Identification of TNFRSF1A as a novel regulator of carfilzomib resistance in multiple myeloma

Multiple myeloma(MM)is a hematological tumor with high mortality and recurrence rate.Carfilzomib is a new-generation proteasome inhibitor that is used as the first-line therapy for MM.However,the development of drug resistance is a pervasive obstacle to treating MM.Therefore,elucidating the drug resistance mechanisms is conducive to the formulation of novel therapeutic therapies.To elucidate the mechanisms of carfilzomib resistance,we retrieved the GSE78069 microarray dataset containing carfilzomib-resistant LP-1 MM cells and parental MM cells.Differential gene expression analyses revealed major alterations in the major histocompatibility complex(MHC)and cell adhesion molecules.The upregulation of the tumor necrosis factor(TNF)receptor superfamily member 1A(TNFRSF1A)gene was accompanied by the downregulation of MHC genes and cell adhesion molecules.Furthermore,to investigate the roles of these genes,we established a carfilzomib-resistant cell model and observed that carfilzomib resistance induced TNFRSF1A overexpression and TNFRSF1A silencing reversed carfilzomib resistance and reactivated the expression of cell adhesion molecules.Furthermore,TNFRSF1A silencing suppressed the tumorigenesis of MM cells in immunocompetent mice,indicating that TNFRSF1A may lead to carfilzomib resistance by dampening antitumor immunity.Furthermore,our results indicated that TNFRSF1A overexpression conferred carfilzomib resistance in MM cells and suppressed the expression of MHC genes and cell adhesion molecules.The suppression of MHC genes and cell adhesion molecules may impair the interaction between immune cells and cancer cells to impair antitumor immunity.Future studies are warranted to further investigate the signaling pathway underlying the regulatory role of TNFRSF1A in MM cells.

SCN1A rs6732655A/T polymorphism:Diagnostic and therapeutic insights for drug-resistant epilepsy

BACKGROUND A significant subset of individuals with epilepsy fails to respond to currently available antiepileptic drugs,resulting in heightened mortality rates,psychosocial challenges,and a diminished quality of life.Genetic factors,particularly within the SCN1A gene,and the pro-inflammatory cytokine response is important in intricating the drug resistance in idiopathic epilepsy cases.In this extended study,we determined the correlation of rs6732655A/T single nucleotide polymorphism to understand the causative association of SCN1A gene with epilepsy drug resistance and inflammatory response.AIM To find the correlation of SCN1A gene rs6732655A/T polymorphism with the drug-resistant epilepsy and inflammatory response.METHODS The study enrolled 100 age and sex-matched patients of both drug-resistant and drug-responsive epilepsy cases.We analysed the rs6732655A/T polymorphism to study its association and causative role in drug-resistant epilepsy cases using restriction fragment length polymorphism technique.The diagnostic performance of interleukin(IL)-1β,IL-6,and high mobility group box 1(HMGB1)protein levels was evaluated in conjunction with genotypic outcome receiver operating characteristic analysis.RESULTS AT and AA genotypes of rs6732655 SCN1A gene polymorphism were associated with higher risk of drug resistance epilepsy.Serum biomarkers IL-6,IL1βand HMGB1 demonstrated diagnostic potential,with cutoff values of 4.63 pg/mL,59.52 pg/mL and 7.99 ng/mL,respectively,offering valuable tools for epilepsy management.Moreover,specific genotypes(AA and AT)were found to be linked to the elevated levels of IL-1βand IL-6 and potentially reflecting increased oxidative stress and neuro-inflammation in drug-resistant cases supporting the previous reported outcome of high inflammatory markers response in drug resistance epilepsy.CONCLUSION SCN1A genotypes AA and AT are linked to higher drug-resistant epilepsy risk.These findings underscore the potential influence of inflammation and genetics on epilepsy treatment resistance.

