Role of serum endotoxin, FGF19, TLR2, TNF-α, IL-12 and IL-10 in NAFLD-associated T2DM pathogenesis: Insights into Th1 bias and protective mechanisms.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) in non-obese patients is pathophysiologically distinct, exhibiting common immunological link with type-2 diabetes mellitus (T2DM). This study aims to delineate the role of Toll-like receptor 2 (TLR2)-mediated immuno-modulation along with its association with fibroblast growth factor receptor 4 (FGFR4) and its ligand fibroblast growth factor 19 (FGF19) in the pathogenesis of NAFLD without or with T2DM. METHODOLOGY: Blood samples were collected from patients with NAFLD (n = 90), NAFLD with T2DM (n = 90) and healthy cohorts (n = 90) with consent and clinical records. Real-time polymerase chain reaction (PCR), enzyme-linked immunoassay (ELIZA) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to analyze messenger ribonucleic acid (mRNA), protein expression and gene polymorphism. RESULTS: The molecular genetic analysis revealed the prevalence of variant allele(A) in FGFR4 gene in both cases compared to controls. The mRNA expression of FGF19 and TLR2 exhibited significant upregulation in NAFLD without T2DM compared to NAFLD with T2DM. Tumor necrosis factor-α (TNF-α) and interleukin-12 (IL-12) showed upregulation in both disease cohorts compared to control while IL-10 showed significant downregulation in NAFLD with T2DM compared to the other two cohorts. Correlation analysis between FGF19 and TLR2 revealed significant positive association in both NAFLD with and without T2DM. The Th1:Th2 ratio showed significant upregulation in NAFLD with T2DM compared to NAFLD without T2DM. CONCLUSION: In conclusion, elevated serum endotoxin levels appear to contribute to NAFLD and T2DM development. Upregulated FGF19 seems to be protective against developing T2DM in NAFLD patients. Higher TLR2, TNF-α and IL-12 expression in NAFLD without T2DM suggests a Th1 bias in its pathogenesis, while reduced IL-10 in NAFLD with T2DM implies a more skewed Th1 state in this condition.

Gut microbial dysbiosis in the pathogenesis of leukemia: an immune-based perspective.

Leukemias are a set of clonal hematopoietic malignant diseases that develop in the bone marrow. Several factors influence leukemia development and progression. Among these, the gut microbiota is a major factor influencing a wide array of its processes. The gut microbial composition is linked to the risk of tumor development and the host's ability to respond to treatment, mostly due to the immune-modulatory effects of their metabolites. Despite such strong evidence, its role in the development of hematologic malignancies still requires attention of investigators worldwide. In this review, we make an effort to discuss the role of host gut microbiota-immune crosstalk in leukemia development and progression. Additionally, we highlight certain recently developed strategies to modify the gut microbial composition that may help to overcome dysbiosis in leukemia patients in the near future.

Oxidative stress-A key determinant of complications and negative outcome in hepatitis E virus infected pregnancies: A comprehensive account involving cases from northeast India.

Regulated oxidative stress (OS) is important during pregnancy. Sporadic studies suggest the significance of deregulated OS in hepatitis E virus (HEV) infected pregnancy, but with limited reactive oxygen species (ROS) or antioxidant markers. The present novel study, therefore, aimed to evaluate the significance of ROS-antioxidant imbalance and resulting altered OS in HEV infected pregnancy complications like preterm delivery (PTD) and outcome. Difference in serum levels of ROS and antioxidant panel of markers were evaluated by ELISA for HEV immunoglobulin M RNA positive genotype 1 cases (including acute [acute viral hepatitis, AVH] and fulminant [fulminant hepatic failure, FHF] cases) and healthy term delivery subjects, and analyzed statistically. Direct ROS marker H2 O2 levels and indirect OS marker for DNA damage 8-hydroxy-2'-deoxyguanosine was significantly increased in HEV-cases compared to controls, and was associated and prognostic factor for PTD and fetal death in HEV cases. A comparatively lower total serum antioxidant capacity was observed in the FHF cases compared to the control subjects and the AVH cases. Glutathione (GSH) levels and superoxide dismutase (SOD) activity were significantly associated with PTD in the FHF sub-cohorts (p = 0.017) and AVH sub-cohorts (p < 0.001), respectively, and was associated with poor prognosis in HEV cases. The serum H2 O2 levels were found to be negatively correlated with SOD activity (p = 0.016) and GSH levels (p = 0.001) in the HEV-AVH cases; and positively correlated with the viral load in HEV cases (p = 0.023). The ROS-antioxidant imbalance resulting OS plays a detrimental associative role in HEV infected pregnancy complications like PTD and adverse pregnancy outcomes; and holds therapeutic significance.

