident macrophages. In the DRG there was a small but insignificant decrease in macrophages.ConclusionsCCL2 is not necessary for macrophage accumulation, myelin clearance, and axon regeneration in the peripheral nervous system. Without CCL2, other CCR2 chemokines, resident macrophage proliferation, and CCR2-independent monocyte recruitment can compensate and allow for normal macrophage accumulation.

CCL2, CCR2 Gene Variants and CCL2, CCR2 Serum Levels Association with Age-Related Macular Degeneration.

Background: Age-related macular degeneration (AMD) is the most common cause of progressive and irreversible blindness in developed countries. Although the pathogenesis is not fully understood, AMD is a multifactorial pathology with an accumulation of inflammatory components and macrophages and a strong genetic predisposition. Our purpose was to investigate the association between early AMD and CCL2 (rs1024611, rs4586, rs2857656) and CCR2 (rs1799865) single nucleotide polymorphisms (SNPs) and CCL2, CCR2 serum levels in a Lithuanian population. Methods: The study included 310 patients with early AMD and 384 healthy subjects. Genotyping of CCL2 rs1024611, rs4586, rs2857656, and CCR2 rs1799865 was performed using a real-time polymerase chain reaction method, while CCL2 and CCR2 chemokines serum concentrations were analyzed using an enzyme-linked immunosorbent assay. Results: We found that the G allele at CCL2 rs1024611 was more prevalent in the early AMD group than in controls (29.2% vs. 24.1%, p = 0.032). Similarly, the C allele in CCL2 rs2857656 is more common in the early AMD group than in controls (29.2% vs. 24.2%, p = 0.037). Binomial logistic regression revealed that each G allele in rs1024611 was associated with 1.3-fold increased odds of developing early AMD under the additive model (OR = 1.322; 95% CI: 1.032–1.697, p = 0.027) as was each C allele in rs2857656 under the additive model (OR = 1.314; 95% CI: 1.025–1.684, p = 0.031). Haplotype analysis revealed that the C-A-G haplotype of CCL2 SNPs was associated with 35% decreased odds of early AMD development. Further analysis showed elevated CCL2 serum levels in the group with early AMD compared to controls (median (IQR): 1181.6 (522.6) pg/mL vs. 879.9 (494.4) pg/mL, p = 0.013); however, there were no differences between CCR2 serum levels within groups. Conclusions: We found the associations between minor alleles at CCL2 rs1024611 and rs2857656, elevated CCL2 serum levels, and early AMD development.

CCR5 activation and endocytosis in circulating tumor-derived cells isolated from the blood of breast cancer patients provide information about clinical outcome

BackgroundCCR5 is a motility chemokine receptor implicated in tumor progression, whose activation and subsequent endocytosis may identify highly aggressive breast cancer cell subtypes likely to spread into the circulatory system.MethodsThe MDA-MB-231 cell line was used to model and visualize CCR5 activation by stimulation with RANTES, in an effort to quantify CCR5 endocytosis from the cell surface to the perinuclear space. CCR5 expression was then examined in tumor-associated cells (TACs), consisting of circulating tumor cells and circulating stromal cells, isolated from the peripheral blood of 54 metastatic breast cancer (mBC) patients to evaluate these CCR5 pooling patterns as they relate to progression and survival over 2 years.ResultsIn MB231 experiments, it was observed that CCR5 formed ~ 1 micron clusters identified as “CCR5 pools” on the surface of the cell, which in the presence of RANTES were endocytosed and translocated to the cell cytoplasm. When TACs from patients were analyzed, CCR5 pools were observed on the cell surface and translocating to the nuclear area, with CCR5 also having a positive statistical correlation between increased numbers of TACs and increased CCR5 pools on the cells. Further, it was determined that patients with very high numbers of CCR5 (> 10 CCR5 pools), specifically in the circulating stromal cells, were associated with worse progression-free survival (hazard ratio = 4.5, p = 0.002) and worse overall survival (hazard ratio = 3.7, p = 0.014).ConclusionsUsing a liquid biopsy approach, we evaluated two populations of tumor-associated cells emanating from primary tumors, with data suggesting that upregulation of the motility chemokine CCR5 in TACs provides clinically relevant opportunities for treating and tracking drug targetable receptors in mBC.