Prox1 Suppresses Proliferation and Drug Resistance of Retinoblastoma Cells via Targeting Notch1

Objective Retinoblastoma(RB)is a prevalent type of eye cancer in youngsters.Prospero homeobox 1(Prox1)is a homeobox transcriptional repressor and downstream target of the proneural gene that is relevant in lymphatic,hepatocyte,pancreatic,heart,lens,retinal,and cancer cells.The goal of this study was to investigate the role of Prox1 in RB cell proliferation and drug resistance,as well as to explore the underlying Notch1 mechanism.Methods Human RB cell lines(SO-RB50 and Y79)and a primary human retinal microvascular endothelial cell line(ACBRI-181)were used in this study.The expression of Prox1 and Notch1 mRNA and protein in RB cells was detected using quantitative real time-polymerase chain reaction(RT-qPCR)and Western blotting.Cell proliferation was assessed after Prox1 overexpression using the Cell Counting Kit-8 and the MTS assay.Drug-resistant cell lines(SO-RB50/vincristine)were generated and treated with Prox1 to investigate the role of Prox1 in drug resistance.We employed pcDNA-Notch1 to overexpress Notch1 to confirm the role of Notch1 in the protective function of Prox1.Finally,a xenograft model was constructed to assess the effect of Prox1 on RB in vivo.Results Prox1 was significantly downregulated in RB cells.Overexpression of Prox1 effectively decreased RB cell growth while increasing the sensitivity of drug-resistant cells to vincristine.Notch1 was involved in Prox1’s regulatory effects.Notch1 was identified as a target gene of Prox1,which was found to be upregulated in RB cells and repressed by increased Prox1 expression.When pcDNA-Notch1 was transfected,the effect of Prox1 overexpression on RB was removed.Furthermore,by downregulating Notch1,Prox1 overexpression slowed tumor development and increased vincristine sensitivity in vivo.Conclusion These data show that Prox1 decreased RB cell proliferation and drug resistance by targeting Notch1,implying that Prox1 could be a potential therapeutic target for RB.

Reversing multidrug resistance by RNA interference through the suppression of MDR1 gene in human hepatoma cells

AIM： To reverse the multidrug resistance （MDR） by RNA interference （RNAi）-mediated MDRI suppression in heparoma cells.METHODS： For reversing MDR by RNAi technology, two different short hairpin RNAs （shRNAs） were designed and constructed into pGenSil-1 plasmid, respectively. They were then transfected into a highly adriarnycin-resistant HepG2 hepatorna cell line （HepG2/ADM）. The RNAi effect on MDR was evaluated by real-time PCR, cell cytotoxicity assay and rhodarnine 123 （Rh123） efflux assy. RESULTS： The stably-transfected clones showed various degrees of reversal of MDR phenotype. Surprisingly, the MDR phenotype was completely reversed in two transfected clones. CONCLUSION： MDR can be reversed by the shRNAmediated MDRI suppression in HepG2/ADM cells, which provides a valuable clue to make multidrug-resistant hepatoma cells sensitive to anti-cancer drugs.

Synergistic Effect of Hyperthermia and Neferine on Reverse Multidrug Resistance in Adriamycin-resistant SGC7901/ADM Gastric Cancer Cells

Multidrug resistance（MDR） plays a major obstacle to successful gastric cancer chemotherapy.The purpose of this study was to investigate the MDR reversal effect and mechanisms of hyperthermia in combination with neferine（Nef） in adriamycin（ADM） resistant human SGC7901/ADM gastric cancer cells.The MDR cells were heated at 42℃ and 45℃ for 30 min alone or combined with 10 μg/mL Nef.The cytotoxic effect of ADM was evaluated by MTT assay.Cellular plasma membrane lipid fluidity was detected by fluorescence polarization technique.Intracellular accumulation of ADM was monitored with high performance liquid chromatography.Mdr-1 mRNA,P-glycoprotein（P-gp）,γH2AX expression and γH2AX foci formation were determined by real-time PCR,Western blot and immunocytochemical staining respectively.It was found that different heating methods induced different cytotoxic effects.Water submerged hyperthermia had the strongest cytotoxicity of ADM and Nef combined with hyperthermia had a synergistic cytotoxicity of ADM in the MDR cells.The water submerged hyperthermia increased the cell membrane fluidity.Both water submerged hyperthermia and Nef increased the intracellular accumulation of ADM.The water submerged hyperthermia and Nef down-regulated the expression of mdr-1 mRNA and P-gp.The water submerged hyperthermia could damage DNA and increase the γH2AX expression of SGC7901/ADM cells.The higher temperature was,the worse effect was.Our results show that combined treatment of hyperthermia with Nef can synergistically reverse MDR in human SGC7901/ADM gastric cancer cells.