Impact of TNF-α profile in recurrent pregnancy loss pathogenesis: A patient based study from Assam.

BACKGROUND: Lacunae exist in understanding the underlying etiology in majority of recurrent pregnancy loss (RPL) cases. Given the significance of regulated immune-modulation in pregnancy, and the central role of pro-inflammatory TNF-α plays in it; this study targeted to appraise the significance of TNF-α profile in RPL pathogenesis in an ethnically distinct population from Assam, India. METHODS: Term delivery, medically terminated pregnancy (MTP) and RPL cases (based on ASRM criteria) were enrolled with no anatomical and chromosomal abnormalities or pathological infections; and blood and/or placenta/product of conceptus (POC) tissue samples were collected with informed consent. Serum level and tissue level TNF-α expression profile were screened using specific molecular tools, and was correlated with TNF-α -308 G/A genotype; for its association with RPL predisposition. RESULTS: A significant gestation specific increase in serum TNF-α levels was observed in MTP cases (19.932 ± 4.407 pg/mL) compared to term delivery subjects (p = 0.001), while a comparable levels were observed with RPL cases (22.709 ± 5.833 pg/mL) (p = 0.646). A site specific (POC) increased expression was observed in RPL compared to MTP cases at both at transcript (6.37 ± 3.714 folds) and protein levels. The TNF-α -308 variant genotype was associated with increased predisposition to RPL (OR = 1.721) compared to MTP as well as significantly increased serum TNF-α levels (p = 0.017); especially in subjects with a homozygous TNF-α -308 A/A genotype. CONCLUSION: Our data emphasizes on the importance of site specific TNF-α expression levels in RPL pathogenesis in the studied population, and underlines its importance in screening, clinical stratification, and therapeutics by molecular targeting using TNF-α inhibitors.

Increased homocysteine mediated oxidative stress as key determinant of hepatitis E virus (HEV) infected pregnancy complication and outcome: A study from Northeast India.

With the background of association of oxidative stress and Hepatitis E virus (HEV) infection in pregnancy complications the present novel study aimed to evaluate the significance of changes in maternal homocysteine levels and the related mechanism(s) in the pathophysiology of HEV related pregnancy complications and negative outcomes. Term delivery (TD, N = 194) and HEV-IgM positive pregnancy cases [N = 109] were enrolled. Serum and placental homocysteine levels were evaluated by ELISA and immunofluorescence and in turn correlated with serum Vitamin B12 levels. Distribution of variant MTHFR C➔T and TYMS1494del6bp genotyping were studied by PCR-RFLP. Differential folate receptor alpha (FR-α) expression in placenta was evaluated by real-time PCR and immunofluorescence respectively. The HEV viral load was significantly higher in both FHF and AVH cases. Higher serum homocysteine levels was associated with preterm delivery (PTD) and fetal death in HEV infected cases and was significantly inversely correlated with serum VitaminB12 levels in HEV cases. Placental homocysteine expression was upregulated in HEV cases, and in cases with negative pregnancy outcome. A Homocysteine level was associated with MTHFR C677T status. Genetic alterations in folate pathway was associated with increased risk of PTD in HEV infected pregnancy cases, disease severity, and negative pregnancy outcome in AVH and FHF groups. FR-α expression was downregulated in placental tissues of HEV infected pregnancy.Placental stress caused by HEV inflicted increased homocysteine due to alterations in maternal vitamin B12 levels and folate pathway components is detrimental mechanism in PTD and negative pregnancy outcome in HEV infected pregnancy cases and holds prognostic and therapeutic significance.

Exploring the p53 connection of cervical cancer pathogenesis involving north-east Indian patients.