CCR2 identifies two subtypes of pathogenic human type 17 Th cells and has a non-redundant role in their transendothelial migration

Abstract We have shown previously that CCR2 is expressed on highly differentiated human Th memory cells as well as mucosal associated invariant T (MAIT) cells. CCR2+ T cells typically co-express a combination of other inflammation-associated chemokine receptors, including CCR6, which is found on all type 17 T cells. We found that the CCR6+ CCR2+ human Th cell population is enriched in cells with a pathogenic cytokine and cytotoxic profile. Single-cell analysis revealed two subtypes of such CCR6+ CCR2+ cells, either biased to produce GM-CSF or IFNg. Pathogenic activity requires migration into inflamed tissue, and to understand the roles for CCR2, CCR6 and other chemokine receptors expressed on these cells in extravasation we used flow chambers containing HUVEC activated with TNFa and/or IFNg. We found that although CCR6, CCR5 and CXCR3 (but not CCR2) were capable of mediating T cell arrest, only CCR2 was active in transendothelial migration. CCR2’s activity was a function of the localization of its principal ligand, CCL2, in endothelial cells. Native CCL2 was found intracellularly but not on the cell surface. Immediate pre-treatment of cells with CCL2 or using HUVEC transduced to express a CCL2-CXCL9 chimeric chemokine that bound to surface glycosaminoglycans enabled CCR2 to mediate T cell arrest. These studies reveal the integration of transendothelial trafficking and a pathogenic effector profile in type 17 human Th cells and demonstrate redundant and non-redundant roles for the multiple chemokine receptors on these cells due to chemokine positioning. Our findings could inform more effective combinatorial inhibition or expression of chemokine receptors/ligands to regulate the migration of highly inflammatory T cells. Supported by NIH

Investigating the Frequency of Delta 32 (∆32) Mutation Related to CCR5 Chemokine Receptor in Patients at Gonabad Health Centers, Iran

Aims Chemokine receptor type 5 is recognized as the main co-receptor in HIV infection. Many studies have shown that the deletion of 32-base pairs (bp) of both alleles of CCR5 leads to the reduction or elimination of the expression of this gene in immune cells and the natural resistance to HIV infection. This study aims to determine the frequency of delta 32 mutations in healthy people, referring to the health center of Gonabad City, Iran, to focus on finding new treatment methods for the treatment of HIV. Methods & Materials In this descriptive cross-sectional study, after obtaining ethical approval and informed consent, blood samples were collected from 293 healthy people along with ethylenediaminetetraacetic acid anticoagulant. Genomic DNA extraction was carried out by the kit, and the investigation of delta 32 (∆32) mutation of the CCR5 gene was investigated via the polymerase chain reaction method. Findings Our results showed that out of 293 people who referred to the health center, 269 people (91.8%) had a healthy gene without mutation (188 bp), 15 people (5.1%) had heterozygous mutations (188 bp and 156 bp), and 9 people (3.07%) had a homozygous mutation on both alleles of CCR5 (156 bp). Conclusion Based on the results, the homozygous mutation rate of Gonabad Region, Iran, in the studied community is higher compared to similar studies, and more epidemiological and molecular studies are needed for better conclusions.

CCR1 antagonist ameliorates experimental autoimmune encephalomyelitis by inhibition of Th9/Th22-related markers in the brain and periphery

Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system. The disease manifestation is associated with the proliferation and activation of lymphocytes and astrocytes, leading to demyelination and neuronal damage. Most of the current therapies are not completely effective, and few target the underlying pathophysiology of MS. T helper 9 (Th9)- and Th22-dominant cells have been proven to play a pathogenic role in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The goal of the present study was to investigate the therapeutic efficacy of J-113863, a novel CCR1 chemokine receptor, on PLP139–151-induced EAE in SJL/J mice. Following induction of EAE, mice were treated with J-113863 (10 mg/kg) or saline intraperitoneally daily from day 14 until day 25, and the clinical score was evaluated. We further investigated the effect of J-113863 on IL-9, IRF4, IL-22, IFN-γ, STAT3, AhR, and IL-17A in CD3+, CD4+, CCR6+, and CCR8+ spleen cells using flow cytometry. We also analyzed the effect of J-113863 on IL-9, IRF4, IL-22, IFN-γ, STAT3, AhR, and IL-17A mRNA expression levels. Our results revealed that J-113863 treatment notably attenuated the severity of clinical scores in EAE mice. J-113863 treatment decreased the percentage expression of CD4+IL-9+, CCR8+IL-9+, CD4+IRF4+, CD3+IL-22+, CCR6+IL-22+, CD3+IFN-γ+, CCR6+IFN-γ+, CD3+STAT3+, CCR6+STAT3+, CD4+IL-17A+, and CCR6+IL-17A+, and increased the percentage of CD3+AhR+, and CCR6+AhR+ cells in the spleen. These results confirmed that J-113863 suppressed Th9/Th22 cells to reduce demyelination in EAE mice, suggesting its potential role as a novel drug candidate for MS treatment.

