PII: S097475591200623
 

Objectives: To investigate the effects of antiretroviral (ARV) drugs on hematological parameters and thymic function in HIV-uninfected newborns of HIV-infected mothers.

Study design: Cross sectional study.

Setting: Chiang-Mai University Hospital, Chiang-Mai, Thailand.

Participants/Patients: 49 HIV-uninfected and 26 HIV-infected pregnancies.

Methods: Cord blood samples of newborns from HIV-uninfected and HIV-infected mothers were collected. Hematological parameters were measured using automatic blood cell count. T-cell receptor excision circles (TRECs) levels in cord blood mononuclear cells (CBMCs), CD4⁺ and CD8⁺ T-cells were quantified using real-time PCR.

Main Outcome Measures: Hemotological parameters and thymic function.

Results: Newborn of HIV-infected mother tended to have lower mean levels of hemoglobin than those of HIV-uninfected mother (137 ± 22 vs 146 ± 17 g/L, P = 0.05). Furthermore, mean of red blood cell (RBC) counts and hematocrit and median of TRECs in CD4⁺ T-cells in the newborns of the former were significantly lower than those of the latter [3.6 ± 0.7 vs 4.8 ± 0.6 x 10¹² cells/L, P <0.001; 0.40 ± 0.07 vs 0.46 ± 0.05 L/L, P <0.001 and 0.53 (IQR: 0.03-5.76) vs 13.20 (IQR: 2.77-27.51) x 10^-3 pg/µL, P = 0.02, respectively].

Conclusion: ARV drugs altered hematological parameters and thymic function (TRECs CD4⁺ T-cells) in HIV-uninfected newborns of HIV-infected mothers.

Key Words: Adverse effects, Antiretroviral drugs, Hematology, HIV, Newborn, Thymic function .

Almost half of the estimated 40 million people living with HIV are women of childbearing age [1]. The risk of these women to transmit HIV to their infants is 15-25% when no precautions are taken [2]. The HIV-mother-to-child transmission (MTCT) rate has dramatically reduced to be less than 2% with antiretroviral (ARV) prophylaxis during pregnancy and labor as well as to the infant [3, 4]. The previous studies showed that Zidovudine (ZDV) which is a potent inhibitor of bone marrow function is associated with hematological abnormalities not only in mothers, but also in newborns, because this drug can cross the placental barrier and negatively affect fetal erythropoiesis [5-8]. Moreover, ZDV-based HAART is commonly associated with a greater negative impact on hematological parameters than ZDV-free regimens [9]. The adverse hematological effects of ARV drugs have been reported in HIV-uninfected infants, especially in their early life [10]. The frequently adverse hematological effects found are anemia, neutropenia, lymphocytopenia and thrombocytopenia [7, 11-13].

The thymus is a primary source of naïve T-cells and plays a key role in establishing and maintaining a peripheral T-cell pool [14]. Thymus reaches its maximum volume by one year of age [15]. A production of naïve T-cells by the thymus can be quantified by measuring T-cell receptor excision circles (TRECs), a DNA fragment formed during T-cell development. These DNA fragments do not replicate during mitosis and are thus diluted during cell division [16]. Previous studies demonstrated that both HIV-proteins and some antiretroviral drugs inhibited progenitor cells and thymic functions, as indicated by the frequency of TRECs [17-19]. However, an evaluation of hematological and immunological toxicity in newborn exposed to maternal ARV drugs administered during pregnancy has been limited. The aims of this study were to measure and compare hematological parameters and TRECs levels in HIV-uninfected newborn of HIV-infected mother receiving ARV drugs for prevention of HIV-MTCT with those of normal control newborn.

This study was conducted at Chiang-Mai University Hospital, Chiang-Mai, Thailand. The protocol was approved by the Faculty of Medicine Ethics Committee, Chiang-Mai University, Chiang-Mai, Thailand. All pregnant women participating in this study had signed a written informed consent. To obtain the subjects, the exclusion criteria for the study were set as follow: women with twin or multiple births, infected with other micro-organisms, and use of psychopharmaceutical drugs, illicit drugs, alcohol and tobacco during gestation. From March to December 2011, 26 HIV-infected and 49 HIV-uninfected pregnant women were enrolled. These HIV-infected women received ARV drugs [ZDV plus Lamivudine (3TC) and Lopinavir/Ritonavir (LPV/r)] during pregnancy and labor every 12 hours with adding of ZDV every 3 hours during labor and delivered vaginally or elective caesarean section. The following data were collected from all women: age, gestational age at delivery and mode of delivery. For the HIV-1 infected women, the following additional data were collected: antiretroviral prophylaxis (type and timing), CD4+ T-cell counts (cells/µL) during pregnancy and plasma HIV-1 RNA viral load measured a week before delivery (log10 copies/mL). All women in our study were given iron and folate supplementation as recommended by the Thai National Guidelines for Pregnancies [20]. Diagnosis for HIV-1 infection in infants born to HIV-1 infected mothers was performed at one and four months of age using DNA PCR (Amplicor HIV-1 DNA assay version 1.5, Roche Molecular Systems Inc., USA).

