A10 Presence and frequency of M184V mutation in the MOBIDIP trial

Abstract The MOBIDIP trial evaluated the simplification by protease (PI/r) monotherapy for HIV infection versus dual therapy and boosted protease inhibitor plus lamivudine (PI/r + 3TC) in controlled patients under second-line regimens. MOBIDIP was interrupted because of a significant number of patients with virological failure (VF) at week 48 (W48) in PI/r (33/133, ∼25%) versus in PI/r + 3TC (4/132, ∼3%). At the time of first-line VF, 96 per cent of patients harbored the M184V mutation. The presence of the M184V mutation was related to a protective effect against VF in the PI/r + 3TC arm. We developed a methodology that allows to determine the frequency of M184V/I mutations in the HIV reverse transcriptase (RT) gene in peripheral blood mononuclear cells (PBMC) obtained before MOBIDIP simplification. Paired-end sequences were obtained from 252 PBMC samples covering the first 855 bp of the RT gene (HXB2: 2485–3405) by MiSeq technology. These sequences were subjected to an in-house Bioinformatics pipeline. The results of our pipeline were compared to the output of PASeq (https://www.paseq.org), an open web-tool for the identification of drug resistance mutations. The M184V mutation was identified at a frequency greater than 1 per cent in 178 individuals (∼71%). The M184I mutation was observed in 34 patients (∼13%), always in the presence of stop codons, and is in agreement with expectations, as this mutation is a known APOBEC-targeted site. Sixty-seven patients (∼27%) had a frequency of the M184V mutation with values greater than 75 per cent. PASeq confirmed the presence of M184V mutation in 173 patients. The frequencies estimated by the PASeq tool and in-house pipeline were correlated up to 99.5 per cent. We found a significant loss of the M184V mutation archived in PBMC between the first-line regimen treatment failure and the beginning of the MOBIDIP trial. In patients under long-term antiretroviral therapy, as in our case, viral sub-populations could be lost, reducing the presence, and frequency of a mutation. In the next step, we will evaluate the association between the presence and frequency of M184V mutation and MOBIDIP results.

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Lamivudine Can Exert a Modest Antiviral Effect against Human Immunodeficiency Virus Type 1 Containing the M184V Mutation

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.2.747-754.2003 ◽

2003 ◽

Vol 47 (2) ◽

pp. 747-754 ◽

Cited By ~ 28

Author(s):

Yudong Quan ◽

Bluma G. Brenner ◽

Maureen Oliveira ◽

Mark A. Wainberg

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Mononuclear Cells ◽

Antiviral Effect ◽

Hiv Reverse Transcriptase ◽

Peripheral Blood Mononuclear ◽

Functional Changes ◽

Immunodeficiency Virus ◽

M184v Mutation ◽

Level Resistance

ABSTRACT The M184V mutation in human immunodeficiency virus (HIV) reverse transcriptase is associated with high-level resistance to both (−)2′,3′-dideoxy-3′-thiacytidine (3TC) and (−)2′,3′-dideoxy-5-fluoro-3′-thiacytidine as well as low-level resistance to 2′,3′-dideoxyinosine, 2′,3′-dideoxycytidine, and abacavir. This mutation is also associated with diminished HIV replicative fitness as well as several functional changes in enzyme activity, including diminutions in polymerase processivity, pyrophosphorylysis, and nucleotide primer unblocking. Despite the fact that M184V encodes up to 1,000-fold resistance to 3TC, we asked whether this drug might still display some antiviral effect in regard to viruses containing this mutation. Cell-free assays revealed that high concentrations of 3TC triphosphate (i.e., >100 μM) could affect chain termination and/or inhibit purified reverse transcriptase containing the M184V substitution. This effect became more pronounced with elongation of reverse transcriptase products. In newly infected cells (i.e., peripheral blood mononuclear cells), we found that the amount of full-length reverse transcriptase product was diminished in the presence of 2 to 10 μM 3TC, although no decrease in the first product of the reverse transcriptase reaction, i.e., minus strong-stop DNA, was observed. In the presence of two other HIV inhibitors, e.g., nevirapine and indinavir, 3TC exerted additive effects in tissue culture at concentrations only marginally higher than the 50% inhibitory concentration (IC50). Reverse transcriptases cloned from clinical isolates harboring M184V in the context of multidrug resistance had similar IC50 values for 3TC triphosphate compared to reverse transcriptase containing only the M184V mutation. These results suggest that viruses containing M184V can retain a higher degree of sensitivity to 3TC than previously assumed.

