Aberrant Dendritic Cell Subsets in Patients with Myasthenia Gravis and Related Clinical Features.

INTRODUCTION: Dendritic cells (DCs) play critical roles in the pathogenesis of myasthenia gravis (MG), and a series of DC-based experimental strategies for MG have recently been developed. However, the definite roles of different DC subsets in the mechanism of MG have scarcely been covered by previous studies. The present study aimed to investigate the levels of three main DC subsets, plasmacytoid DCs (pDCs) (CD303 positive) and two distinct subsets of conventional DCs (cDCs), namely CD1c+ cDCs and CD141+ cDCs, in MG patients and analyze related clinical features. METHODS: From January 2016 to December 2020, 160 newly diagnosed MG patients and matched healthy controls (n = 160) were included in the study, and their clinical data were collected. The blood samples from MG patients before treatment and controls were collected for flow cytometry analysis. A total of 14 MG thymoma, 24 control thymoma, and 3 thymic cysts were used to immunostain the DC subsets. RESULTS: The flow cytometry analysis showed a significantly higher frequency of circulating pDCs, CD1c+ cDCs, and CD141+ cDCs in MG patients than in healthy controls (p < 0.001 for all). Patients with early-onset MG (<50 years old) had a lower frequency of circulating pDCs but a higher frequency of circulating CD1c+ cDCs than those with late-onset MG (≥50 years old) (p = 0.014 and p = 0.025, respectively). The frequency of circulating pDCs was positively associated with the clinical severity of late-onset MG patients (r = 0.613, p < 0.001). 64.3% (9/14) of MG thymoma is of type B2 under the World Health Organization classification, which is higher than that in control thymoma (33.3%, 8/24) (p = 0.019). For type B2 thymoma, there were significantly more pDCs but fewer CD1c+ cDCs in MG thymoma than in the controls. CONCLUSION: The distribution of aberrant pDCs, CD1c+ cDCs, and CD141+ cDCs in MG patients displayed age- and thymoma-related differences, which may contribute to the impaired immune tolerance and lead to the onset of MG.

Asymmetric limb weakness in Guillain-Barré syndrome: Three case reports.

BACKGROUND: Guillain-Barré syndrome (GBS) is an autoimmune-mediated peripheral neuropathy characterized by symmetric weakness. Asymmetric weakness in GBS is uncommon and may be easily confused with other differential diagnoses. We herein present three cases of asymmetric GBS and review the literature on this atypical subtype of GBS in order to describe the characteristics of asymmetric GBS and to provide experience for clinicians. CASE SUMMARY: Different from patients in the previous reports, our patients showed persistent asymmetric limb weakness from the onset to recovery phase. All three patients were serologically positive for antecedent infections. Two of the three cases had IgG antibodies against ganglioside GM1. Two patients received immunotherapy including intravenous immunoglobulin and plasma exchange, while one patient received only supportive treatment. Autoantibodies against gangliosides, asymmetry of congenital development of blood-nerve barrier and limb use may contribute to the development of asymmetric limb weakness in GBS. CONCLUSION: Asymmetric GBS may be a rare clinical variant and should be considered when a patient develops acute and progressive asymmetric limb weakness. The differences in clinical features and prognosis between asymmetric GBS and classic GBS deserve further investigation in a large study.

[Severe encephalitis caused by infection of Rickettsia felis: a case report].

Rickettsia felis is a Gram-negative prokaryotic organism that is obligatorically parasitic in cells. Cat fleas are the main vector of Rickettsia felis. Clinical symptoms of human infection with Rickettsia felis include fever, fatigue, headache, macular papules, and eschar. There are few reports of serious complications or deaths due to infection of Rickettsia felis. A confirmed case of severe encephalitis caused by Rickettsia felis infection was admitted to neurology department of Affiliated Hospital of Jining Medical University on January 29, 2020. After comprehensive treatment of antiviral by acyclovir, mannitol dehydration to reduce intracranial pressure, human immunoglobulin to regulateimmunity, minocycline hydrochloride capsule, levofloxacin mesylate and sodium chloride against Rickettsia felis infection, control of seizure and pulmonary infection, the patient's condition was improved and then discharged. By summarizing the experience and lessons in the treatment of this patient, we hope to remind everyone to strengthen the understanding of Rickettsia felis infection.

