RESUMO
BACKGROUND: We investigated an ultrasensitive prostate-specific antigen (uPSA) immunoassay (MesoScale; lower limit of detection (LLD) of 0.0035 pg/mL) to monitor patients with prostate cancer (PCa) following radical prostatectomy (RP) and to examine whether changes in PSA in the conventionally undetectable range (<1 pg/mL) can predict biochemical relapse (BCR). METHODS: We measured uPSA in serial serum samples (N = 100) collected from 20 RP cases with a third-generation ELISA (LLD of 1 pg/mL) and the fifth-generation MesoScale assay. We analyzed the PSA nadir changes to classify patients into BCR or non-BCR groups, observed the trends in PSA kinetics, and associated BCR status with clinicohistopathological features. RESULTS: The ELISA could quantify PSA in only 38% of the RP samples, detecting BCR in 7 of 20 patients with PCa. The MesoScale assay quantified PSA in all samples, showing 8 of 20 patients with BCR. However, there was no significant difference between the median time to BCR detection based on ELISA (1016 days) compared with MesoScale data (949 days). Gleason scores were higher in the BCR groups compared with non-BCR. There was no significant difference for other clinicohistopathological parameters. CONCLUSIONS: The uPSA MesoScale technology could track miniscule changes in serum PSA in the range of 0.003-1 pg/mL in all RP cases. However, PSA kinetics and nadir at concentrations <2 pg/mL fluctuated, and increases below this range could not reliably suggest signs of BCR. Instead, ultrasensitive fifth-generation PSA assays may hold clinical potential for measuring the low concentrations of PSA in women for various medical contexts.
Assuntos
Imunoensaio/métodos , Antígeno Prostático Específico/sangue , Prostatectomia , Neoplasias da Próstata/sangue , Idoso , Seguimentos , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Antígeno Prostático Específico/imunologiaRESUMO
BACKGROUND: Polycystic ovarian syndrome (PCOS) is a common cause of reproductive and metabolic dysfunction. We hypothesized that serum prostate-specific antigen (PSA) may constitute a new biomarker for hyperandrogenism in PCOS. METHODS: We conducted a cross-sectional study of 45 women with PCOS and 40 controls. Serum from these women was analyzed for androgenic steroids and for complexed PSA (cPSA) and free PSA (fPSA) with a novel fifth- generation assay with a sensitivity of ~10 fg/mL for cPSA and 140 fg/mL for fPSA. RESULTS: cPSA and fPSA levels were about three times higher in PCOS compared to controls. However, in PCOS, cPSA and fPSA did not differ according to waist-to-hip ratio, Ferriman-Gallwey score, or degree of hyperandrogenemia or oligo-ovulation. In PCOS and control women, serum cPSA and fPSA levels were highly correlated with each other, and with free and total testosterone levels, but not with other hormones. Adjusting for age, body mass index (BMI) and race, cPSA was significantly associated with PCOS, with an odds ratio (OR) of 5.67 (95% confidence interval [CI]: 1.86, 22.0). The OR of PCOS for fPSA was 7.04 (95% CI: 1.65, 40.4). A multivariate model that included age, BMI, race and cPSA yielded an area-under-the-receiver-operating-characteristic curve of 0.89. CONCLUSIONS: Serum cPSA and fPSA are novel biomarkers for hyperandrogenism in PCOS and may have value for disease diagnosis.