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Evidence for late stage compartmentalization of HIV-1 resistance mutations between lymph node and peripheral blood mononuclear cells

AIDS ◽

10.1097/00002030-200010200-00008 ◽

2000 ◽

Vol 14 (15) ◽

pp. 2273-2281 ◽

Cited By ~ 27

Author(s):

Da' N. Haddad ◽

Christopher Birch ◽

Tracy Middleton ◽

Dominic E. Dwyer ◽

Anthony L. Cunningham ◽

...

Keyword(s):

Lymph Node ◽

Peripheral Blood Mononuclear Cells ◽

Late Stage ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Resistance Mutations ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells ◽

Hiv 1

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Lenalidomide IS Able to Restore Immune SYSTEM IN MULTIPLE MYELOMA PATIENTS.

Blood ◽

10.1182/blood.v114.22.4909.4909 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4909-4909 ◽

Cited By ~ 3

Author(s):

Annalisa Chiarenza ◽

Nunziatina Parrinello ◽

Piera La Cava ◽

Eleonora Spina ◽

Daniele Tibullo ◽

...

Keyword(s):

Multiple Myeloma ◽

Immune System ◽

T Cell ◽

T Lymphocytes ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Gradient Centrifugation ◽

Target Cells ◽

First Line ◽

Number Of Patients

Abstract Abstract 4909 LENALIDOMIDE IS ABLE TO RESTORE IMMUNE SYSTEM IN MULTIPLE MYELOMA PATIENTS Annalisa Chiarenza, Nunziatina Parrinello, Piera La Cava, Eleonora Spina, Daniele Tibullo, Cesarina Giallongo, Maide Cavalli, Alessandra Romano, Paolo Fiumara, Giuseppe A. Palumbo, Francesco Di Raimondo Background Multiple myeloma (MM) is a malignant plasma-cell proliferative disorder associated with dysfunctional T-cell responses. The immunomodulatory Thal derivative (IMiD) CC-5013 (lenalidomide) appears to be a promising agent for the treatment of myeloma. Although the exact antitumor mechanism of action of lenalidomide is unknown, a number of mechanisms are postulated to be responsible for it's activity (inhibition of angiogenesis, direct antiproliferative and proapoptotic effects on MM cells, suppression of pro-inflammatory cytokines, modulation of myeloma-stromal cells adhesive interactions). In addition, it has been demonstrated that lenalidomide in vitro is able to enhance T cell proliferation and to promotes ADCC. In this study we evaluated if MM patients have a deficit of T-reg (CD4+, CD25+, and FOXP3+) and of T lymphocytes bearing CD200 (a tolerogenic molecule) and the effect of lenalidomide treatment on these parameters. In addition, we investigated whether lenalidomide could improve ex vivo the ADCC against myeloma cells. Materials and methods Eight patients with previously untreated MM (median age 56 years) were treated with lenalidomide plus dexamethasone as first line therapy. Lenalidomide was given orally 25 mg daily on days 1 to 21 of a 28-day cycle. Dexamethasone was given orally 40 mg daily on days 1, 8, 15, 22 of each cycle. All patients were evaluable for response and toxicity. Peripheral blood mononuclear cells (PBMNc) were obtained from MM patients using density gradient centrifugation (Fycoll) under sterile condictions, at the beginning of treatment and after 4 cycles of therapy. The percentage of T-reg (CD4+CD25+FOXP3+) and the expression of CD200 on T- lymphocytes were evaluated by cytometry. Twelve healthy subjects were used as control. Moreover, PBMNc (effector cells, E) were incubated with MM cells line ARH-77 (target cells, T), previously labelled with CFDA,SE (carboxyfluorescein diacetate, succinimidyl ester) as a tracing fluorescent marker, in culture medium (RPMI-1640, 10%FCS, 1%penicillin/streptomycin) at different concentration (T/E ratio 1:20, 1:40). After 18-24 h co-colture cells were analyzed by flow cytometry and MM plasma cells cytotoxicity was calculated as the percentage of positive CFDA,SE/propidium cells. Myeloma cell viability was determined by tripan blue esclusion and apoptosis was also evaluated using Annexin V/propidium assay. Two MM patients treated in first line with a combination of Velcade, Thalidomide and Dexamethasone (VTD) were used as control and the experiments were performed in duplicate. Results MM patients have a significantly lower rate of CD4+/CD25+/FOXP3+ and CD200+/CD3+ than normal (28,3±14,9/mmc and 37,8±24,7 /mmc vs 79,3±27,8 and 79,5± 48,9)(p=0,0001 and p=0,01 respectively). In our study, lenalidomide treatment resulted in an increase both of Treg cells and T-lymphocytes espressing CD200. This improvement is not statistically significant probably due to the low number of patients examined (tab I). More important, we observed that PBMC derived from patients treated with lenalidomide showed an increase ability to kill a target MM cell line compared to PBMC collected at diagnosis (CFDA,SE/propidium cells 11% vs 68%). This effect was more prominent in patients treated with lenalidomide than in MM patients treated with VTD (CFDA,SE/propidium cells 12% vs 39%), Fig.1. Conclusions Our data emphasize the role of lenalidomide in modulating the endogenous tumor-specific immune response and underline the anti-myeloma activity of these new class of drugs. Disclosures No relevant conflicts of interest to declare.