Mechanosensitive ACKR4 scavenges CCR7 chemokines to facilitate Tcell de-adhesion and passive transport by flow in inflamed afferent lymphatics.

T cell migration via afferent lymphatics to draining lymph nodes (dLNs) depends on expression of CCR7 in Tcells and CCL21 in the lymphatic vasculature. Once Tcells have entered lymphatic capillaries, they slowly migrate into contracting collecting vessels. Here, lymph flow picks up, inducing Tcell detachment and rapid transport to the dLNs. We find that the atypical chemokine receptor 4 (ACKR4), which binds and internalizes CCL19 and CCL21, is induced by lymph flow in endothelial cells lining lymphatic collectors, enabling them to scavenge these chemokines. In the absence of ACKR4, migration of Tcells to dLNs in TPA-induced inflammation is significantly reduced. While entry into capillaries is not impaired, Tcells accumulate in the ACKR4-deficient dermal collecting vessel segments. Overall, our findings identify an ACKR4-mediated mechanism by which lymphatic collectors facilitate the detachment of lymph-borne Tcells in inflammation and their transition from crawling to free-flow toward the dLNs.

CCR2 monocytes repair cerebrovascular damage caused by chronic social defeat stress

Immune surveillance of the brain plays an important role in health and disease. Peripheral leukocytes patrol blood–brain barrier interfaces, and after injury, monocytes cross the cerebrovasculature and follow a pattern of pro- and anti-inflammatory activity leading to tissue repair. We have shown that chronic social defeat (CSD) causes scattered vasculature disruptions. Here, we assessed CCR2+ monocyte trafficking to the vascular injury sites in Ccr2wt/rfp reporter mice both during CSD and one week following CSD cessation. We found that CSD for 14 days induced microhemorrhages where plasma fibrinogen leaked into perivascular spaces, but it did not affect the distribution or density of CCR2rfp+ monocytes in the brain. However, after recovery from CSD, many vascularly adhered CCR2+ cells were detected, and gene expression of the CCR2 chemokine receptor ligands CCL7 and CCL12, but not CCL2, was elevated in endothelial cells. Adhered CCR2+ cells were mostly the non-classical, anti-inflammatory Ly6Clo type, and they phagocytosed fibrinogen in perivascular spaces. In CCR2-deficient Ccr2rfp/rfp mice, fibrinogen levels remained elevated in recovery. Fibrinogen infused intracerebroventricularly induced CCR2+ cells to adhere to the vasculature and phagocytose perivascular fibrinogen in Ccr2wt/rfp but not Ccr2rfp/rfp mice. Depletion of monocytes with clodronate liposomes during CSD recovery prevented fibrinogen clearance and blocked behavioral recovery. We hypothesize that peripheral CCR2+ monocytes are not elevated in the brain on day 14 at the end of CSD and do not contribute to its behavioral effects at that time, but in recovery following cessation of stress, they enter the brain and exert restorative functions mediating vascular repair and normalization of behavior.

Preparation of a stable CCL5·CCR5·Gi signaling complex for Cryo-EM analysis.

The numerous chemokines and their cognate G protein-coupled chemokine receptors on the surface of leukocytes form a complex signaling network, which regulates the immune response and also other key physiological processes. Currently only a very limited number of structures of chemokine•chemokine receptor complexes have been solved. More structures are needed for the understanding of their mechanism of action and the rational design of drugs against these highly relevant therapeutic targets. Recently, we have determined the cryo-EM structure of the human wild-type CCR5 chemokine receptor, which is also the HIV-1 coreceptor, in its active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist CCR5 chemokine ligands. In this chapter, we present a detailed protocol for the preparation of the active agonist chemokine•CCR5•Gi complex for cryo-EM studies including quality controls and caveats. As such the protocol may serve as starting point for structural and biophysical studies of other chemokine•chemokine receptor complexes.