Assuntos
Imunoensaio , Medições Luminescentes , Síndrome do Ovário Policístico/diagnóstico , Antígeno Prostático Específico/sangue , Adulto , Área Sob a Curva , Biomarcadores/sangue , Índice de Massa Corporal , Estudos Transversais , Feminino , Humanos , Análise Multivariada , Razão de Chances , Curva ROC , Kit de Reagentes para DiagnósticoRESUMO
OBJECTIVES: HIV-1 reverse transcriptase (RT) mutations associated with antiviral drug resistance have been extensively characterized in the enzyme polymerase domain. Recent studies, however, have verified the involvement of the RT C-terminal domains (connection and RNase H) in drug resistance to RT inhibitors. In this work, we have characterized the correlation of recently described C-terminal domain mutations with thymidine analogue mutations (TAMs), as well as their phenotypic impact on susceptibility to zidovudine and nevirapine. METHODS: HIV-1 RT sequences from Brazilian patients and from public sequence databases for which the C-terminal RT domains and treatment status were also available were retrieved and analysed for the association of C-terminal mutations and the presence of TAMs and treatment status. Several C-terminal RT mutations previously characterized were introduced by site-directed mutagenesis into an HIV-1 subtype B molecular clone in a wild-type, TAM-1 or TAM-2 pathway context. Mutants were tested for drug susceptibility to the prototypic drugs zidovudine and nevirapine. RESULTS: Subtype B-infected patient database analysis showed that mutations N348I, A360V/T, T377M and D488E were found to be selected independently of TAMs, whereas mutations R358K, G359S, A371V, A400T, K451R and K512R increased in frequency with the number of TAMs in a dose-dependent fashion. Phenotypic analysis of C-terminal mutations showed that N348I, T369V and A371V conferred reduced susceptibility to zidovudine in the context of the TAM-1 and/or TAM-2 pathway, and also conferred dual resistance to nevirapine. Other mutations, such as D488E and Q547K, showed TAM-specific enhancement of resistance to zidovudine. Finally, mutation G359S displayed a zidovudine hypersusceptibility phenotype, both per se and when combined with A371V. CONCLUSIONS: This study demonstrates that distinct RT C-terminal mutations can act as primary or secondary drug resistance mutations, and are associated in a complex array of phenotypes with RT polymerase domain mutations.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , Timidina/análogos & derivados , Substituição de Aminoácidos , Brasil , Infecções por HIV/virologia , HIV-1/enzimologia , HIV-1/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , Nevirapina/farmacologia , Estrutura Terciária de Proteína/genética , Timidina/farmacologia , Zidovudina/farmacologiaRESUMO
Recently, mutations in the connection subdomain (CN) and RNase H domain of HIV-1 reverse transcriptase (RT) were observed to exhibit dual resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). To elucidate the mechanism by which CN and RH mutations confer resistance to NNRTIs, we hypothesized that these mutations reduce RNase H cleavage and provide more time for the NNRTI to dissociate from the RT, resulting in the resumption of DNA synthesis and enhanced NNRTI resistance. We observed that the effect of the reduction in RNase H cleavage on NNRTI resistance is dependent upon the affinity of each NNRTI to the RT and further influenced by the presence of NNRTI-binding pocket (BP) mutants. D549N, Q475A, and Y501A mutants, which reduce RNase H cleavage, enhance resistance to nevirapine (NVP) and delavirdine (DLV), but not to efavirenz (EFV) and etravirine (ETR), consistent with their increase in affinity for RT. Combining the D549N mutant with NNRTI BP mutants further increases NNRTI resistance from 3- to 30-fold, supporting the role of NNRTI-RT affinity in our NNRTI resistance model. We also demonstrated that CNs from treatment-experienced patients, previously reported to enhance NRTI resistance, also reduce RNase H cleavage and enhance NNRTI resistance in the context of the patient RT pol domain or a wild-type pol domain. Together, these results confirm key predictions of our NNRTI resistance model and provide support for a unifying mechanism by which CN and RH mutations can exhibit dual NRTI and NNRTI resistance.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Mutação de Sentido Incorreto , Inibidores da Transcriptase Reversa/farmacologia , Transcriptase Reversa do HIV/genética , Humanos , Ribonuclease H/metabolismoRESUMO
We previously proposed that mutations in the connection subdomain (cn) of HIV-1 reverse transcriptase increase AZT resistance by altering the balance between nucleotide excision and template RNA degradation. To test the predictions of this model, we analyzed the effects of previously identified cn mutations in combination with thymidine analog mutations (D67N, K70R, T215Y, and K219Q) on in vitro RNase H activity and AZT monophosphate (AZTMP) excision. We found that cn mutations G335C/D, N348I, A360I/V, V365I, and A376S decreased primary and secondary RNase H cleavages. The patient-derived cns increased ATP- and PPi-mediated AZTMP excision on an RNA template compared with a DNA template. One of 5 cns caused an increase in ATP-mediated AZTMP excision on a DNA template, whereas three cns showed a higher ratio of ATP- to PPi-mediated excision, indicating that some cn mutations also affect excision on a DNA substrate. Overall, the results strongly support the model that cn mutations increase AZT resistance by reducing template RNA degradation, thereby providing additional time for RT to excise AZTMP.
