Variation In Expression Levels Research Articles

Quantitative expression evaluation of PRAME gene in osteosarcoma.

In a previous study, our group observed that 68% of the osteosarcoma (OS) samples presented PRAME (Preferentially Expressed Antigen in Melanoma) gene expression. In this work, we propose to investigate quantitatively gene expression of PRAME in distinct patients groups. 61 osteosarcoma samples, from 3 distinct patients groups were selected for this study: (1) Patients younger than 10 years old at diagnosis, (2) Patients that had poor evolution of disease and (3) Patients that were in remission of disease and had treatment with no intercurrences) PRAME gene expression levels were obtained using quantitative Real-Time Polymerase Chain Reaction method (qRT-PCR). Clinical parameters were collected from patient's medical charts. Results demonstrated an increase in PRAME gene expression in all samples, with high variation in expression levels, when considering all samples and when analyzed in each group. In addition, no statistical difference was found when considering clinical data collected or patients groups. PRAME gene expression quantitative investigation did not bring any complementary information beyond of what had already been observed in other qualitative investigations published by our group, there is no relation between PRAME gene expression levels and disease evolution. However, the findings in this work contribute for validation PRAME gene expression as a good biomarker to OS, which, in the future, may allow identification circulating tumor cell or molecules to contribute with early diagnostic of metastasis, a genuine problem in OS that determinate flattening in survival curves.

Phenobarbital, a hepatic metabolic enzyme inducer, inhibits preneoplastic hepatic lesions with expression of selective autophagy receptor p62 and ER-phagy receptor FAM134B in high-fat diet-fed rats through the inhibition of ER stress.

We investigated the role of endoplasmic reticulum (ER)-phagy in NAFLD-related hepatocarcinogenesis in high-fat diet (HFD)-fed and/or phenobarbital (PB)-treated rats by clustering the expression levels of the selective autophagy receptor p62 and the ER-phagy-specific receptor FAM134B in preneoplastic hepatic lesions. We obtained four clusters with variable expression levels of p62 and FAM134B in preneoplastic lesions, and a variable population of clusters in each group. PB administration increased the clusters with high expression levels of p62 while HFD feeding increased the clusters with high expression levels of both p62 and FAM134B. The areas of preneoplastic lesions of these clusters were significantly increased than those of other clusters with low expression levels of p62 and FAM134B. The combination of HFD feeding with PB counteracted the effects of each other, and the cluster composition was similar to that in the control group. The results were associated with decreased gene expression of ER stress, inflammatory cytokine, autophagy, and increased expression of antioxidant enzyme. The present study demonstrated that clustering analysis is useful for understanding the role of autophagy in each preneoplastic lesion, and that HFD feeding increased preneoplastic lesions through the inhibition of ER-phagy, which was cancelled with PB administration through the induction of ER-phagy.

Mutational Bias and Natural Selection Driving the Synonymous Codon Usage of Single-Exon Genes in Rice (Oryza sativa L.)

The relative abundance of single-exon genes (SEGs) in higher plants is perplexing. Uncovering the synonymous codon usage pattern of SEGs will benefit for further understanding their underlying evolutionary mechanism in plants. Using internal correspondence analysis (ICA), we reveal a significant difference in synonymous codon usage between SEGs and multiple-exon genes (MEGs) in rice. But the effect is weak, accounting for only 2.61% of the total codon usage variability. SEGs and MEGs contain remarkably different base compositions, and are under clearly differential selective constraints, with the former having higher GC content, and evolving relatively faster during evolution. In the group of SEGs, the variability in synonymous codon usage among genes is partially due to the variations in GC content, gene function, and gene expression level, which accounts for 22.03%, 5.99%, and 3.32% of the total codon usage variability, respectively. Therefore, mutational bias and natural selection should work on affecting the synonymous codon usage of SEGs in rice. These findings may deepen our knowledge for the mechanisms of origination, differentiation and regulation of SEGs in plants.