ENANTIOSELECTIVE SYNTHESIS OF (S)-(-)-1-(4-FLUOROPHENYL)ETHANOL

S)-(–)-1-(4-Fluorophenyl)ethanolis an intermediate in the synthesis of an antagonist of the CCR5 chemokine receptor that can be protective against HIV infection. Along with o-fluoro-substituted analogs, (S)-(–)-1-(4-fluorophenyl)ethanol can be used to study the role of substituents in the chiral recognition of molecular complexes, and (R)-(+)-1-(4-fluorophenyl)ethanol is a com-ponent of an antimalarial drug and a γ-secretase modulator necessary for the treatment of Alzhei-mer's disease.We proposed an approach to the synthesis of (S)-(-)-1-(4-fluorophenyl)ethanol based on the enantioselective reduction of prochiral 1-(4-fluorophenyl)ethanone catalyzed by Daucus carota cells. We found the conditions that make it possible to obtain (S)-(–)-1-(4-fluorophenyl)eth-anol by bioreduction of 1-(4-fluorophenyl)ethanone in the presence of a D. carota biocatalyst for 48 h with 55% yield (98% ee).We also studied the possibility of optimizing the transformation of 1-(4-fluorophenyl)ethanone in a reaction medium containing various exogenous reducing agents(ethanol, isopropanol, or glucose), which are known to significantly affect the product yield and the enantioselectivity of the process. Biotransformation of 1-(4-fluorophenyl)ethanone in the pres-ence of ethanol (1-5%) gives (S)-(-)-1-(4-fluorophenyl)ethanol with 7-85% yields and optical purity 10-98% ee.Enantioselective reduction of the initial ketone in the presence of isopropanol (1-5%) does not lead to a significant increase in the yield and optical purity of S-alcohol (7-57% (18-98% ee).In the case of biotransformation of 1-(4-fluorophenyl)ethanone in the presence of an equimo-lar amount of glucosefor 48 hthe yield of (S)-(-)-1-(4-fluorophenyl)ethanol is 66% (98% ee).Fur-ther transformation of the initial ketone (144 h) both in the absence of an exogenous reducing agent and in the presence of an equimolar amount of glucose leads to an increase in the yield of alcohol to 73% and 76%, respectively, however, the optical purity of the alcohol decreases to 62% ee and 80% ee, which suggests the stereoinversion.

Decreased MIP-3α Production from Antigen-Activated PBMCs in Symptomatic HIV-Infected Subjects.

CD4+ CCR6+ T cells are highly susceptible to HIV infection, and a high cytokine producing CCR6+ T cell subset is selectively lost during HIV infection. The CCR6 chemokine MIP-3α (CCL20) is produced at sites of infection in SIV animal models. Recently, we have shown that MIP-3α inhibits HIV replication. This inhibition of HIV infection is mediated by CCR6 signaling and eventuates in increased APOBEC3G expression. Since there are few existing reports on the role of MIP-3α in health or disease, we studied its production by PBMCs from HIV-seronegative and HIV+ subjects. We evaluated the ability of PBMCs to produce MIP-3α in response to antigen stimulation using cells obtained from two groups: one composed of HIV-seronegative subjects (n = 16) and the other composed of HIV+ subjects (n = 58), some asymptomatic and some with clinically defined AIDS. Antigens included fragment C of the tetanus toxin, Candida albicans, whole-inactivated HIV, and HIV p24. MIP-3α was detected by ELISA in tissue culture supernatants of antigen-stimulated PBMCs. MIP-3α production by antigen-stimulated PBMCs was readily measured for HIV-negative subjects and for HIV-seropositive asymptomatic subjects, but not for patients with AIDS. These results suggest that subversion of the MIP-3α-CCR6 axis by HIV during the course of infection contributes to the loss of immune function that eventually leads to AIDS.

Tre-P-08 - Granulomatous rash associated with mogamulizumab mimicking mycosis fungoides: a case series

Mogamulizumab, a humanized monoclonal antibody targeting the CCR4 chemokine receptor, has recently been approved for the treatment of cutaneous epidermotropic T lymphomas. Rash has been reported as one of the side effects. We report six original cases of granulomatous mogamulizumab-associated rash on photo exposed areas clinically mimicking disease progression, which poses a challenge for clinicians.