Assuntos
Farmacorresistência Viral/genética , Transcriptase Reversa do HIV/genética , Modelos Biológicos , Mutação/genética , RNA/metabolismo , Zidovudina/metabolismo , Linhagem Celular , Clonagem Molecular , Primers do DNA/genética , Humanos , MutagêneseRESUMO
We previously shown that mutations in the connection (CN) subdomain of human immunodeficiency virus type 1 (HIV-1) subtype B reverse transcriptase (RT) increase 3'-azido-3'-deoxythymidine (AZT) resistance in the context of thymidine analog mutations (TAMs) by affecting the balance between polymerization and RNase H activity. To determine whether this balance affects drug resistance in other HIV-1 subtypes, recombinant subtype CRF01_AE was analyzed. Interestingly, CRF01_AE containing TAMs exhibited 64-fold higher AZT resistance relative to wild-type B, whereas AZT resistance of subtype B containing the same TAMs was 13-fold higher, which in turn correlated with higher levels of AZT-monophosphate (AZTMP) excision on both RNA and DNA templates. The high level of AZT resistance exhibited by CRF01_AE was primarily associated with the T400 residue in wild-type subtype AE CN subdomain. An A400T substitution in subtype B enhanced AZT resistance, increased AZTMP excision on both RNA and DNA templates, and reduced RNase H cleavage. Replacing the T400 residue in CRF01_AE with alanine restored AZT sensitivity and reduced AZTMP excision on both RNA and DNA templates, suggesting that the T400 residue increases AZT resistance in CRF01_AE at least in part by directly increasing the efficiency of AZTMP excision. These results show for the first time that CRF01_AE exhibits higher levels of AZT resistance in the presence of TAMs and that this resistance is primarily associated with T400. Our results also show that mixing the RT polymerase, CN, and RNase H domains from different subtypes can underestimate AZT resistance levels, and they emphasize the need to develop subtype-specific genotypic and phenotypic assays to provide more accurate estimates of clinical drug resistance.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Transcriptase Reversa do HIV/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Zidovudina/farmacologia , Linhagem Celular , Reparo do DNA , Genótipo , Transcriptase Reversa do HIV/genética , HIV-1/classificação , HIV-1/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação Puntual , RNA Viral/metabolismo , Ribonuclease H/metabolismo , Timidina/genética , Proteínas Virais/metabolismoRESUMO
BACKGROUND: We hypothesize that prostate specific antigen (PSA), a protein that it is under regulation by androgens, may be differentially expressed in female elite athletes in comparison to control women. METHODS: We conducted a cross-sectional study of 106 female athletes and 114 sedentary age-matched controls. Serum from these women was analyzed for complexed prostate specific antigen (cPSA) and free prostate specific antigen (fPSA), by fifth generation assays with limits of detection of around 6 and 140 fg/mL, respectively. A panel of estrogens, androgens and progesterone in the same serum was also quantified by tandem mass spectrometry. Results: Both components of serum PSA (cPSA and fPSA) were lower in the elite athletes vs the control group (P=0.033 and 0.013, respectively). Furthermore, estrone (p=0.003) and estradiol (p=0.004) were significantly lower, and dehydroepiandrosterone (p=0.095) and 5-androstene-3ß, 17ß-diol (p=0.084) tended to be higher in the athletes vs controls. Oral