A clinically feasible diagnostic spectro-histology built on SERS-nanotags for multiplex detection and grading of breast cancer biomarkers

Simultaneous detection of multiple biomarkers is always an obstacle in immunohistochemical (IHC) analysis. Herein, a straightforward spectroscopy-driven histopathologic approach has emerged as a paradigm of Raman-label (RL) nanoparticle probes for multiplex recognition of pertinent biomarkers in heterogeneous breast cancer. The nanoprobes are constructed by sequential incorporation of signature RL and target specific antibodies on gold nanoparticles, which are coined as Raman-Label surface enhanced Raman scattering (RL-SERS)-nanotags to evaluate simultaneous recognition of clinically relevant breast cancer biomarkers i.e., estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor2 (HER2). As a foot-step assessment, breast cancer cell lines having varied expression levels of the triple biomarkers are investigated. Subsequently, the optimized detection strategy using RL-SERS-nanotags is subjected to clinically confirmed, retrospective formalin-fixed paraffin embedded (FFPE) breast cancer tissue samples to fish out the quick response of singleplex, duplex as well as triplex biomarkers in a single tissue specimen by adopting a ratiometric signature RL-SERS analysis which enabled to minimize the false negative and positive results. Significantly, sensitivity and specificity of 95% and 92% for singleplex, 88% and 85% for duplex, and 75% and 67% for triplex biomarker has been achieved by assessing specific Raman fingerprints of the respective SERS-tags. Furthermore, a semi-quantitative evaluation of HER2 grading between 4+/2+/1+ tissue samples was also achieved by the Raman intensity profiling of the SERS-tag, which is fully in agreement with the expensive fluorescent in situ hybridization analysis. Additionally, the practical diagnostic applicability of RL-SERS-tags has been achieved by large area SERS imaging of areas covering 0.5–5 mm2 within 45 min. These findings unveil an accurate, inexpensive and multiplex diagnostic modality envisaging large-scale multi-centric clinical validation.

Stability evaluation and validation of appropriate reference genes for real-time PCR expression analysis of immune genes in the rohu (Labeo rohita) skin following argulosis

Argulosis is one of the most unrestrained economically significant freshwater fish ectoparasitic diseases. Proper selection or normalization of the best reference gene governs the accuracy of results of gene expression studies using real-time PCR. Earlier studies in rohu carp (Labeo rohita) have used reference genes without proper validation. Here, seven candidate reference genes viz., acidic ribosomal protein (ARP0), glyceraldehyde 3-phosphate dehydrogenase, RNA polymerase II (RPo), elongation factor1α (EF1α), α- tubulin (AT), ribosomal protein L 10, and β-actin were evaluated using four algorithms (geNorm, BestKeeper, NormFinder and ∆Ct) followed by a comprehensive gene expression analysis using skin tissue of rohu at varied time points of experimental Argulus siamensis infection. ARP0 and EF1α were found to be the most stable whereas RPo and AT were considered as least stable genes based on basal expression level and variation in expression levels. Validation of candidate reference genes was undertaken by looking into the expression of six immune-related genes using the two most stable and two least stable genes as housekeeping genes in Argulus-infected rohu skin at different time points of infection. An increased expression of immune genes indicated the role of inflammation and the immune modulation process at the site of attachment of parasites in governing infection.

STR-based feature extraction and selection for genetic feature discovery in neurological disease genes

Gene expression, often determined by single nucleotide polymorphisms, short repeated sequences known as short tandem repeats (STRs), structural variants, and environmental factors, provides means for an organism to produce gene products necessary to live. Variation in expression levels, sometimes known as enrichment patterns, has been associated with disease progression. Thus, the STR enrichment patterns have recently gained interest as potential genetic markers for disease progression. However, to the best of our knowledge, we are unaware of any study that evaluates and explores STRs, particularly trinucleotide sequences, as machine learning features for classifying neurological disease genes for the purpose of discovering genetic features. Thus, in this paper, we proposed a new metric and a novel feature extraction and selection algorithm based on statistically significant STR-based features and their respective enrichment patterns to create a statistically significant feature set. The proposed new metric has shown that the neurological disease family genes have a non-random AA, AT, TA, TG, and TT enrichment pattern. This is an important result, as it supports prior research that has established that certain trinucleotides, such as AAT, ATA, ATT, TAT, and TTA, are favored during protein misfolding. In contrast, trinucleotides, such as TAA, TAG, and TGA, are favored during premature termination codon mutations as they are stop codons. This suggests that the metric has the potential to identify patterns that may be genetic features in a sample of neurological genes. Moreover, the practical performance and high prediction results of the statistically significant STR-based feature set indicate that variations in STR enrichment patterns can distinguish neurological disease genes. In conclusion, the proposed approach may have the potential to discover differential genetic features for other diseases.