Distinctive Drug-resistant Mutation Profiles and Interpretations of HIV-1 Proviral DNA Revealed by Deep Sequencing in Reverse Transcriptase

Objective To investigate distinctive features in drug-resistant mutations （DRMs） and interpretations for reverse transcriptase inhibitors （RTIs） between proviral DNA and paired viral RNA in HIV-l-infected patients. Methods Forty-three HIV-l-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA. Results Compared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M1841 and M2301 were more prevalent in proviral DNA than in viral RNA （Fisher＇s exact test, P〈0.05）. Considering ＇majority resistant variants＇, 15 samples （19.48%） showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering ＇minority resistant variants＇, 22 samples （28.57%） were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA. Conclusion Compared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.

THE AMPLIFICATION AND EXPRESSION OF MDR1 GENE IN ADRIAMYCINE RESISTANT CELL LINE OF COLON CANCER CELL HR8348

P-glycoprotein plays an important role in highly drug resistant cells. But its high expression cannot be acheived by chemotherapy. In order to study the effect of P-glycoprotein on clinical tumors, we established a low ADM resistant colon cancer cell line HR/ADM and determined the amplification and expression of mdr-1 gene. The GLC/ADM showed a resistant pattern similar to classical MDR and the transcription of mdr-1 gene determined by RT-PCR increased. The immunocytochemical analysis showed strong positive staining with monoclonal antibody. The gene amplification of mdr-1 was clearly demonstrated by southernblot. Our results suggested that moderate expression of P-glycoprotein might be enough for a high resistantpattern.

PPP1R14A is Associated with Immunotherapy Resistance in Head and Neck Squamous Cell Carcinoma Identified by Single-Cell and Bulk RNA-Sequencing

Objective To identify nivolumab resistance-related genes in patients with head and neck squamous cell carcinoma(HNSCC)using single-cell and bulk RNA-sequencing data.Methods The single-cell and bulk RNA-sequencing data downloaded from the Gene Expression Omnibus database were analyzed to screen out differentially expressed genes(DEGs)between nivolumab resistant and nivolumab sensitive patients using R software.The Least Absolute Shrinkage Selection Operator(LASSO)regression and Recursive Feature Elimination(RFE)algorithm were performed to identify key genes associated with nivolumab resistance.Functional enrichment of DEGs was analyzed with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses.The relationships of key genes with immune cell infiltration,differentation trajectory,dynamic gene expression profiles,and ligand-receptor interaction were explored.Results We found 83 DEGs.They were mainly enriched in T-cell differentiation,PD-1 and PD-L1 checkpoint,and T-cell receptor pathways.Among six key genes identified using machine learning algorithms,only PPP1R14A gene was differentially expressed between the nivolumab resistant and nivolumab sensitive groups both before and after immunotherapy(P<0.05).The high PPP1R14A gene expression group had lower immune score(P<0.01),higher expression of immunosuppressive factors(such as PDCD1,CTLA4,and PDCD1LG2)(r>0,P<0.05),lower differentiation of infiltrated immune cells(P<0.05),and a higher degree of interaction between HLA and CD4(P<0.05).Conclusions PPP1R14A gene is closely associated with resistance to nivolumab in HNSCC patients.Therefore,PPP1R14A may be a target to ameliorate nivolumab resistance of HNSCC patients.