contraceptive use was similar between groups and significantly associated with increased cPSA and fPSA in athletes (p= 0.046 and 0.009, respectively). PSA fractions were not significantly associated with progesterone changes. The Spearman correlation between cPSA and fPSA in both athletes and controls was 0.75 (P < 0.0001) and 0.64 (P < 0.0001), respectively. Conclusions: Elite athletes have lower complexed and free PSA, higher levels of androgen precursors and lower levels of estrogen in their serum than sedentary control women. ABBREVIATIONS: cPSA, complexed PSA; fPSA, free PSA; PCOS, polycystic ovarian syndrome; E1, estrone; E2, estradiol; DHEA, dehydroepiandrosterone, Testo, testosterone; DHT, dihydrotestosterone; PROG, progesterone; Delta 4, androstenedione; Delta 5, androst-5-ene-3ß, 17ß-diol; BMD, body mineral density; LLOQ, lower limit of quantification; ULOQ, upper limit of quantification; LOD, limit of detection; ACT, α 1-antichymotrypsin.
RESUMO
Currently, nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) are two classes of antiretroviral agents that are approved for treatment of HIV-1 infection. Since both NRTIs and NNRTIs target the polymerase (pol) domain of reverse transcriptase (RT), most genotypic analysis for drug resistance is limited to the first ~300 amino acids of RT. However, recent studies have demonstrated that mutations in the C-terminal domain of RT, specifically the connection subdomain and RNase H domain, can also increase resistance to both NRTIs and NNRTIs. In this review we will present the potential mechanisms by which mutations in the C-terminal domain of RT influence NRTI and NNRTI susceptibility, summarize the prevalence of the mutations in these regions of RT identified to date, and discuss their importance to clinical drug resistance.
RESUMO
We recently observed that mutations in the human immunodeficiency type 1 (HIV-1) reverse transcriptase (RT) connection domain significantly increase 3'-azido-3'-deoxythymidine (AZT) resistance up to 536 times over wild-type (WT) RT in the presence of thymidine analog resistance mutations (TAMs). These mutations also decreased RT template switching, suggesting that they altered the balance between nucleotide excision and template RNA degradation, which in turn increased AZT resistance. Several residues in the HIV-1 connection domain contact the primer strand and form an RNase H primer grip structure that helps to position the primer-template at the RNase H and polymerase active sites. To test the hypothesis that connection domain mutations enhanced AZT resistance by influencing the RNase H primer grip, we determined the effects of alanine substitutions in RNase H primer grip residues on nucleoside RT inhibitor resistance in the context of a WT, TAM-containing, or K65R-containing polymerase domain. Ten of the 11 RNase H primer grip mutations increased AZT resistance 20 to 243 times above WT levels in the context of a TAM-containing polymerase domain. Furthermore, all mutations in the RNase H primer grip decreased template switching, suggesting that they reduced RNase H activity. These results demonstrate that mutations in the RNase H primer grip region can significantly enhance AZT resistance and support the hypothesis that mutations in the connection and RNase H domains can increase resistance by altering the RNase H primer grip region, changing interactions between RT and the template-primer complex and/or shifting the balance between the polymerase and RNase H activities.