BACKGROUND: As per WHO, Cervical cancer (CaCx) is a global issue, being the fourth common cancer in women with incidence rate of 13.1 per 1 lakh women globally and accounting for 311000 deaths in the year 2018 itself globally. The molecular pathogenesis in Human papillomavirus (HPV) infected cases is inconclusive. The detection of molecular factors leading to progression of CaCx can be important in the diagnosis and management of the disease. p53 a known tumor suppressor gene having a regulative role in cell cycle has been highlighted as key factor in the prevention of cancer but its significance in CaCx cases has been variably documented. The present study therefore targeted to evaluate the significance of p53 profile in CaCx cases in ethnically distinct northeast Indian population. METHODS: Blood and Tissue samples (N = 85) of cervical cancer patients were collected and screening for HPV was performed using PCR. Thereafter the differential mRNA expression(qPCR), Immunohistochemistry, Mutation (PCR direct sequencing method) of p53 was studied. Further p53 epigenetic profiling was done by Methylation specific PCR (MS-PCR) and western blotting by using p53 acetylation specific antibodies. RESULTS: Our findings revealed that the downregulation of p53 was associated with the progression of disease and the variation in downregulation based on p53 polymorphism was observed. Further hypermethylation and deacetylation of p53 was also found to be associated with the pathogenesis of CaCx. The downregulated expression and hypermethylation of p53 in lower grade of CaCx, together established its association with the progression of CaCx from lower to severe grade. CONCLUSION: Therefore, in CaCx patients of northeast Indian population, malfunctioning of p53 is found to have significant role in cervical cancer progression.

Impact of modulation of telomerase and cancer stem-cell marker OCT4 axis in cervical cancer pathogenesis with underlying HPV16 infection.

Lacunae exist in the molecular event(s) specificity associated with cervical cancer (CaCx) pathogenesis. The present study aimed to evaluate the significance of telomerase-cervical cancer stem cells (CSCs) modulation in CaCx pathogenesis with underlying HPV16 infection. The study included HPV16 positive cases only (N = 65) of the total enrolled cases from Northeast India. The analysis of viral load and the differential messenger RNA expression of E6, E7, hTERT, hTR, and cancer stem-cell markers was studied by real-time polymerase chain reaction. Further the protein and colocalization study for E6, hTERT, and oct4 was performed by immunofluorescence. The real-time polymerase chain reaction based analysis showed an upregulation of HPV16 viral oncoprotein E6 and E7, and telomerase component hTERT and hTR expression and their correlation in CaCx susceptibility and severity. The hTERT expression correlated with viral load; while the E6 and telomerase protein expression colocalized in the nucleus. The CSCs marker octamer-binding transcription factor 4 (OCT4) was significantly upregulated in CaCx cases, was associated with CaCx susceptibility and severity, and colocalized with E6 expression in the nucleus as revealed from the immunofluorescence studies. To conclude, the telomerase-OCT4 axis modulation holds key in HPV16 CaCx pathogenesis mediated by HPV16 E6 viral oncoprotein expression, and underlines its potential for therapeutic targeting.

Deregulated TNF-Alpha Levels Along with HPV Genotype 16 Infection Are Associated with Pathogenesis of Cervical Neoplasia in Northeast Indian Patients.

Multiple factors are associated with human papillomavirus (HPV) infection related cervical anomalies and its progression to cervical carcinoma (CaCx), but data vary with respect to the underlying HPV genotype and with population being studied. No data are available regarding the role of immunological imbalance in HPV infected CaCx pathogenesis from Northeast India, which has an ethnically distinct population, and was aimed to be addressed through this study. The study included 76 CaCx cases, 25 cervical intraepithelial neoplasia (CIN) cases, and 50 healthy female controls. HPV screening and genotyping were performed by PCR. Differential expression of tumor necrosis factor alpha (TNF-α) was studied at serum level by enzyme-linked immunosorbent assay and tissue level by immunohistochemistry and messenger RNA (mRNA) level by real-time PCR. The data were correlated with interferon gamma (IFN-γ) and NF-κßp65 levels at protein level, as well as HPV16 E6 and E7 expression at transcript level statistically. HPV infection and HPV16 genotype were predominant in the studied cohort. TNF-α was found to be downregulated at both mRNA and protein levels in CaCx cases compared to controls; and the gradient downregulation correlated with progression of the disease from normal→CIN→CaCx. TNF-α expression correlated with insufficient modulation of both IFN-γ and NF-κßp65. The HPV16 E6 and E7 transcripts were found to be sharply upregulated in CaCx cases strongly inversely correlated with the TNF-α expression. Significant role of TNF-α downregulation associated with insufficient IFN-γ and total NF-κßp65 modulation and the resulting significant upregulation of viral transcripts E6 and E7 are key to the HPV16 infection mediated CaCx pathogenesis in northeast Indian patients.