Low-Density Lipoprotein Pathway Is a Ubiquitous Metabolic Vulnerability in High Grade Glioma Amenable for Nanotherapeutic Delivery

Metabolic reprogramming, through increased uptake of cholesterol in the form of low-density lipoproteins (LDL), is one way by which cancer cells, including high grade gliomas (HGG), maintain their rapid growth. In this study, we determined LDL receptor (LDLR) expression in HGGs using immunohistochemistry on tissue microarrays from intra- and inter tumour regions of 36 adult and 133 paediatric patients to confirm LDLR as a therapeutic target. Additionally, we analysed expression levels in three representative cell line models to confirm their future utility to test LDLR-targeted nanoparticle uptake, retention, and cytotoxicity. Our data show widespread LDLR expression in adult and paediatric cohorts, but with significant intra-tumour variation observed between the core and either rim or invasive regions of adult HGG. Expression was independent of paediatric tumour grade or identified clinicopathological factors. LDLR-expressing tumour cells localized preferentially within perivascular niches, also with significant adult intra-tumour variation. We demonstrated variable levels of LDLR expression in all cell lines, confirming their suitability as models to test LDLR-targeted nanotherapy delivery. Overall, our study reveals the LDLR pathway as a ubiquitous metabolic vulnerability in high grade gliomas across all ages, amenable to future consideration of LDL-mediated nanoparticle/drug delivery to potentially circumvent tumour heterogeneity.

BnaMPK3s promote organ size by interacting with BnaARF2s in Brassica napus.

BnaMPK3s promote organ size by interacting with BnaARF2s in Brassica napus.

LCN2-Fungal siderophore-iron binding and uptake leads to oxidative stress and cell death in hepatocellular carcinoma cell line HepG2

Microorganisms produce non-ribosomal peptides called siderophores for the purpose of iron acquisition. Mammalian immune system is well-known for producing small secretory proteins called lipocalins upon bacterial infection. These proteins sequester siderophores produced by invading bacterial pathogens rendering them unable to acquire iron from the host. However, this is not their sole function. In addition to transferrin and lactoferrin, lipocalins are also known to transport siderophore-bound iron to the host cells. While binding of bacterial siderophores with human lipocalin is well studied, binding of the fungal counterpart is still not confirmed and fully understood. Apart from pathogen-affected cells, developing cancerous cells also show varying expression level of different proteins including those involved in iron transport. The possibility of exogenous fungal siderophore-mediated iron transport via lipocalin and its receptor in mammalian cells has not yet been explored much. In present investigation we have checked differential expression of human lipocalin, LCN2 in hepatocellular carcinoma cell lines HepG2 as well as its normal counterpart WRL-68 and computationally determined the feasibility of LCN2 binding with fungal siderophore. Further in case of a stable complex being formed, whether this complex has the ability to transport iron through its specific receptor was assessed. Also, we have tried to explore possible mechanism of fungal-siderophore mediated oxidative stress leading to significant cell death in cancerous cells. This study will thus be useful towards finding a new way of treating hepatocellular carcinoma via inducing siderophore-mediated cell death in cancerous cells. Communicated by Ramaswamy H. Sarma

Genome-Wide Analysis of ATP Binding Cassette (ABC) Transporters in Peach (Prunus persica) and Identification of a Gene PpABCC1 Involved in Anthocyanin Accumulation.

The ATP-binding cassette (ABC) transporter family is a large and diverse protein superfamily that plays various roles in plant growth and development. Although the ABC transporters are known to aid in the transport of a wide range of substrates across biological membranes, their role in anthocyanin transport remains elusive. In this study, we identified a total of 132 putative ABC genes in the peach genome, and they were phylogenetically classified into eight subfamilies. Variations in spatial and temporal gene expression levels resulted in differential expression patterns of PpABC family members in various tissues of peach. PpABCC1 was identified as the most likely candidate gene essential for anthocyanin accumulation in peach. Transient overexpression of PpABCC1 caused a significant increase in anthocyanin accumulation in tobacco leaves and peach fruit, whereas virus-induced gene silencing of PpABCC1 in the blood-fleshed peach resulted in a significant decrease in anthocyanin accumulation. The PpABCC1 promoter contained an MYB binding cis-element, and it could be activated by anthocyanin-activator PpMYB10.1 based on yeast one-hybrid and dual luciferase assays. Thus, it seems that PpABCC1 plays a crucial role in anthocyanin accumulation in peach. Our results provide a new insight into the vacuolar transport of anthocyanins in peach.