Assuntos
Substituição de Aminoácidos , Farmacorresistência Viral/genética , Transcriptase Reversa do HIV/química , HIV-1/enzimologia , Mutação de Sentido Incorreto , RNA/química , Ribonuclease H/química , Linhagem Celular , Transcriptase Reversa do HIV/genética , HIV-1/genética , Humanos , Estrutura Quaternária de Proteína , RNA/genética , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Ribonuclease H/genética , Zidovudina/química , Zidovudina/farmacologiaRESUMO
Replication of human immunodeficiency virus type 1 (HIV-1), like all organisms, involves synthesis of a minus-strand and a plus-strand of nucleic acid. Currently available PCR methods cannot distinguish between the two strands of nucleic acids. To carry out detailed analysis of HIV-1 reverse transcription from infected cells, we have developed a novel strand-specific amplification (SSA) assay using single-stranded padlock probes that are specifically hybridized to a target strand, ligated, and quantified for sensitive analysis of the kinetics of HIV-1 reverse transcription in cells. Using SSA, we have determined for the first time the ex vivo rates of HIV-1 minus-strand DNA synthesis in 293T and human primary CD4(+) T cells ( approximately 68 to 70 nucleotides/min). We also determined the rates of minus-strand DNA transfer ( approximately 4 min), plus-strand DNA transfer ( approximately 26 min), and initiation of plus-strand DNA synthesis ( approximately 9 min) in 293T cells. Additionally, our results indicate that plus-strand DNA synthesis is initiated at multiple sites and that several reverse transcriptase inhibitors influence the kinetics of minus-strand DNA synthesis differently, providing insights into their mechanism of inhibition. The SSA technology provides a novel approach to analyzing DNA replication processes and should facilitate the development of new antiretroviral drugs that target specific steps in HIV-1 reverse transcription.
Assuntos
DNA de Cadeia Simples/fisiologia , HIV-1/fisiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Transcrição Reversa/fisiologia , Replicação Viral , Sequência de Bases , Linfócitos T CD4-Positivos/fisiologia , Linhagem Celular , Sondas de DNA/genética , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese , OligonucleotídeosRESUMO
We previously proposed that a balance between nucleotide excision and template RNA degradation plays an important role in nucleoside reverse transcriptase inhibitor (NRTI) resistance. To explore the predictions of this concept, we analyzed the role of patient-derived C-terminal domains of HIV-1 reverse transcriptase (RT) in NRTI resistance. We found that when the polymerase domain contained previously described thymidine analog resistance mutations, mutations in the connection domain increased resistance to 3'-azido-3'-deoxythymidine (AZT) from 11-fold to as much as 536-fold over wild-type RT. Mutational analysis showed that amino acid substitutions E312Q, G335C/D, N348I, A360I/V, V365I, and A376S were associated strongly with the observed increase in AZT resistance; several of these mutations also decreased RT template switching, suggesting that they alter the predicted balance between nucleotide excision and template RNA degradation. These results indicate that mutations in the C-terminal domain of RT significantly enhance clinical NRTI resistance and should be considered in genotypic and phenotypic drug resistance studies.
Assuntos
Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/genética , Mutação , Zidovudina/farmacologia , Sequência de Aminoácidos , Farmacorresistência Viral , Transcriptase Reversa do HIV/química , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Replicação ViralRESUMO
The RNase H primer grip of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contacts the DNA primer strand and positions the template strand near the RNase H active site, influencing RNase H cleavage efficiency and specificity. Sequence alignments show that 6 of the 11 residues that constitute the RNase H primer grip have functional equivalents in murine leukemia virus (MLV) RT. We previously showed that a Y586F substitution in the MLV RNase H primer grip resulted in a 17-fold increase in substitutions within 18 nucleotides of adenine-thymine tracts, which are associated with a bent DNA conformation. To further determine the effects of the MLV RNase H primer grip on replication fidelity and viral replication, we performed additional mutational analysis. Using either beta-galactosidase (lacZ) or green fluorescent protein (GFP) reporter genes, we found that S557A, A558V, and Q559L substitutions resulted in statistically significant increases in viral mutation rates, ranging from 2.1- to 3.8-fold. DNA sequencing analysis of nonfluorescent GFP clones indicated that the mutations in RNase H primer grip significantly increased the frequency of deletions between the primer-binding site (PBS) and sequences downstream of the PBS. In addition, quantitative real-time PCR analysis of reverse transcription products revealed that the mutant RTs were substantially inefficient in plus-strand DNA transfer relative to the wild-type control. These results indicate that the MLV RNase H primer grip is an important determinant of in vivo fidelity of DNA synthesis and suggest that the mutant RT was unable to copy through the DNA-RNA junction of the minus-strand DNA and the tRNA because of its bent conformation resulting in error-prone plus-strand DNA transfer.