Roles of the tumor microenvironment in the resistance to programmed cell death protein 1 inhibitors in patients with gastric cancer

Despite the continuous developments and advancements in the treatment of gastric cancer(GC),which is one of the most prevalent types of cancer in China,the overall survival is still poor for most patients with advanced GC.In recent years,with the progress in tumor immunology research,attention has shifted toward immunotherapy as a therapeutic approach for GC.Programmed cell death protein 1(PD-1)inhibitors,as novel immunosuppressive medications,have been widely utilized in the treatment of GC.However,many patients are still resistant to PD-1 inhibitors and experience recurrence in the advanced stages of PD-1 immunotherapy.To reduce the occurrence of drug resistance and recurrence in GC patients receiving PD-1 immunotherapy,to maximize the clinical activity of immunosuppressive drugs,and to elicit a lasting immune response,it is essential to research the tumor microenvironment mechanisms leading to PD-1 inhibitor resistance in GC patients.This article reviews the progress in studying the factors influencing the resistance to PD-1 inhibitors in the GC tumor microenvironment,aiming to provide insights and a basis for reducing resistance to PD-1 inhibitors for GC patients in the future.

Paclitaxel-induced stress granules increase LINE-1 mRNA stability to promote drug resistance in breast cancer cells

Abnormal expression of long interspersed element-1(LINE-1)has been implicated in drug resistance,while our previous study showed that chemotherapy drug paclitaxel(PTX)increased LINE-1 level with unknown mechanism.Bioinformatics analysis suggested the regulation of LINE-1 mRNA by drug-induced stress granules(SGs).This study aimed to explore whether and how SGs are involved in drug-induced LINE-1 increase and thereby promotes drug resistance of triple negative breast cancer(TNBC)cells.We demonstrated that SGs increased LINE-1 expression by recruiting and stabilizing LINE-1 mRNA under drug stress,thereby adapting TNBC cells to chemotherapy drugs.Moreover,LINE-1 inhibitor efavirenz(EFV)could inhibit drug-induced SG to destabilize LINE-1.Our study provides the first evidence of the regulation of LINE-1 by SGs that could be an important survival mechanism for cancer cells exposed to chemotherapy drugs.The findings provide a useful clue for developing new chemotherapeutic strategies against TNBCs.

Chitosan/pshRNA Plasmid Nanoparticles Targeting MDR1 Gene Reverse Paclitaxel Resistance in Ovarian Cancer Cells

In order to investigate the effect of chitosan/pshRNA plasmid nanoparticles targeting MDR1 genes on the resistance of A2780/TS cells to paclitaxel, chitosan/pshRNA plasmid nanoparti- cles were synthesized by means of a complex coacervation technique and transfected into A2780/TS cells. The cells transfected with MDRl-targeted chitosan/pshRNA plasmid nanoparticles were experimental cells and the cells transfected with chitosan/pGPU6/GFP/Neo no-load plasmid nanoparticles served as negative control cells. Morphological features of the nanoparticles were observed under transmission electron microscope （TEM）. MDR1 mRNA expression was assessed by RT-PCR. Half-inhibitory concentration （IC50） ofpaclitaxel for A2780/TS cells was determined by MTT method. TEM showed that the nanoparticles were round-shaped, smooth in surface and the diameters varied from 80 to 120 nm. The MDR1 mRNA in the transfected cells was significantly decreased by 17.6%, 27.8% and 52.6% on the post-transfection day 2, 4 and 7 when compared with that in A2780/TS cells control （P〈0.05）. MTT assay revealed that the relative reversal efficiency was increased over time and was 29.6%, 51.2% and 61.3% respectively in the transfected cells 2, 4, 7 days after transfection and IC_50 （0.197±0.003, 0.144±0.001, 0.120±0.004） were decreased with difference being significant when compared with that in A2780/TS （0.269±0.003） cells control （P〈0.05）. It was concluded that chitosan/pshRNA plasmid nanoparticles targeting MDR1 can effectively reverse the paclitaxel resistance in A2780/TS cells in a time-dependent manner.

Prevalence and patterns of drug-resistance mutations among HIV-1 patients infected with CRF07_BC strains in Sichuan province,China

