Pranee Leechanachai

Modulation of P-glycoprotein expression and function by curcumin in multidrug-resistant human KB cells

Multidrug resistance (MDR) is a phenomenon that is often associated with decreased intracellular drug accumulation in the tumor cells of a patient, resulting from enhanced drug efflux. It is often related to the overexpression of P-glycoprotein (Pgp) on the surface of tumor cells, thereby reducing drug cytotoxicity. In this study, curcumin was tested for its potential ability to modulate the expression and function of Pgp in the multidrug-resistant human cervical carcinoma cell line KB-V1. Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) showed that treatment with 1, 5, and 10 microM curcumin for up to 72hr was able to significantly lower Pgp expression in KB-V1 cells. Curcumin (1-10 microM) decreased Pgp expression in a concentration-dependent manner and was also found to have the same effect on MDR1 mRNA levels. The effect of curcumin on Pgp function was demonstrated by rhodamine 123 (Rh123) accumulation and efflux in Pgp-expressing KB-V1 cells. Curcumin increased Rh123 accumulation in a concentration-dependent manner (1-55 microM) and inhibited the efflux of Rh123 from these cells, but did not affect the efflux of Rh123 from the wild-type drug-sensitive KB-3-1 cells. Treatment of drug-resistant KB-V1 cells with curcumin increased their sensitivity to vinblastine, which was consistent with an increased intracellular accumulation of Rh123. In addition, curcumin inhibited verapamil-stimulated ATPase activity and the photoaffinity labeling of Pgp with the prazosin analog [125I]iodoarylazidoprazosin in a concentration-dependent manner, demonstrating that curcumin interacts directly with the transporter. Thus, curcumin seems to be able to modulate the in vitro expression and function of Pgp in multidrug-resistant human KB-V1 cells. In summary, this study describes the duel modulation of MDR1 expression and Pgp function by the phytochemical curcumin, which may be an attractive new agent for the chemosensitization of cancer cells.

Early HIV-1 diagnosis using in-house real-time PCR amplification on dried blood spots for infants in remote and resource-limited settings.

Background:In resource-limited settings, most perinatally HIV-1-infected infants do not receive timely antiretroviral therapy because early HIV-1 diagnosis is not available or affordable. Objective:To assess the performance of a low-cost in-house real-time polymerase chain reaction (PCR) assay to detect HIV-1 DNA in infant dried blood spots (DBS). Methods:One thousand three hundred nineteen DBS collected throughout Thailand from non-breast-fed infants born to HIV-1-infected mothers were shipped at room temperature to a central laboratory.In-house real-time DNA PCR results were compared with Roche Amplicor HIV-1 DNA test (Version 1.5) results. In addition, we verified the Roche test performance on DBS sampled from 1218 other infants using as reference HIV serology result at 18 months of age. Results:Real-time DNA PCR and Roche DNA PCR results were 100% concordant. Compared with HIV serology results, the Roche test sensitivity was 98.6% (95% confidence interval: 92.6% to 100.0%) and its specificity at 4 months of age was 99.7% (95% confidence interval: 99.2% to 99.9%). Conclusions:In-house real-time PCR performed as well as the Roche test in detecting HIV-1 DNA on DBS in Thailand. Combined use of DBS and real-time PCR assays is a reliable and affordable tool to expand access to early HIV-1 diagnosis in remote and resource-limited settings, enabling timely treatment for HIV-1-infected infants.