Associative role of TYMS6bpdel polymorphism and resulting hyperhomocysteinemia in the pathogenesis of preterm delivery and associated complications: A study from Northeast India.

Aberrations including genetic alterations in folate pathway are detrimental in multiple disease pathogenesis, including pregnancy. The present study is based on the screening of the associative role of TYMS 14946bp deletion(del) polymorphism and associated hyperhomocysteinemia in susceptibility to preterm delivery (PTD), which is strongly associated with neonatal mortality and morbidity. METHODS: A total of 209 PTD cases {extremely preterm (n=22), very preterm (n=43) and moderately preterm (n=144)} and 194 term delivery cases were evaluated for TYMS 14946bp deletion and its association with preterm delivery, pregnancy outcome, baby birth weight and homocysteine estimation. RESULTS: The results showed that the distribution of TYMS 14946bp del/del genotype significantly increased the risk of PTD [OR=2.801, p=0.002] and is associated with fetal death. The TYMS 6bp ins/del and 6bp del/del genotype was associated with low birth weight (LBW) compared to 6bp ins/ins genotype in both term and PTD groups, and in case of very (p=0.024) and moderately (p=0.045) sub-cohorts of PTD significantly. Elevated serum homocysteine levels were significantly associated with PTD (p<0.001) and fetal death (p=0.013); and was also found to significantly correlate with TYMS 14946bp del/del genotype in all the pregnancy cases (p=0.008). TYMS 6bp del/del genotype was associated with higher homocysteine levels compared to ins/ins (p=0.005) and ins/del (p=0.062) genotypes within the PTD group. CONCLUSION: The study provides crucial information regarding the importance of TYMS6bpdel/del genotype and associated hyperhomocysteinemia in susceptibility to PTD, fetal death and LBW; and thus indicating their prognostic significance of TYMS 6bp del/del genotype in PTD which is of clinical importance.

Preterm delivery and associated negative pregnancy outcome - A tale of faulty progesterone receptor signalling pathway and linked derailed immunomodulation: A study from Northeast India.

Preterm delivery (PTD) is one of the potent contributor of neonatal mortality and morbidity, and the underlying cause in some situation is elusive. This study attempts to delineate the association of deregulation in progesterone receptor (PR) pathway and deleterious immune responses in predisposing patients to PTD in Northeast India, a region with high rate of PTD cases. A total of 109 cases of PTD and 100 term delivery cases were enrolled with all clinical details. The PTD cases were stratified based on gestation age at delivery. The differential expression of PR and key downstream effectors and cytokines were evaluated for correlation with PTD susceptibility, gestational period, and pregnancy outcome. The results indicated a sharp downregulation in PR expression is associated with PTD susceptibility, lower gestational period and negative pregnancy outcome. The PR downstream effector PIBF was also found to be downregulated in PTD, and is associated with gestational period and negative pregnancy outcome. The downregulation of PR and PIBF expression was found to correlate with a predominant Th1 state with higher CD56+NK cell counts and pro-inflammatory burst lead by hyper TNF-α, NF-kB and IFNγ expression, and complicated by lower IL10 expression, contributing to PTD as well as negative pregnancy outcome in the PTD cases. TNF-α expression in placenta inversely correlated with placental PR expression. To conclude, deregulation in PR pathway is a hallmark of preterm delivery and negative pregnancy outcome. Differential expression of several markers such as PR, PIBF and TNF-α has prognostic significance, and hence is of clinical significance.

MTHFR (C677T) polymorphism and PR (PROGINS) mutation as genetic factors for preterm delivery, fetal death and low birth weight: A Northeast Indian population based study.