Little information is available on the prevalence of drug-resistance mutations in patients harboring the human immunodeficiency virus type 1(HIV-1) circulating recombinant form(CRF)07_BC variant in Sichuan, China. This study examined 375 plasma samples from patients with HIV-1 who were infected with the CRF07_BC strain, including 104 drug-naive participants and 271 in whom antiretroviral therapy(ART) had failed. Only one participant in the drug-naive group had a drug-resistance mutation(M46L), compared with 31.73% of those in whom ART had failed. Further analysis showed that 19.56% of strains contained mutations conferring resistance to non-nucleoside reverse transcriptase inhibitors(NNRTIs) alone, 0.74% were resistant to nucleoside reverse transcriptase inhibitors(NRTIs) alone, and 11.44% were dual-resistant to both NRTIs and NNRTIs. The most common mutation in the ART-failure group was M184V(35.88%), K103N(45.01%), Y181C(17.33%), and G190S/A(15.88%). The percentages of HIV-1 strains resistant to lamivudine, emtricitabine, efavirenz, etravirine, and nevirapine were 10.70%, 10.70%, 28.04%, 7.75%, and 26.20%, respectively. To explore site variants possibly related to drug resistance, variations in the ancestor/consensus CRF07_BC sequences from the therapy-naive and ART-failure groups were compared, and seven mutations at six positions were identified as being significantly differently distributed between the two groups(p<0.05). Detailed sequence data will provide information on CRF07_BC genetic characterizations, and improve our understanding of antiretroviral susceptibility and the evolution of drug-resistance mutations. This will be valuable in developing and implementing local public-health approaches for HIV drug-resistance prevention and treatment.

Expression of multidrug resistance 1 gene and C3435T genetic polymorphism in peripheral blood of patients with intractable epilepsy

BACKGROUND： Increased expression of multidrug resistance 1 （MDR1） mRNA in peripheral blood of patients with intractable epilepsy is not due to epilepsy drugs, but epilepsy behavior. Monitoring MDR1 expression in peripheral blood is a target for MDR1 gene evaluation. OBJECTIVE： To investigate the influence of antiepileptic drugs and seizures on MDR expression in intractable epilepsy, and to analyze the genetic polymorphisms of C3435T in the MDRl gene. DESIGN, TIME AND SETTING： Factorial designs and comparative observations at the experimental center of the Affiliated Hospital of Qingdao Medical College, Qingdao University between October 2003 and October 2004. PARTICIPANTS： A total of 120 subjects were recruited from the epilepsy clinical department of the Affiliated Hospital of Qingdao Medical College. Four groups （n = 30） were classified according to statistical factorial design： intractable epilepsy, treatment response, no treatment, and normal control groups. METHODS： One-step semi-quantitative reverse-transcription polymerase chain reaction technology was used to test expressions of the MDR1 gene in 120 subjects. C3435T polymorphisms in intractable epilepsy group and normal control groups were analyzed by polymerase chain reaction-restriction fragment length polymorphism. MAIN OUTCOME MEASURES： Expression of MDR1 mRNA in the four groups, and C3435T genetic polymorphisms in intractable epilepsy and normal control groups. RESULTS： MDRl gene expression was increased in the intractable epilepsy group, due to the factor seizures, but not the antiepileptic drugs. However, the interaction between the two factors was not statistically significant. Of the 30 subjects in the intractable epilepsy group, the following genotypes were exhibited： 3 （10%） C/C genotype, 9 （30%） C/T genotype, and 18 （60%） T/T genotype at the site of C3435T, while 4 （13%）, 10 （33%）, and 16 （53%） subjects were determined to express these genotypes in the normal control group, respectively. C and T allele frequency were 25% and 75% in the intractable epilepsy group, and 30% and 70% in the normal control group, respectively. However, there was no statistical difference between the groups. CONCLUSION： Results demonstrated that seizures, not antiepileptic drugs, induced MDR1 gene expression in intractable epilepsy. Genetic polymorphisms of C3435T in the MDR1 gene did not contribute to the development of multidrug resistance in patients with intractable epilepsy.

Study of HIV-1 Drug Resistance in Patients Receiving Free Antiretroviral Therapy in China