Separation of CD4⁺ and CD8⁺ T-cells: It was performed from CBMCs of the 15 HIV-uninfected newborns of HIV-infected mothers and only 12 HIV-uninfected newborns of HIV-uninfected mothers. Frozen CBMCs were thawed and washed twice in cold phosphate-buffered saline solution. CD4⁺ and CD8⁺ T-cells were separated using a magnetic cell separator (EasySep, STEMCELL Technologies, USA) according to manufacturers’ instructions. The separated CD4⁺ and CD8⁺ T-cells cells were count on hemacytometer under light microscope using Turk’s solution.

DNA was extracted from 1.5×10⁶ cells of CBMCs, separated CD4⁺and CD8⁺ T-cells using the NucleoSpin kit (Macherey-Nagel, KG., Duren, Germany) according to manufacturers’ instructions and was stored at -20^oC until used. TRECs analysis was performed by quantitative real-time PCR as described by Ometto, et al. [21] with slightly modification. The DNA amplification was carried out in a 25 µL reaction mixture containing 5 µL DNA sample or sterile distilled water as a no template control, 1×real-time PCR Master Mix (Thermo Scientific ABsolute^TM QPCR ROX Mix, Surrey, UK), 400 nM each primer (forward, 5'-CACATCCCTTTCAACCATGCT-3'; reverse, 5'-GCCAGCTGCAGGGTTTAGG-3' : GenBank sequence accession number DQ858179.1) and 200 nM of the fluorogenic probe (5'-ACACCTCTGGTTTTT GTAAAGG TGCCCAC T-3') conjugated with FAM (6-carboxy-fluorescein) at the 5'-end, and TAMRA (6-carboxy-tetramethilrhodamine) at the 3'-end. The PCR primers and the fluorogenic probe were specifically designed for the detection of human TRECs. The amplification was performed in a Rotor-Gene 6000™ (Corbett Research; Mortlake, New South Wales, Australia). The mixture was preheated at 95°C for 15 min, followed by 50 cycles at 95°C for 15 sec and 60°C for 1 min. A cycle threshold (C_T) is defined as the PCR cycle at which an increase in the fluorescence above the baseline signal is first detected. The C_T value is inversely related to the copy number of the target sequence. TRECs concentrations were calculated from a standard curve of a plasmid clone containing TRECs which run in parallel with the test. All samples and TRECs plasmid were run in duplicate. TRECs level in CBMCs was presented as concentration of TRECs per 1.5 x 10⁶ CBMCs while those in CD4⁺ and CD8⁺ T-cell was presented as concentration of TRECs per cell.

TABLE I Characteristics of HIV-infected and Uninfected Mothers and Their Newborns

TABLE II Hematological Parameters of Newborns of HIV-infected and Uninfected Mothers  

No significant difference in median of TRECs levels in CBMCs (Fig. 1a) and in CD8⁺ T-cell (Fig. 1b) was found between newborns of HIV-infected mothers and uninfected mothers. However, TRECs levels in CD4⁺ T-cell (Fig. 1c) in newborns of HIV-infected mothers were significantly lower than those of HIV-uninfected mothers.

Fig. 1 T-cell receptor excision circles (TRECs) levels of newborns of HIV-infected and uninfected mothers. (a) TRECs in CBMCs, (b) TRECs CD8+ T-cell, (c) TRECs CD4+ T-cell. TRECs levels in CBMCs were analyzed from 26 and 49 newborns of HIV-infected and uninfected mothers, respectively while TRECs CD8+ and CD4+ T-cell were analyzed from 15 and 12 newborns of HIV-infected and uninfected mothers, respectively.