DELongSeq for efficient detection of differential isoform expression from long-read RNA-seq data.

Conventional gene expression quantification approaches, such as microarrays or quantitative PCR, have similar variations of estimates for all genes. However, next-generation short-read or long-read sequencing use read counts to estimate expression levels with much wider dynamic ranges. In addition to the accuracy of estimated isoform expression, efficiency, which measures the degree of estimation uncertainty, is also an important factor for downstream analysis. Instead of read count, we present DELongSeq, which employs information matrix of EM algorithm to quantify uncertainty of isoform expression estimates to improve estimation efficiency. DELongSeq uses random-effect regression model for the analysis of DE isoform, in that within-study variation represents variable precision in isoform expression estimation and between-study variation represents variation in isoform expression levels across samples. More importantly, DELongSeq allows 1 case versus 1 control comparison of differential expression, which has specific application scenarios in precision medicine (such as before versus after treatment, or tumor versus stromal tissues). Through extensive simulations and analysis of several RNA-Seq datasets, we show that the uncertainty quantification approach is computationally reliable, and can improve the power of differential expression (DE) analysis of isoforms or genes. In summary, DELongSeq allows for efficient detection of differential isoform/gene expression from long-read RNA-Seq data.

A Mechanism Exploration for the Yi-Fei-San-Jie Formula against Non-Small-Cell Lung Cancer Based on UPLC-MS/MS, Network Pharmacology, and In Silico Verification.

Non-small-cell lung cancer (NSCLC) is one of the most prevalent cancers worldwide. A Yi-Fei-San-Jie formula (YFSJF), widely used in NSCLC treatment in south China, has been validated in clinical studies. However, the pharmacological mechanism behind it remains unclear. In this study, 73 compounds were identified using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), with 58 enrolled in network pharmacology. The protein-protein interaction network, functional enrichment analysis, and compound-target-pathway network were constructed using 74 overlapping targets from 58 drugs and NSCLC. YFSJF has many targets and pathways in the fight against NSCLC. PIK3R1, PIK3CA, and AKT1 were identified as key targets, and the PI3K/AKT pathway was identified as the key pathway. According to the Human Protein Atlas (THPA) database and the Kaplan-Meier Online website, the three key targets had varying expression levels in normal and abnormal tissues and were linked to prognosis. Molecular docking and dynamics simulations verified that hub compounds have a strong affinity with three critical targets. This study revealed multiple compounds, targets, and pathways for YFSJF against NSCLC and suggested that YFSJF might inhibit PIK3R1, PIK3CA, and AKT1 to suppress the PI3K/AKT pathway and play its pharmacological role.

ADGR: Admixture-Informed Differential Gene Regulation.

The regulatory elements in proximal and distal regions of genes are involved in the regulation of gene expression. Risk alleles in intronic and intergenic regions may alter gene expression by modifying the binding affinity and stability of diverse DNA-binding proteins implicated in gene expression regulation. By focusing on the local ancestral structure of coding and regulatory regions using the paired whole-genome sequence and tissue-wide transcriptome datasets from the Genotype-Tissue Expression project, we investigated the impact of genetic variants, in aggregate, on tissue-specific gene expression regulation. Local ancestral origins of the coding region, immediate and distant upstream regions, and distal regulatory region were determined using RFMix with the reference panel from the 1000 Genomes Project. For each tissue, inter-individual variation of gene expression levels explained by concordant or discordant local ancestry between coding and regulatory regions was estimated. Compared to European, African descent showed more frequent change in local ancestral structure, with shorter haplotype blocks. The expression level of the Adenosine Deaminase Like (ADAL) gene was significantly associated with admixed ancestral structure in the regulatory region across multiple tissue types. Further validations are required to understand the impact of the local ancestral structure of regulatory regions on gene expression regulation in humans and other species.

Transcripts of the Prostate Cancer-Associated Gene ANO7 Are Retained in the Nuclei of Prostatic Epithelial Cells.