To investigate the prevalence of drug-resistance mutations,resistance to antiretroviral drugs,and the subsequent virological response to therapy in treatment-naive and antiretroviral-treated patients infected with HIV/AIDS in Henan,China,a total of 431 plasma samples were collected in Queshan county between 2003 and 2004,from patients undergoing the antiretroviral regimen Zidovudine + Didanosine + Nevirapine(Azt+Ddi+Nvp).Personal information was collected by face to face interview.Viral load and genotypic drug resistance were tested.Drug resistance mutation data were obtained by analyzing patient-derived sequences through the HIVdb Program(http://hivdb.stanford.edu).Overall,38.5% of treatment-naive patients had undetectable plasma viral load(VL),the rate significantly increased to 61.9% in 0 to 6 months treatment patients(mean 3 months)(P<0.005)but again significantly decrease to 38.6% in 6 to 12 months treatment patients(mean 9 months)(P<0.001)and 40.0% in patients receiving more than 12 months treatment(mean 16 months)(P<0.005).The prevalence of drug resistance in patients who had a detectable VL and available sequences were 7.0%,48.6%,70.8%,72.3% in treatment-na?ve,0 to 6 months treatment,6 to 12 months treatment,and treatment for greater than 12 months patients,respectively.No mutation associated with resistance to Protease inhibitor(PI)was detected in this study.Nucleoside RT inhibitor(NRTI)mutations always emerged after non-nucleoside RT inhibitor(NNRTI)mutations,and were only found in patients treated for more than 6 months,with a frequency less than 5%,with the exception of mutation T215Y(12.8%,6/47)which occurred in patients treated for more than 12 months.NNRTI mutations emerged quickly after therapy begun,and increased significantly in patients treated for more than 6 months(P<0.005),and the most frequent mutations were K103N,V106A,Y181C,G190A.There had been optimal viral suppression in patients undergoing treatment for less than 6 months in Queshan,Henan.The drug resistance strains were highly prevalent in antiretroviral-treated patients,and increased with the continuation of therapy,with many patients encountering virological failure after 6 months therapy.

Detection and clinical significance of multidrug resistance-1 mRNA in bone marrow cells in children with acute lymphoblastic leukemia by real-time fluorescence quantitative RT-PCR

Objective： Multidrug resistance（MDR） is one of the most important reasons for treatment failure and recurrence of acute leukemia. Its manifestations are different in children with acute lymphoblastic leukemia（ALL） which may be due to different detection methods. This study was to detect the expression of MDR1 mRNA in bone marrow cells of children with ALL by real-time fluorescence- quantitative reverse transcription polymerase-chain reaction（FQ-RT-PCR）, and combine minimal residual desease（MRD） detection by flow cytometry（FCM） and to study their relationship with treatment response and prognosis of ALL. Methods：The MDR1 mRNA levels in bone marrow cells from 67 children with ALL[28 had newly diagnosed disease, 27 had achieved complete remission（CR）, 12 recurrent] and 22 children without leukemia were detected by FQ-RT-PCR. MRD was detected by FCM. The patients were observed for 9-101 months, with a median of 64 months. Results：Standard curves of human MDR1 and GAPDH genes were constructed successfully. MDR1 mRNA was detected in all children with a positive rate of 100%. The mRNA level of MDR1 was similar among the newly diagnosed ALL group, CR group, and control group（P 〉 0.05）, but significantly higher in the recurrence group than that in newly diagnosed disease group and control group（0.50 ± 0.55 vs. 0.09 ± 0.26 and 0.12 ± 0.23, P〈 0.05）. 54 ALL patients were followed up, and it was found that MDR1 mRNA level was significantly higher in ALL patients within 3 years duration than that of ALL patients with 3-6 years and over 6 years duration（0.63 ± 0.56 vs. 0.11 ± 0.12 and 0.04 ± 0.06, P〈 0.01）. For the 28 children with newly diagnosed disease, the MDR1 mRNA level was similar between WBC 〉 50 ~ 109 group and WBC〈50 × 10^9 group（P〉 0.05）. In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD〉10a group than that in MRD〈10a group（0.39 ± 0.47 vs. 0.03 ± 0.03, P 〈 0.05）. Conclusion：The sensitivity and specificity of FQ-RT-PCR in detecting MDR1 mRNA in bone marrowy cells of children with ALL patients are high. MDR1 mRNA is expressed in children with and without leukemia. MDR1 mRNA is highly expressed in the CR ALL patients with high MRD, recurrence and short duration（within 3 years）. Monitoring MRD and the MDR1 mRNA level might be helpful for individual treatment.