UNLABELLED: Preterm delivery (PTD) is one of the most significant contributors to neonatal mortality, morbidity, and long-term adverse consequences for health; with highest prevalence reported from India. The incidence of PTD is alarmingly very high in Northeast India. The objective of the present study is to evaluate the associative role of MTHFR gene polymorphism and progesterone receptor (PR) gene mutation (PROGINS) in susceptibility to PTD, negative pregnancy outcome and low birth weights (LBW) in Northeast Indian population. METHODS: A total of 209 PTD cases {extreme preterm (< 28 weeks of gestation, n = 22), very preterm (28-32 weeks of gestation, n = 43) and moderate preterm (32-37 weeks of gestation, n = 144) and 194 term delivery cases were studied for MTHFR C677T polymorphism and PR (PROGINS) gene mutation. Statistical analysis was performed using SPSS software. RESULTS: Distribution of MTHFR and PR mutation was higher in PTD cases. Presence of MTHFR C677T polymorphism was significantly associated and resulted in the increased risk of PTD (p < 0.001), negative pregnancy outcome (p < 0.001) and LBW (p = 0.001); more significantly in extreme and very preterm cases. Presence of PR mutation (PROGINS) also resulted in increased risk of PTD and negative pregnancy outcome; but importantly was found to increase the risk of LBW significantly in case of very preterm (p < 0.001) and moderately preterm (p < 0.001) delivery cases. CONCLUSIONS: Both MTHFR C677T polymorphism and PR (PROGINS) mutation are evident genetic risk factors associated with the susceptibility of PTD, negative pregnancy outcome and LBW. MTHFR C677T may be used as a prognostic marker to stratify subpopulation of pregnancy cases predisposed to PTD; thereby controlling the risks associated with PTD.

Evidence of extrahepatic replication of hepatitis E virus in human placenta.

The incidence and severity of hepatitis E virus (HEV) infection in pregnant women is high in developing countries. Transplacental transmission of HEV in the third trimester of pregnancy has been found to be associated with high fetal mortality. Based on this evidence and in the absence of reports on HEV replication in extrahepatic sites, this study was carried out to investigate if HEV replication occurs in the placenta of infected mothers. The study included 68 acute viral hepatitis (AVH) and 22 acute liver failure (ALF) pregnant patients. Viral RNA was extracted from blood and placenta. HEV replication in placenta was confirmed by a replicative negative-strand-specific reverse transcriptase PCR. Viral load was estimated by real-time PCR. Immunohistochemical studies were also carried out for in situ detection of HEV in placental tissue sections. Replicative HEV RNA was detectable only in the placenta in ALF and AVH cases and not in blood samples. Positive staining of placental tissue sections with HEV antibody against the viral structural protein ORF3 was observed. HEV replication in placenta also correlated with fetal and maternal mortality in ALF patients. It is demonstrated for the first time that HEV replication occurs in human placenta and that placenta may be a site of extrahepatic replication of HEV in humans.

Role of polymorphic N-acetyl transferase2 and cytochrome P4502E1 gene in antituberculosis treatment-induced hepatitis.

BACKGROUND AND AIM: Antituberculosis drugs, isoniazid and rifampicin, in combination, are known to develop drug-induced hepatotoxicity (DIH). A higher risk of DIH during antituberculosis treatment (ATT) has been reported in the Indian subcontinent compared to its Western counterparts. The role of genetic factors in a higher incidence of ATT hepatotoxicity in the Indian population is still unclear. The present study was aimed at investigating the role of the N-acetyltransferase2 (NAT2) and cytochrome P4502E1 (CYP2E1) gene polymorphisms in ATT hepatotoxicity. METHODS: The study population included 218 pulmonary tuberculosis patients who were started on ATT and followed up for the occurrence of ATT-induced hepatitis. The genetic polymorphisms of the NAT2 and CYP2E1 genes were studied by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The occurrence of DIH was 18.8% (41/218). There was a higher prevalence of NAT2 slow-acetylator genotypes in DIH (70.73%) compared to non-DIH (44.63%; P < 0.05). The frequency of the NAT2*5/*7 and NAT2*6/*7 genotypes was significantly higher in DIH than non-DIH (19.51% vs 6.78%, and 19.51% vs 5.08%). No association of the CYP2E1 RsaI polymorphism could be demonstrated with DIH. However, the DraI C/D genotype of the CYP2E1 gene was mostly prevalent in DIH (85.37%), compared to non-DIH (64.41%) (P < 0.05). Slow-acetylator status and the CYP2E1 C/D or C/C genotype together showed a higher frequency in DIH (65.85%) compared to non-DIH (28.81%) (P < 0.0001). CONCLUSION: The study demonstrates for the first time a possible association between the DraI polymorphism of the CYP2E1 gene and the risk of ATT hepatotoxicity. The genotyping of the NAT2 and CYP2E1 genes could possibly identify the groups at highest risk of developing ATT-induced hepatitis prior to medication.