Viral causes of unexplained anterior uveitis in Thailand

AimsTo assess the possible role of virus infection in patients with unexplained anterior uveitis (AU).MethodsIntraocular fluid and plasma samples of 30 HIV-negative AU patients who were unresponsive or poorly responsive to topical steroid therapy were analyzed for nucleic acid of cytomegalovirus (CMV), herpes simplex virus (HSV), and varicella zoster virus (VZV) by real-time polymerase chain reaction (PCR) and for intraocular antibodies against these viruses by Goldmann–Witmer coefficient (GWC) analysis. Of these 30 cases, 21 were tested for rubella virus by GWC analysis, 16 of which also had PCR assessment of aqueous for rubella virus.ResultsViral uveitis determined by either real-time PCR and/or GWC was documented in 20 out of 30 patients (67%). Of 30 paired samples tested by both methods for HSV, CMV, and VZV, 15 showed positive results (CMV (10), HSV (4), and VZV (1)). Real-time PCR was positive in 8/15 (53%), whereas GWC was positive in 10/15 (67%). Out of 10 CMV-positive patients, four had endotheliitis, two had Posner–Schlossman syndrome, and one Fuchs heterochromic uveitis syndrome (FHUS). Five out of 21 (24%) samples tested by GWC for Rubella virus were positive, three of which exhibited clinical features of FHUS.ConclusionsOur results indicate that CMV is a major cause of AU in Thailand and show that FHUS can be caused by both CMV and Rubella virus.

A study of seven rule-based algorithms for the interpretation of HIV-1 genotypic resistance data in Thailand.

Since the free therapy program was started by the Thai government, the number of patients infected by HIV-1 with access to antiretroviral drugs has increased. The selection of effective interpretation algorithms for antiretroviral drug resistance has become even more important for clinical management. In this retrospective study, the level of agreement was evaluated in 721 antiretroviral-therapy failing HIV-1 subjects. Regarding genetic diversity, about 89% was recognized as non-B variants (CRF01_AE). The level of complete concordant interpretation score in all seven algorithms was recognized in non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) (67%), but not in nucleoside reverse transcriptase inhibitors (NRTIs) (52%). Over 10% of the major discordance score with TRUGENE was revealed in didanosine (Agence Nationale de Recherches sur le SIDA[ANRS]; Detroit Medical Centre [DMC]), abacavir (ANRS; Centre Hospitalier de Luxembourg [CHL]), and also with delavirdine, indinavir and amprenavir (Grupo de Aconselhamento Virológico [GAV]). A good to excellent agreement range of kappa scores was detected for most antiretroviral drugs. However, poor agreement with the TRUGENE system (k<0.40) was seen in the ANRS system with didanosine, abacavir and lopinavir; GAV system in indinavir and amprenavir; and DMC system in ritonavir. These might be an option for resource limited countries when selecting the use of a low cost or free algorithm interpretation, which has excellent agreement as the U.S. Food and Drug Administration (FDA)-approved TRUGENE commercial system.

Intraocular and plasma HIV-1 RNA loads and HIV uveitis.

Objective:The objective of this study was to analyze human immunodeficiency virus (HIV) dynamics across the blood–retinal barrier and to determine whether the high levels of HIV in the eye are associated with any ocular disorders in HIV-infected patients. Design:This study included a prospective case series of 40 HIV-positive patients with uveitis. Intervention:Clinical and laboratory examinations included plasma and intraocular HIV-1 RNA loads as well as the clinical manifestations of uveitis. Results:Intraocular HIV-1 RNA was detected in 32% (13/40) of HIV-positive patients with uveitis. Intraocular HIV-1 RNA loads were associated with high HIV-1 RNA plasma loads (P < 0.001) and not being on HAART therapy (P = 0.005). In addition, detectable intraocular HIV-1 RNA levels were higher in patients with the absence of retinal lesions (P = 0.008). In three patients, the HIV load in the eye largely exceeded that of plasma. These three patients had all bilateral anterior uveitis and/or vitritis without retinal lesions and exhibited no evidence of other intraocular infectious agents causing uveitis than HIV itself. Conclusion:The eye can form a sanctuary where HIV might replicate and cause an inflammatory reaction.

Development of TaqMan real-time polymerase chain reaction for the detection and identification of Penicillium marneffei.