HIV-1 Subtype Diversity and Factors Affecting Drug Resistance among Patients with Virologic Failure in Antiretroviral Therapy in Hainan Province,China,2014–2020

Objective This study aimed to determine the HIV-1 subtype distribution and HIV drug resistance(HIVDR)in patients with ART failure from 2014 to 2020 in Hainan,China.Methods A 7-year cross-sectional study was conducted among HIV/AIDS patients with ART failure in Hainan.We used online subtyping tools and the maximum likelihood phylogenetic tree to confirm the HIV subtypes with pol sequences.Drug resistance mutations(DRMs)were analyzed using the Stanford University HIV Drug Resistance Database.Results A total of 307 HIV-infected patients with ART failure were included,and 241 available pol sequences were obtained.Among 241 patients,CRF01_AE accounted for 68.88%,followed by CRF07_BC(17.00%)and eight other subtypes(14.12%).The overall prevalence of HIVDR was 61.41%,and the HIVDR against non-nucleoside reverse transcriptase inhibitors(NNRTIs),nucleotide reverse transcriptase inhibitors(NRTIs),and protease inhibitors(PIs)were 59.75%,45.64%,and 2.49%,respectively.Unemployed patients,hypoimmunity or opportunistic infections in individuals,and samples from 2017 to 2020 increased the odd ratios of HIVDR.Also,HIVDR was less likely to affect female patients.The common DRMs to NNRTIs were K103N(21.99%)and Y181C(20.33%),and M184V(28.21%)and K65R(19.09%)were the main DRMs against NRTIs.Conclusion The present study highlights the HIV-1 subtype diversity in Hainan and the importance of HIVDR surveillance over a long period.

Utility of a Relatively Affordable In-House HIV-1 Genotyping Assay for Drug Resistance Testing among Non B HIV-1 Infected Drug Naive Patients in Nigeria

Background: The introduction of antiretroviral (ARV) in resource-limited settings has increased life expectancy among non-B HIV-1 infected individuals. We used a validated In-house genotyping assay to characterize non-B HIV-1 and to determine drug resistance mutations among treatment-naive patients. Methods: Plasma samples from 105 HIV-1 infected drug-naive adult patients attending a tertiary hospital Jos, Nigeria were subjected to HIV-1 RNA extraction, reverse transcription amplification, and population-based sequencing of the partial pol gene on the ABI 3130xl genetic analyzer. Subtyping and phylogenetic analyses were performed by REGA Subtyping Tool v2.0 and MEGA v5.0 respectively. Drug resistance profiles were evaluated according to IAS-USA 2013 drug resistance mutations list. Result: One hundred samples (95.2%) were successfully genotyped. The distribution of the non-B HIV-1 subtypes were;CRF02_AG-48%, G-41.0%, CRF06_cpx-6.0%, and A-5.0%. Ten percent of the isolates had at least one major drug resistance mutation in the pol gene. The drug-class specific resistance prevalences were 6.0% for NRTIs;M41L-1.0%, K65KR-1.0%, M184IM-1.0%, M184V-2.0%, and T215ADNT-1%, 8.0% for NNRTIs;K103N-2%, 1.0% for K101E, E138A, G190A, P225HP, Y181I, Y188L, Y181C including protease inhibitors’ Q58E (1.0%). Conclusion: HIV-1 was heterogeneously distributed;CRF02_AG and G predominate and some known major mutations associated with NRTIs and NNRTIs were determined. The In-house assay is suitable for both characterization of non-B HIV-1 subtypes and detection of drug resistance at a significant lower cost than available commercial genotyping assays. This finding underscores the need to consider use of low-cost In-house genotyping assay as an alternative in resource-limited settings with non-B HIV-1 epidemic.