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Reiter’s syndrome - disease of young men: Analysis of 312 patients

Medicinski pregled ◽

10.2298/mpns1408222b ◽

2014 ◽

Vol 67 (7-8) ◽

pp. 222-230 ◽

Cited By ~ 13

Author(s):

Jaroslav Bojovic ◽

Natasa Strelic ◽

Ljiljana Pavlica

Keyword(s):

Synovial Fluid ◽

Human Leukocyte Antigen ◽

Reactive Arthritis ◽

Mononuclear Cells ◽

Clinical Manifestations ◽

Human Leukocyte ◽

Multisystem Disease ◽

Peripheral Blood Mononuclear ◽

Leukocyte Antigen ◽

Number Of Patients

Introduction. Reiter?s syndrome is reactive arthritis occurring after acute urogenital (urethritis, cervicitis) or enterocolitis infections. The associated ophthalmological and/or mucocutaneous changes are full clinical manifestations of this disease. This paper was aimed at analyzing clinical and radiological characteristics and findings of possible etiological factors and protocol for Reiter?s syndrome therapy. Material and Methods. Of 312 patients included in the study, 279 were men and 33 were women, the ratio between them being 8.5:1. The disease was diagnosed based on clinical evidence of two basic characteristics of Reiter?s syndrome: arthritis preceded by acute urogenital or enteral infection. Results. Urogenital and enterocolitic form of disease was found in 242 (77.5%) and 52 (16.5%) patients, respectively; whereas the initial cause was not discovered in 18 patients (6%). Three or two main signs of Reiter?s syndrome were present in approximately the same number of patients (41.7% and 44.2%), whereas all four signs of disease were present in 14.1% of the patients. Acute or sub-acute form was present in 40.5%, while recurrent and chronic disease was diagnosed in 31% and 28.5% of the patients, respectively. The most frequent clinical manifestation of this disease was on the locomotor system as asymmetrical oligoarthritis localized in lower extremities, present in 69.4% of the patients. Chlamydia trachomatis was found in the synovial fluid in 54% of patients (20/37), ureaplasma or mycoplasma was isolated in the synovial tissue of 73.1% of patients (30/41) and in the peripheral blood mononuclear cells in 93.2% of patients (41/44). Human leukocyte antigen B27 was present in 83.3% of patients. Conclusion. Reiter?s syndrome is a multisystem disease, predominantly occurring in human leukocyte antigen B27 positive young males. The fact that the causative agents are found in the synovial membrane or synovial fluid is indicative of infectious rather than reactive arthritis.

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Downregulation of CD45 Signaling in COVID-19 Patients Is Reversed by C24D, a Novel CD45 Targeting Peptide

Frontiers in Medicine ◽

10.3389/fmed.2021.675963 ◽

2021 ◽

Vol 8 ◽

Author(s):

Danny Alon ◽

Yossi Paitan ◽

Eyal Robinson ◽

Nirit Ganor ◽

Julia Lipovetsky ◽

...