Penicillium marneffei is a dimorphic fungus, which is endemic in Southeast Asia and responsible for emerging opportunistic infections. Diagnosis of penicilliosis may be difficult when few yeast cells are present, while a gold standard diagnosis technique requires long‐term culture. In order to provide a more rapid and accurate diagnosis, we developed a TaqMan real‐time PCR to detect and identify P. marneffei DNA coding for 5.8S rRNA in purified yeast DNA and clinical samples. All P. marneffei DNA preparations could be detected using specific primers and TaqMan probe. The assay has a sensitivity to detect at least 10 yeast cells in seeded blood. Moreover, it can detect P. marneffei DNA in peripheral blood samples and blood‐culture bottles. Therefore, the real‐time PCR assay may represent a potential tool for early diagnosis of penicilliosis marneffei.

The diagnostic value of intraocular fluid analysis by polymerase chain reaction in Thai patients with uveitis

Uveitis is a major cause of severe visual impairment throughout the world and can be initiated by various infectious and non-infectious causes. Early recognition of specific infections is important as the treatment with antimicrobial agents might stop the progression or even cure the eye disease. To determine the infectious causes of uveitis in Thailand, intraocular fluid samples of 100 HIV-negative patients and 47 HIV-positive patients with uveitis were examined using real-time PCR analysis for herpes simplex virus, varicella zoster virus, cytomegalovirus and Toxoplasma gondii. Positive PCR results were found in 33/100 (33%) HIV-negative patients and in 33/47 (70%) HIV-positive patients with uveitis. In Thailand, cytomegalovirus was identified as the most frequent cause of infectious uveitis in both HIV-negative and HIV-positive patients (49 and 91%, respectively). PCR analysis of intraocular samples in uveitis was a valuable diagnostic assay. The pattern of uveitis observed in the Far East differs from that found in the West.

Analysis of Real-Time SYBR-Polymerase Chain Reaction Cycle Threshold for Diagnosis of the α-Thalassemia-1 Southeast Asian Type Deletion: Application to Carrier Screening and Prenatal Diagnosis of Hb Bart's Hydrops Fetalis

Without gel electrophoresis and specific probes, the two tubes real-time SYBR-polymerase chain reaction (SYBR-PCR) was setup by using different primer sets: P1/P2 for the detection of wild type α-globin gene alleles and P1/P3 for detection of the allele bearing the Southeast Asian (SEA) type (– –SEA) deletion. Analyses of the cycle threshold (CT) values obtained by each primer set together with a delta-cycle threshold (ΔCT) and CT ratio, showed that lower CT values generated by primer sets P1/P2 and P1/P3 were observed in normal and Hb Barts hydrops fetalis subjects, respectively. In heterozygous subjects the CT values generated by both sets of primers were similar to each other. There was no overlapping of ΔCT and CT ratio between normal, heterozygous and Hb Barts hydrops fetalis subjects. Therefore, the two tubes real-time SYBR-PCR could represent a rapid, cost effective, high-throughput assay for screening of carriers and prenatal diagnosis of α-thalassemia-1 (α-thal-1) with the SEA type (– –SEA) deletion.

Increased Hb A2 Values in an HIV-1-Infected Patient Receiving Antiretroviral Drugs: A Pitfall for Thalassemia Antenatal Diagnosis

We report a human immunodeficiency virus-1 (HIV-1)-infected couple, where the woman in the 11th week of gestation, carried a Hb E trait. She and her spouse were referred to the hemoglobinopathy counselors. Her spouses blood was subsequently tested and showed an increased Hb A2 value. However, his red cell indices and osmotic fragility test were different from those found in β-thalassemia (β-thal) carriers. The β-thal genes were investigated further and no mutations were observed. Therefore, it is unlikely that he is a β-thal carrier and the increased Hb A2 value is a result of receiving antiretroviral drugs. As antenatal thalassemia screening becomes more widespread, measuring the Hb A2 values should be taken in all HIV-1-infected couples before the initiation of antiretroviral drugs to rule out misdiagnosis of β-thal. However, if these tests are not available, the results of the red cell indices and osmotic fragility test should be considered as they may provide great value for β-thal investigations.

Effect of haematological alterations on thalassaemia investigation in HIV‐1‐infected Thai patients receiving antiretroviral therapy

To evaluate the effect of haematological alterations resulting from antiretroviral therapy (ART) on the diagnosis of thalassaemia carriers in HIV‐1‐infected Thai patients.