HIV-1 Drug Resistance Mutations in Patients Failing 1st Line Therapy in a Comprehensive Care Center in Nairobi, Kenya

Background: HIV-1 drug resistance is an emerging challenge for HIV-1 infected clients who are on antiretroviral therapy (ART). In Kenya, as in many other developing countries, ART is now accesible to clients who need it. However, they must be done a CD4 test first and if the count is <300, then ART is commenced. With the initiation of ART comes the challenge of adherence to medication, a factor that is impacted greatly by the understanding of the client of the importance?of adherence and the financial ability to keep their appointments, especially if the clients come from a distant location. Objective: To identify HIV-1 drug resistance mutations inclientsfailing1st line antiretroviral therapy in Nairobi, Kenya. Methodology: A cross sectional study was carried out where whole blood samples were collected from clients attending a HIV care and treatment clinic in Nairobi. Clients who had been on ART for more than 6 months and had a viral load greater than 1000 were enrolled in the study. A total of 52 client samples were successfully sequenced in the reverse transcriptase region and analyzed. Results: After analysis of the generated sequences, it was seen that 43 (82.6%) of the clients had HIV-1 drug resistance mutations conferring resistance to one or more nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse-transcriptase inhibitors (NNRTIs). Majority of the clients (46%) were infected with HIV-1 subtype A viruses. Conclusion: The findings of the study showed that a significant proportion of the clients on ART had developed resistance mutations to one or more drugs that are used as 1st line therapy in Kenya. There is need for continuous education of the population on importance of adherence to medication. There is also need for clinicians to be trained on using viral load and HIV drug resistance testing, where available, as methods of monitoring treatment failure so that clients can be switched to alternative medication immediately the need arises, so as to improve their treatment outcomes.

Autocrine Production of Interleukin-6: A Mechanism of Interleukin-6 Independence in Dexamethasone-Resistant 7TD1 Murine Myeloma Cells

Several factors could contribute to proliferation of multiple myeloma (MM) cells independent of interleukin-6 (IL6) in the later stages of the disease. Our previous studies established a dexamethasone-resistant 7TD1 cell line (7TD1-Dxm) and have shown that one mechanism of resistance to dexamethasone is due to inhibition of cytochrome c release. We have also observed that 7TD1-Dxm cells proliferate independently of externally-added IL6. This study therefore aimed to elucidate the mechanisms responsible for IL6-independent proliferation in 7TD1-Dxm cells. Our results indicated that 7TD1-Dxm cells produced IL6 in an autocrine fashion. We have observed that dexamethasone-resistant 7TD1 cells become dexamethasone-resistant and IL6-independent for proliferation concomitantly. This strongly suggests that production of IL6 by 7TD1-Dxm cells may play an important role in the development of dexamethasone resistance. Consequently, further investigation of the molecular mechanisms responsible for IL6 production may be helpful in delineating the mechanisms leading to dexamethasone resistance.

Drug Resistance Mutations and Genetic Diversity in Patients Treated for HIV Type 1 Infection in Rural Care Centers in Togo

Introduction: Access to antiretroviral treatment (ART) in resource-limited countries has increased signif-icantly but scaling up ART into rural areas is more recent and information on treatment outcome in rural areas is still very limited. We reported here virological outcome and drug resistance in ART in rural settings in Togo. Methods: HIV-1 infected adults (≥18 years) and infants were enrolled in routine medical visit at 12 on first-line ART in three HIV care centers. Epidemiological and demographic information and data on ART history were collected. Viral load (VL) was determined and genotypic drug resistance testing was performed on all samples with viral load above 1000 copies/ml. Results: 102 adult patients and 27 infants were consecutively enrolled. Virological failure was observed in 28 (21.5%) patients. For 25/28 patients, sequencing was successful and drug resistance mutations were observed in 23 (92%) of them. The global prevalence of drug resistance in the study population was thus at least 17.8% (23/129), with 7 (6.9%) patients infected with HIV strains that are resistant to two of the three first-line antiretroviral (ARVs) drugs and 9 (8.3%) to all three first-line ARVs. As expected, the observed drug resistance mutations were mainly associated with the drugs used in first line regimens, zidovudine, lamivudine and effavirenz/nevirapine but several patients accumulated high numbers of mutations and developed also cross-resistance to abacavir, didanosine or the new non-nucleoside reverse transcriptase inhibitor drugs, like etravirine and rilpivirine. Conclusion: The observations on ART treatment outcome from ART clinics in rural areas are the same as observed in previous observations in Lomé, the capital city. Although access to viral load will improve treatment outcome, better programme management and implementation of actions to improve factors as patient adherence, drugs stock-outs and lost to follow-up are also essential.