Biocomputational Screening and Pharmacological Studies of Gymnema sylvester in Contrast to Human CCR5 and CXCR4 Chemokine Receptors

In last twenty years DNA, profiling has become an obligatory technology, which has brought scientists towards forensic identification. Thousands of STR markers are present in human genome but only core set of loci are selected for forensic DNA and human identification. STRs have become famous in forensic labs because even low amount and degraded form can be easily typed. STRs are found in prokaryotes and eukaryotes, including humans. They appear scattered almost evenly throughout the human genome, resulting in about 3% of the genome. However, their distribution in chromosomes is not quite uniform. Usually, STRs occur in the noncoding regions, only about 8% are located in the coding regions. In humans, chromosome 19 has the highest density of STRs. On an average, one STR occurs per 2,000 bp in the human genome. On the basis of repeat units, STRs can be differentiated into different types. The repeats of STR markers are highly variable in human population. STR marker evaluation very precisely figures out individual humans at the molecular level even from very small quantities. STR locus in the identification selected is D7S820 from NIST (National Institute of Standards and Technology). Conditions for STR with the changes in temperature, magnesium ion concentration, primer and setting up, PCR of the marker used to carry out. PCR product is inspected of agarose gel. The results showed that the STR locus being investigated is detectable by PCR. PCR showed that the detection of primer and temperature conditions measured by using the fixed amount of magnesium, D7S820 locus bands are weaker than expected. Using a buffer and setting magnesium condition towards changes in primer and temperature, addition of Taq polymerase at a temperature of 94°C, bands become visible desirably.

Early Effects of HTLV-1 Infection on the Activation, Exhaustion, and Differentiation of T-Cells in Humanized NSG Mice.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy of CD4+ T-cells associated with HTLV-1 infection. In this study, we used the model of immunodeficient NSG mice reconstituted with a functional human immune system (HIS) to investigate early events in HTLV-1 pathogenesis. Upon infection, human T-cells rapidly increased in the blood and lymphoid tissues, particularly CD4+CD25+ T-cells. Proliferation of CD4+ T-cells in the spleen and mesenteric lymph nodes (MLN) correlated with HTLV-1 proviral load and CD25 expression. In addition, splenomegaly, a common feature of ATLL in humans, was also observed. CD4+ and CD8+ T-cells predominantly displayed an effector memory phenotype (CD45RA−CCR7−) and expressed CXCR3 and CCR5 chemokine receptors, suggesting the polarization into a Th1 phenotype. Activated CD8+ T-cells expressed granzyme B and perforin; however, the interferon-γ response by these cells was limited, possibly due to elevated PD-1 expression and increased frequency of CD4+FoxP3+ regulatory T-cells in MLN. Thus, HTLV-1-infected HIS-NSG mice reproduced several characteristics of infection in humans, and it may be helpful to investigate ATLL-related events and to perform preclinical studies. Moreover, aspects of chronic infection were already present at early stages in this experimental model. Collectively, we suggest that HTLV-1 infection modulates host immune responses to favor viral persistence.

Biocomputational Screening and Pharmacological Studies of Gymnema sylvester in Contrast to Human CCR5 and CXCR4 Chemokine Receptors

Viral diseases have remained a major global threat regardless of significant advances in therapeutic and treatment strategies. The advancement of viral resistance to drugs, along with several side effects, has resulted in critical medical issues, especially when administered in combination for extended treatment periods. Natural product-based therapies for the health of humans are associated with lower toxicity and fewer side effects. Virtual in-silico screening has been shown to be effective in addressing the unique challenges of antiviral drug development, making the process quicker, safer and less expensive. Novel ligands are evolving as potential agents, which are efficient in blocking virus–cell fusion to surpass the main disadvantages of traditional Highly Active Antiretroviral (HAART) drugs used in HIV-1 infection. The plant Gymnema sylvestre contains various bioactive compounds with medicinal properties. The HIV entry process can be blocked during the binding activity of glycoprotein gp120 viral envelope to one of the two receptors CXCR4 or CCR5 and binding to both the CD4 receptors. The significance of chemokine receptors as an anti- HIV target depends on the fact that they are involved in the entry of the virus into the cell at the early stages. Thus, the HIV/coreceptor relationship has emerged as a key focus for developing new antiviral strategies for the prevention and also treatment of HIV infection. In the present study, the phytochemicals selected from Gymnema sylvestre were subjected to ADME analysis. This was followed by the molecular docking analysis and MD simulation to estimate the selected ligands drug likeness, which may serve as new leads for the cure of HIV-1 infections.