Prostate cancer affects millions of men globally. The prostate cancer-associated gene ANO7 is downregulated in advanced prostate cancer, whereas benign tissue and low-grade cancer display varying expression levels. In this study, we assess the spatial correlation between ANO7 mRNA and protein using fluorescent in situ hybridization and immunohistochemistry for the detection of mRNA and protein in parallel sections of tissue microarrays prepared from radical prostatectomy samples. We show that ANO7 mRNA and protein expression correlate in prostate tissue. Furthermore, we show that ANO7 mRNA is enriched in the nuclei of the luminal cells at 89% in benign ducts and low-grade cancer, and at 78% in high-grade cancer. The nuclear enrichment of ANO7 mRNA was validated in prostate cancer cell lines 22Rv1 and MDA PCa 2b using droplet digital polymerase chain reaction (ddPCR) on RNA isolated from nuclear and cytoplasmic fractions of the cells. The nuclear enrichment of ANO7 mRNA was compared to the nuclearly-enriched lncRNA MALAT1, confirming the surprisingly high nuclear retention of ANO7 mRNA. ANO7 has been suggested to be used as a diagnostic marker and a target for immunotherapy, but a full comprehension of its role in prostate cancer progression is currently lacking. Our results contribute to a better understanding of the dynamics of ANO7 expression in prostatic tissue.

Anti-cancer potential of substituted "amino-alkyl-rhodamine" derivatives against MCF-7 human breast cancer cell line.

Breast cancer is the most prevalent diagnosed cancer among women and the main cause of morbidity and mortality. As for breast cancer, MCF-7 cells are an important candidate since they are widely utilized in research for estrogen receptor (ER)-positive breast cancer cell assays, and various sub-clones have been identified to reflect different classes of ER-positive tumors with varied levels of nuclear receptor expression. Rhodamines and its derivatives have shown a great interest over the past two decades due to their excellent structural and spectroscopic properties. Rhodamine derivatives have been widely investigated for their mitochondrial targeting and chemotherapeutic properties. Rhodamine derivatives, in particular, have been widely investigated for their therapeutic properties. In this regard, several studies have shown that rhodamine dye derivatives have promising in vitro and in vivo therapeutic efficacy. The present study deals with potential anticancer activity of few synthesized rhodamine derivatives against MCF-7 cell lines.

Altered Expression of the HLA-G and IL10RB Genes in Placental Tissue of Women with Recurrent Pregnancy Loss.

Several lines of evidence strongly suggest that the contribution of human leukocyte antigen-G (HLA-G) and interleukin 10 receptor (IL10R) to maternal immunological tolerance toward paternal alloantigens of the embryo limits the activation and function of the maternal immune system. This study is aimed to assess the varia-tion of the mRNA expression levels of HLA-G and IL10RB genes in placental tissue of women with recurrent pregnancy loss (RPL). Placental tissue samples were collected from 78 women with a history of at least two consecutive miscarriages and 40 healthy women with no history of pregnancy loss. The expression of HLA-G and IL10RB in placental tissue specimens was evaluated by the quantitative real-time PCR (qPCR) method. Moreover, the correlation be-tween the expression levels of these genes and clinicopathological parameters was analyzed. The results showed that the expression of HLA-G was down-regulated in placental tissues samples of RPL patients compared to healthy subjects, while the expression of IL10RB was up-regulated, but none of them was statistically significant (p-value > 0.05). The mRNA expression levels of HLA-G and IL10RB in placental tissue of RPL patients were negatively correlated with age and number of miscarriages (p-value > 0.05). A significant positive correlation was observed between the expression levels of HLA-G and IL10RB in women with RPL (p-value < 0.05). The altered expression of HLA-G and IL10RB in placental tissue may contribute to the pathogenesis of RPL and therefore serve as potential therapeutic targets for its prevention.