Keyword(s):

Immune System ◽

Signaling Pathway ◽

Intracellular Signaling ◽

Mononuclear Cells ◽

Tyrosine Phosphatase ◽

Cell Receptor ◽

Peripheral Blood Mononuclear ◽

Intracellular Signals ◽

Number Of Patients ◽

Targeting Peptide

CD45, the predominant transmembrane tyrosine phosphatase in leukocytes, is required for the efficient induction of T cell receptor signaling and activation. We recently reported that the CD45-intracellular signals in peripheral blood mononuclear cells (PBMCs) of triple negative breast cancer (TNBC) patients are inhibited. We also reported that C24D, an immune modulating therapeutic peptide, binds to CD45 on immune-suppressed cells and resets the functionality of the immune system via the CD45 signaling pathway. Various studies have demonstrated that also viruses can interfere with the functions of CD45 and that patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are immune-suppressed. Given the similarity between the role of CD45 in viral immune suppression and our findings on TNBC, we hypothesized that the C24D peptide may have a similar “immune-resetting” effect on PBMCs from COVID-19 patients as it did on PBMCs from TNBC patients. We tested this hypothesis by comparing the CD45/TCR intracellular signaling in PBMCs from ten COVID-19 patients vs. PBMCs from ten healthy volunteers. Herein, we report our findings, demonstrating the immune reactivating effect of C24D via the phosphorylation of the tyrosine 505 and 394 in Lck, the tyrosine 493 in ZAP-70 and the tyrosine 172 in VAV-1 proteins in the CD45 signaling pathway. Despite the relatively small number of patients in this report, the results demonstrate that C24D rescued CD45 signaling. Given the central role played by CD45 in the immune system, we suggest CD45 as a potential therapeutic target.

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Increased Capacity Of Monocytes From Patients With Multiple Myeloma Treated With Thalidomide Or Lenalidomide To Generate Tissue Factor

Blood ◽

10.1182/blood.v122.21.3628.3628 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3628-3628

Author(s):

Abraham Kornberg ◽

Marina Izak ◽

Yossi Cohen

Keyword(s):

Multiple Myeloma ◽

Tissue Factor ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Line Treatment ◽

First Line ◽

Peripheral Blood Mononuclear ◽

Before And After

Abstract Treatment of multiple myeloma (MM) with thalidomide or lenalidomide is associated with increased incidence of thrombosis in contrast to treatment with bortezomib. The mechanism of thrombosis is unknown. Monocytes generate potent tissue factor (TF), the main activator of coagulation, in response to various stimuli (endotoxin, cytokins and others) and in diseases with increased incidence of thrombosis. We investigated the capacity of monocytes from patients with MM to generate TF in relation to different modalities of treatments and activation of coagulation. Peripheral blood mononuclear cells (PBMC) were isolated on Ficoll-Hypaque centrifugation and monocytes by adhesion. TF activity was assayed by modified PT using the cells as source of TF and TF antigen by ELISA, in endotoxin stimulated (10 ug/ml) and unstimulated PBMC ( all patients) and purified monocytes (50% of patients), before and after 3-4 courses of treatment. Monocytes were identified by anti CD14 and Plasma Fragment1.2 was assayed by ELISA. No TF activity@ (X10-5 U/monocyte) TF antigen@ (X10-5 pg/monocyte) Plasma fragment 1.2@ (nM) Endotoxin - + - + Bortezomib based treat* 12 before 2.2 4.8 22 64 0.28 after 1.8 4.2 18 57 0.36 Thalidomide based treat* 4 before 2.4 5.5 26 60 0.24 after 1.8 34.8# 20 440# 2.48# Lenalidomide based treat^ 6 before 2.1 6.2 28 62 0.30 after 4.1 58.6# 16 312# 3.42# @Mean *First line treatment ^Second line treatment # P<0.05 One patient on thalidomide and 2 on lenalidomide developed thrombosis. The results show that TF was low in unstimulated monocytes from all patients before treatment. It was increased mildely in stimulated monocytes from all patients before treatment and also after treatment with bortezomib (X 2-3). TF in stimulated monocytes and also plasma fragment1.2 were increased significantly after treatment with thalidomide and lenalidomide (x 14-22 and X 10). The phenomenon of enhanced capacity of monocytes to generate potent TF after treatment with thalidomide and lenalidomide but not bortezomib, and the correlation with activation of coagulation suggest a role of monocyte TF in thrombus formation by these drugs. The mechanism of the enhanced capacity is unknown and may be attributed to the immunomodulatory effect of the drugs. Disclosures: No relevant conflicts of interest to declare.