Amino terminal recognition by a CCR6 chemokine receptor antibody blocks CCL20 signaling and IL-17 expression via \u03b2-arrestin

BackgroundCCR6 chemokine receptor is an important target in inflammatory diseases. Th17 cells express CCR6 and a number of inflammatory cytokines, including IL-17 and IL-22, which are involved in the propagation of inflammatory immune responses. CCR6 antagonist would be a potential treatment for inflammatory diseases such as psoriasis or rheumatoid arthritis. The aim of this study is to develop an antagonistic monoclonal antibody (mAb) against human CCR6 receptor (hCCR6).ResultsWe generate monoclonal antibodies against hCCR6 immunizing Balb/c mice with hCCR6 overexpressing cells. The antibodies were tested by flow cytometry for specific binding to hCCR6, cloned by limiting dilution and resulted in the isolation and purification monoclonal antibody 1C6. By ELISA and flow cytometry, was determined that the antibody obtained binds to hCCR6 N-terminal domain. The ability of 1C6 to neutralize hCCR6 signaling was tested and we determined that 1C6 antibody were able to block response in β-arrestin recruitment assay with IC50 10.23 nM, but did not inhibit calcium mobilization. In addition, we found in a chemotaxis assay that 1C6 reduces the migration of hCCR6 cells to their ligand CCL20. Finally, we determined by RT-qPCR that the expression of IL-17A in Th17 cells treated with 1C6 was inhibited.ConclusionsIn the present study, we applied whole cell immunization for successfully obtain an antibody that is capable to neutralize hCCR6 signaling and to reduce hCCR6 cells migration and IL-17 expression. These results provide an efficient approach to obtain therapeutic potential antibodies in the treatment of CCR6-mediated inflammatory diseases.

Dual role of CCL3 and follicular regulatory T cells in the control of germinal center response

Abstract Follicular regulatory T cells (Tfrs) have been reported to play multiple roles in the control of B cells response. They repress foreign antigen-specific germinal center (GC) B cells at the peak of GC response. At the same time, under some conditions they promote GC B cell cycling in IL-10 dependent fashion and ensure optimal affinity maturation. Our previous studies suggested that CCL3 produced by GC centrocytes (CCs) promoted their direct contacts with Tfr cells and negative regulation of GC B cells at the peak of GC response. However, which fraction of CCs upregulates CCL3 expression, which receptors on Tfrs may respond to CCL3, and the long-term effect of CCL3 on GCs has been unclear. Based on the single cell and bulk qPCR analysis we found that CCL3 is upregulated in about 10% of CCs that express Myc and are undergoing positive selection. qPCR and transwell analysis revealed expression and synergistic involvement of CCR5 and CCR1 chemokine receptors on Tfr cells in their chemotaxis to CCL3. Interestingly, both an adoptive transfer and mixed bone marrow chimeras models suggest that after the peak of GC response B cell-intrinsic production of CCL3 promotes their prolonged participation in GCs and B cell affinity maturation, as well as better memory and plasmablast response. We also showed that both the negative and positive regulation of GC B cells by CCL3 was dependent on the presence of Tregs. To summarize, our studies suggest that in addition to the role of CCL3 and Tregs in the modest repression of foreign antigen-specific B cells at the peak of GC response, it is also important for optimal selection of B cells in GCs. Further studies are underway to determine the role of Tfr cells specifically in the negative and positive CCL3-mediated regulation of GCs.

Immune Trait Shifts in Association With Tobacco Smoking: A Study in Healthy Women.

Tobacco smoking is known to impact circulating levels of major immune cells populations, but its effect on specific immune cell subsets remains poorly understood. Here, using high-resolution data from 223 healthy women (25 current and 198 never smokers), we investigated the association between smoking status and 35,651 immune traits capturing immune cell subset frequencies. Our results confirmed that active tobacco smoking is associated with increased frequencies of circulating CD8+ T cells expressing the CD25 activation marker. Moreover, we identified novel associations between smoking status and relative abundances of CD8+ CD25+ memory T cells, CD8+ memory T cells expressing the CCR4 chemokine receptor, and CD4+CD8+ (double-positive) CD25+ T cells. We also observed, in current smokers, a decrease in the relative frequencies of CD4+ T cells expressing the CD38 activation marker and an increase in class-switched memory B cell isotypes IgA, IgG, and IgE. Finally, using data from 135 former female smokers, we showed that the relative frequencies of immune traits associated with active smoking are usually completely restored after smoking cessation, with the exception of subsets of CD8+ and CD8+ memory T cells, which persist partially altered. Our results are consistent with previous findings and provide further evidence on how tobacco smoking shapes leukocyte cell subsets proportion toward chronic inflammation.