Assuntos
DNA Viral/biossíntese , Vírus da Leucemia Murina/enzimologia , Mutação , DNA Polimerase Dirigida por RNA/química , Ribonuclease H/genética , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Primers do DNA , DNA Viral/genética , Cães , Deleção de Genes , Humanos , Vírus da Leucemia Murina/genética , Camundongos , Dados de Sequência Molecular , RNA Viral/biossíntese , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Relação Estrutura-Atividade , Replicação ViralRESUMO
Understanding the mechanisms of HIV-1 drug resistance is critical for developing more effective antiretroviral agents and therapies. Based on our previously described dynamic copy-choice mechanism for retroviral recombination and our observations that nucleoside reverse transcriptase inhibitors (NRTIs) increase the frequency of reverse transcriptase template switching, we propose that an equilibrium exists between (i) NRTI incorporation, NRTI excision, and resumption of DNA synthesis and (ii) degradation of the RNA template by RNase H activity, leading to dissociation of the template-primer and abrogation of HIV-1 replication. As predicted by this model, mutations in the RNase H domain that reduced the rate of RNA degradation conferred high-level resistance to 3'-azido-3'-deoxythymidine and 2,3-didehydro-2,3-dideoxythymidine by as much as 180- and 10-fold, respectively, by increasing the time available for excision of incorporated NRTIs from terminated primers. These results provide insights into the mechanism by which NRTIs inhibit HIV-1 replication and imply that mutations in RNase H could significantly contribute to drug resistance either alone or in combination with NRTI-resistance mutations in reverse transcriptase.
Assuntos
HIV-1/fisiologia , Nucleosídeos/química , Ribonuclease H/metabolismo , Replicação Viral , Antivirais/metabolismo , Linhagem Celular , Reparo do DNA , Farmacorresistência Viral , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/genética , Humanos , Nucleosídeos/metabolismo , Recombinação Genética , Inibidores da Transcriptase Reversa/metabolismo , Zidovudina/farmacologiaRESUMO
Template-switching events during reverse transcription are necessary for completion of retroviral replication and recombination. Structural determinants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that influence its template-switching frequency are not known. To identify determinants of HIV-1 RT that affect the frequency of template switching, we developed an in vivo assay in which RT template-switching events during viral replication resulted in functional reconstitution of the green fluorescent protein gene. A survey of single amino acid substitutions near the polymerase active site or deoxynucleoside triphosphate-binding site of HIV-1 RT indicated that several substitutions increased the rate of RT template switching. Several mutations associated with resistance to antiviral nucleoside analogs (K65R, L74V, E89G, Q151N, and M184I) dramatically increased RT template-switching frequencies by two- to sixfold in a single replication cycle. In contrast, substitutions in the RNase H domain (H539N, D549N) decreased the frequency of RT template switching by twofold. Depletion of intracellular nucleotide pools by hydroxyurea treatment of cells used as targets for infection resulted in a 1.8-fold increase in the frequency of RT template switching. These results indicate that the dynamic steady state between polymerase and RNase H activities is an important determinant of HIV-1 RT template switching and establish that HIV-1 recombination occurs by the previously described dynamic copy choice mechanism. These results also indicate that mutations conferring resistance to antiviral drugs can increase the frequency of RT template switching and may influence the rate of retroviral recombination and viral evolution.