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Mutations in the HIV-1 envelope glycoprotein can broadly rescue blocks at multiple steps in the virus replication cycle

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1820333116 ◽

2019 ◽

Vol 116 (18) ◽

pp. 9040-9049 ◽

Cited By ~ 16

Author(s):

Rachel Van Duyne ◽

Lillian S. Kuo ◽

Phuong Pham ◽

Ken Fujii ◽

Eric O. Freed

Keyword(s):

Virus Replication ◽

Mononuclear Cells ◽

De Novo ◽

Virus Assembly ◽

Human Peripheral Blood ◽

Free Particle ◽

Host Protein ◽

Resistance Mutations ◽

Peripheral Blood Mononuclear ◽

Hiv 1

The p6 domain of HIV-1 Gag contains highly conserved peptide motifs that recruit host machinery to sites of virus assembly, thereby promoting particle release from the infected cell. We previously reported that mutations in the YPXnL motif of p6, which binds the host protein Alix, severely impair HIV-1 replication. Propagation of the p6–Alix binding site mutants in the Jurkat T cell line led to the emergence of viral revertants containing compensatory mutations not in Gag but in Vpu and the envelope (Env) glycoprotein subunits gp120 and gp41. The Env compensatory mutants replicate in Jurkat T cells and primary human peripheral blood mononuclear cells, despite exhibiting severe defects in cell-free particle infectivity and Env-mediated fusogenicity. Remarkably, the Env compensatory mutants can also rescue a replication-delayed integrase (IN) mutant, and exhibit reduced sensitivity to the IN inhibitor Dolutegravir (DTG), demonstrating that they confer a global replication advantage. In addition, confirming the ability of Env mutants to confer escape from DTG, we performed de novo selection for DTG resistance and observed resistance mutations in Env. These results identify amino acid substitutions in Env that confer broad escape from defects in virus replication imposed by either mutations in the HIV-1 genome or by an antiretroviral inhibitor. We attribute this phenotype to the ability of the Env mutants to mediate highly efficient cell-to-cell transmission, resulting in an increase in the multiplicity of infection. These findings have broad implications for our understanding of Env function and the evolution of HIV-1 drug resistance.

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Expression of SLC5A5 in Circulating Tumor Cells May Distinguish Follicular Thyroid Carcinomas from Adenomas: Implications for Blood-Based Preoperative Diagnosis

Journal of Clinical Medicine ◽

10.3390/jcm8020257 ◽

2019 ◽

Vol 8 (2) ◽

pp. 257 ◽

Cited By ~ 1

Author(s):

Hyeon-Gun Jee ◽

Byoung-Ae Kim ◽

Minjun Kim ◽

Hyeong Yu ◽

June Choi ◽

...

Keyword(s):

Thyroid Nodule ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Peripheral Blood ◽

Preoperative Diagnosis ◽

Thyroid Nodules ◽

Mononuclear Cells ◽

Area Under The Curve ◽

Peripheral Blood Mononuclear ◽

Number Of Patients

Preoperative diagnosis of thyroid nodules reduces unnecessary surgery. Circulating tumor cells (CTCs) may contain information of primary tumor(s). We asked whether the peripheral blood expression of genes specific for circulating tumor cells (CTCs) differentiates benign thyroid nodules from malignant nodules. Peripheral blood mononuclear cells from thyroid nodule patients (n = 20) were isolated preoperatively and the expression of seven CTC-associated genes was measured in patients with thyroid nodule(s) (n = 20). Among the tested genes, the expression of SLC5A5 and LGALS3 were validated in a larger number of patients (n = 64) and our results show that SLC5A5 expression differentiated follicular adenomas from follicular carcinomas (area under the curve (AUC) = 0.831). The expression of SLC5A5 in CTCs may preoperatively distinguish thyroid follicular adenomas from follicular carcinomas.

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