Continental vs. tropical breed: Immunity comparison under heat stress conditions utilizing qRT-PCR technique

Understanding the responses of animals to seasonal heat stress on the genomic level has led to the identification of genes implicated in thermal stress reaction mechanisms. In this study, the relative gene expression of Interleukin-10 (IL-10), an anti-inflammatory cytokine and biomarker for heat stress-mediated immune modulation, was observed during the summer and winter seasons in continental and tropical sheep breeds, namely, Hungarian Indigenous Tsigai and White Dorper, employing quantitative real-time polymerase chain reaction (qRT-PCR). Temperature-humidity indices (THI) were calculated to assess heat stress levels. The results indicate that IL-10 CT-values were significantly higher during the summer, when heat stress prevails, in both sheep breeds compared to winter. While the White Dorper exhibited a higher numerical value for the summer relative gene expression ratio (16.2) compared to the Hungarian Indigenous Tsigai (12.3), no significant differences in CT values were observed between breeds or among sexes. These findings suggest the immune-adaptive characteristics of the two sheep breeds during seasonal heat stress. The variation of IL-10 gene expression levels between the two breeds can be attributed to their geographical origins; the White Dorper emerging from arid subtropical South Africa and the Hungarian Indigenous Tsigai Sheep thriving in the seasonally harsh Carpathian climate for centuries.

Sampling strategies for sugarcane using either clonal replicates or diverse genotypes can bias the conclusions of RNA-Seq studies.

A key procedure for ensuring statistical confidence in differential gene expression analyses is to use biological replicates to compare distinct groups. Biological replicates allow the estimation of the residual variation in the gene expression levels among samples of a given experimental condition. In sugarcane, it is possible to obtain an estimate of residual variability at two levels: among samples of distinct genotypes of the same experimental treatment, or clonal replicates of the same genotype. The sequencing costs are often a limitation to leveraging both these levels in the same study, stressing the relevance of efforts to determine an appropriate experimental design. We aim to investigate this question by comparing the transcriptional profiles of young sugarcane culms with different sucrose levels using both sampling strategies. Our results show that clonal replicates provided enough statistical power to identify nearly three times more deferentially expressed genes than the more diverse strategy. However, it resulted in potentially less meaningful biological results, because many of the significant genes were likely related to the particular genotype of choice, rather than representing a common expression profile for the compared groups. This study supports the development of sound experimental designs in new studies regarding differential expression for sugarcane.

In-Cell Western Assay in Ferroptosis.

Ferroptosis is a regulated form of cell death caused by the excessive accumulation of iron-dependent lipid peroxidation. It has been implicated in various pathological processes and diseases, and its modulation involves multiple proteins associated with iron and lipid metabolism. To better understand these mechanisms and monitor the ferroptosis process, there is a need for reliable and high-throughput methods to evaluate variations in protein expression levels. In-Cell Western assays provide a simple and rapid assay method for detecting biomarkers and signaling proteins in whole cells using antibodies. This assay involves seeding cells in microtiter plates, followed by fixation/permeabilization and subsequent labeling with primary antibodies and infrared-conjugated secondary antibodies. In this chapter, we introduce the protocol for the In-Cell Western assay for detecting intracellular proteins during ferroptosis.

Inflammation induced by excessive expression of CD4 T-cell lymphocytes & proinflammatory cytokines results in bacterial dissemination in Mycobacterium tuberculosis-infected guinea pig model.

As CD4+ and CD8+ T lymphocyte numbers decline, the conventional, localized forms of tuberculosis shift to the atypical, disseminated forms. Variations in lymphocyte and immune cell expression levels affect how tuberculosis manifests in disseminated forms. Understanding the relationship between lymphocyte counts (CD4+ and CD8+) and pro-inflammatory cytokines such as tumour necrosis factor-alpha, interleukin-12 and interferon, we may therefore be able to shed light on how infections spread and suggest potential biomarkers for these immune factors. In this study, 15 guinea pigs were infected with Mycobacterium tuberculosis (M.tb) H37Rv strain and grouped into three groups of five each for further investigation. Serum samples and bronchoalveolar lavage (BAL) fluid were examined for the expression of pro-inflammatory cytokines and T-cell subsets in guinea pigs infected with pulmonary tuberculosis and disseminated tuberculosis. We found that M.tb escapes macrophages due to pro-inflammatory cytokine dysregulation. Despite the protective immunity created by T-cells and cytokines, M.tb bacilli may spread to other organs due to inflammation induced by these immune components. A high number of T-cells and stimulated cytokine production are involved in triggering inflammation after necrotic tissue develops and tuberculosis spreads. Our findings imply that increased bacilli in the spleen at the eighth week of infection may be caused by the overexpression of CD4+ T-cell lymphocyte subsets and cytokines that generated inflammation during the fourth week of infection. This is a pilot study with a small sample size and less assertive inference. Larger studies would be helpful to validate the results of the present investigation.