High viral load and deregulation of the progesterone receptor signaling pathway: association with hepatitis E-related poor pregnancy outcome.

BACKGROUND & AIMS: Hepatitis E virus (HEV) infection is associated with high maternal and fetal mortalities. A prospective study was undertaken to evaluate the role of viral and host factors in HEV related pregnancy outcomes. METHODS: The study included HEV infected pregnancy cases; acute viral hepatitis (AVH), n=100 and fulminant hepatic failure (FHF), n=43, and healthy pregnancy cases, n=50. HEV genotypes and viremia were studied by nucleotide sequencing and real time PCR, respectively. Progesterone receptor (PR) gene mutations (PROGINS) were studied by PCR, PR expression at the mRNA and protein levels in the placenta were studied by semi-quantitative RT-PCR and immunohistochemistry, respectively. Progesterone induced blocking factor (PIBF) expression was studied by RT-PCR in blood. Serum interleukin-10 (IL-10) and interleukin-12 (IL-12) levels were assayed by ELISA. RESULTS: HEV viral load was significantly higher in FHF than AVH (p<0.001) and in cases with fetal mortality in AVH (p=0.001) and FHF (p=0.018). PROGINS were predominant in FHF compared to AVH (p=0.26) and showed reduced mRNA and protein expression. The risk of fetal mortality in AVH was two times higher (OR, 2.190; CI, 0.303-15.85) and maternal and fetal mortalities in FHF were 4-fold (OR, 4.0; CI, 0.363-44.113) increased in PROGINS carriers. PR and PIBF expression was lower in AVH and even lower in FHF compared to healthy controls. The higher IL-12/IL-10 ratio observed in FHF compared to other groups correlated with fetal mortality in AVH and FHF (p<0.001). CONCLUSIONS: In conclusion, reduced expression of PR and PIBF, a higher IL-12/IL-10 ratio, and a high viral load results in poor pregnancy outcome in Hepatitis E.

Role of CYP2E1 gene polymorphisms association with hepatitis risk in Northeast India.

AIM: To investigate hepatitis virus, genetic and environmental factors, and their interactions in predisposing patients to liver diseases in Northeast India. METHODS: A total of 104 jaundice patients and 124 community controls were included. Serological analysis was performed by routine enzyme-linked immunosorbent assay, and nucleic acid testing for hepatitis viruses was done by polymerase chain reaction (PCR), followed by PCR direct sequencing for viral genotyping. Cytochrome P450 2E1 (CYP2E1) polymorphism was studied by PCR-restriction fragment length polymorphism. Nitrite and volatile nitrosamines in indigenous foods consumed routinely by the Northeast Indian ethnic population were estimated by Griess's reagent and GC-MS, respectively. RESULTS: Hepatitis A virus (HAV) infection was predominantly prevalent (36.5%) in our cohort, followed by hepatitis B virus (HBV), hepatitis E virus (HEV) and hepatitis C virus. HBV genotype D and HEV genotype 1 were the most dominant. CYP2E1 c1/c2 genotype frequency was comparatively higher in alcoholic (P < 0.0001, OR = 30.5) and cryptogenic (P = 0.014, OR = 8.714) patients, and was associated with significantly higher hepatitis risk (P = 0.0.007, OR = 6.489). Mutant C allele of Cyp2E1 DraI frequency was comparatively higher in HAV (P = 0.006), alcoholic (P = 0.003) and cryptogenic (P = 0.014) cases, and was associated with overall hepatitis risk (P = 0.026, OR = 5.083). Indigenous foods, Gundruk, Kharoli, betel leaf and nuts were found to have the highest nitrite content. CONCLUSION: Apart from viral factors, CYP2E1 polymorphism might be associated with increased risk of liver diseases in Northeast India. Indigenous foods that contain nitrite and nitrosamine might be an associated risk factor.