The current study showed that ARV drugs (ZDV plus 3TC and LPV/r) administered to HIV-infected mother for prevention of HIV-MTCT altered the hematological parameters of newborns. Furthermore, the thymic function of these newborns was also impaired as indicated in the decrease of TRECs CD4⁺ T-cell. The previous study showed that maternal derived HIV-proteins diffusing across the placental barrier during pregnancy could reduce thymic function [19, 22]. In addition, both HIV-proteins and ARV drugs are known to inhibit progenitor cell function [17, 18]. However, in present study, the effects of HIV-proteins on thymic function might be less than those of ARV drugs since maternal viral loads in all HIV-infected mothers measured at one week before delivery were less than 40 copies/mL.

Our data are reassuring, ARV prophylaxis dose seem to significantly alter hematological indices because the mean MCV in newborns of HIV-infected mother was significantly higher than those of HIV-uninfected mothers. Moreover, some newborns (31%) of HIV-infected mother had MCV higher than the normal upper limit value (120 fL). In present study, all HIV-infected mother received ZDV and 3TC, which have been reported to induce macrocytic anemia [10, 23]. Antiretroviral drugs are routinely prescribed during the second trimester, in which hematopoiesis and lymphopoiesis are active, i.e., hepatic hematopoiesis and lymphopoiesis, spleen development, thymic education and bone marrow development. The administration of ARV drugs during the critical window of hematopoiesis and lymphopoiesis may affect the generation of these precursors [12]. Therefore, an impairement of hematopoiesis and lymphopoiesis may have contributed to the hematopoietic alteration and the reduction of thymic output, respectively. The decrease of CD4+-TRECs levels observed in the present study was consistent with the previous study by Clerici, et al. [22] that showed CD4⁺/45RA/62⁺ (naïve lymphocytes) in HIV-uninfected newborns of HIV-infected mothers received ZDV for prevention of HIV-MTCT were significantly lower than those of newborns of HIV-uninfected mothers. In contrast, Kolte, et al. [24] showed that thymic size but not thymic function (TRECs CD4⁺ T-cell) in HIV-uninfected newborns of HIV-infected mothers received ARV drugs [ZDV/ plus 3TC and LPV/r or Nevirapine (NVP)] for prevention of HIV-MTCT was significantly lower than those of HIV-uninfected mothers [24]. While our cohorts were newborns, Kolte’s cohorts were children with age of 15 months, that was probably when the side effect of ARV drugs resolved. Moreover, the maternal ethnicities between the two groups of children were different [24]. There are many parameters that have been shown to be associated with the hematological variables such as maternal ethnicity, drug use, maternal CD4⁺ T-cell count at delivery, mode of delivery and also infant gestation age, birthweight and sex [25, 26]. In the current study, these factors were controlled by matching of maternal ethnicity, maternal age at delivery, gestational age, mode of delivery, fetal sex and birthweight between the test group and control group.

The present study has a limitation in the limited volume of cord blood collected, thus levels of TRECs in CD4⁺ and CD8⁺ T-cells could be analyzed in only 12 and 15 samples of newborns of HIV-uninfected and infected mothers, respectively. Moreover, it was impossible to analyze the levels of TRECs in memory or naïve CD4⁺ and CD8⁺ T-cell sub-populations (CD45RO⁺ and CD45RA⁺), which are the immune resources. Although, the hemoglobin and hematocrit in newborns of HIV-infected mothers were significantly lower than those of HIV-uninfected mothers. We also found that, mean levels of these two hematological parameters in both groups were lower than normal range levels. These lower levels might have caused from the hematologic genetic disorders such as thalassemia and G-6-PD deficiency, frequently found in Thai population [28]. However, the hematologic genetic disorders were not used as a variable factor in our study.

In summary, our study indicates that ARV drugs (ZDV plus 3TC and LPV/r) for prevention of HIV-MTCH altered the hematological parameters and impaired thymic function (TRECs CD4⁺ T-cell) in newborns of HIV-infected mothers. These phenomena may impact the quality of life including growth, development, vaccination responses and susceptibility to infections of infants. Therefore the long-term effects of these drugs in larger population are needed to be clarified.

28. Tanphaichitr VS, Mahasandana C, Suvatte V, Yodthong S, Pung-amritt P, Seeloem J. Prevalence of hemoglobin E, alpha-thalassemia and glucose-6-phosphate dehydrogenase deficiency in 1,000 cord bloods studied in Bangkok. Southeast Asian J Trop Med Public Health. 1995;26:271-4.