Guillain-Barré syndrome in Eastern China: A study of 595 patients.

BACKGROUND AND PURPOSE: This study aimed to investigate geographical differences in the clinical features of Guillain-Barré syndrome (GBS) between patients from our region in Eastern China and patients from other areas. METHODS: A total of 595 patients fulfilling the diagnostic criteria ​for GBS or its variants were included from two large hospitals located in Eastern China. Data collection included demographics, antecedent events, clinical presentation and signs, electrophysiological subtypes, treatment, complications during hospitalization, clinical severity at nadir, and outcome at 12 months, and these data were compared to data from a study conducted in Southern China and the Europe/Americas section of the International GBS Outcome Study. RESULTS: The median (interquartile range) age of patients was 50 (36-61) years, the ratio of men to women was 1.2, and 49% of patients had antecedent events. Patients in our region of Eastern China had pure motor predominant GBS (158/340, 46%) and 30% (103/340) had complications during hospitalization. Patients aged over 60 years had a lower frequency of antecedent infections and single, axonal subtypes, but higher disability scores at entry, nadir, and 12 months. When compared with the Europe/Americas data, our patients had a lower frequency of antecedent infection (46% vs. 63%), cranial nerve involvement (43% vs. 49%), sensory deficits (45% vs. 69%), pain (19% vs. 57%) and mechanical ventilation (11% vs. 17%), but a higher frequency of axonal subtype (35% vs. 6%). There was a higher frequency of patients with antecedent gastroenteritis (16% vs. 8%), mechanical ventilation (11% vs. 8%) and axonal subtypes (35% vs. 19%) in our region in Eastern China than in Southern China. CONCLUSIONS: Patients with GBS in Eastern China showed significant clinical heterogeneity and differences when compared to other geographic areas.

Co-occurrence of Moyamoya syndrome and Kartagener syndrome caused by the mutation of DNAH5 and DNAH11: a case report.

BACKGROUND: Kartagener syndrome is an autosomal recessive inherited disorder of primary ciliary dyskinesia. Moyamoya syndrome refers to a moyamoya angiopathy associated with other neurological and/or extra-neurological symptoms, or due to a well identified acquired or inherited cause. We herein reported a case of a 48-year-old woman who was favored the diagnosis of Kartagener syndrome and moyamoya syndrome. The whole genome sequencing and bioinformatics analysis showed a homozygotic nonsense mutation in the dynein, axonemal, heavy chain (DNAH) 5 gene, and heterozygotic missense mutation in the DNAH11 gene. This is the first report of the co-occurrence of the two rare diseases. CASE PRESENTATION: A case of a 48-year-old woman was presented with hemiplegia and slurred speech. The magnetic resonance imaging of the brain confirmed acute cerebral infarction in the right basal ganglia region, semi-oval center, insular lobe, and frontal parietal lobe. The electrocardiogram showed inverted "P" waves in L1 and AVL on left-sided chest leads and computed tomography scan of the chest showed bronchiectasis changes, cardiac shadow and apex on the right side, and situs inversus of aortic arch position. The digital subtraction angiography showed inversion of the aortic arch, and bilateral internal carotid arteries are occluded from the ophthalmic segment. The clinical, radiological, and laboratory findings made the diagnosis of Kartagener syndrome and moyamoya syndrome. The whole genome sequencing and bioinformatics analysis showed a homozygotic nonsense mutation in DNAH5 gene, and heterozygotic missense mutation in the DNAH11 gene. CONCLUSION: The combined mutation of DNAH5 and DNAH11 may lead to the overlapping dysfunction of motile and nonmotile cilia, which contribute to the co-occurrence of Kartagener syndrome and moyamoya syndrome. Our report deserves further confirm by more case reports.

Antecedent infections in Guillain-Barré syndrome: a single-center, prospective study.

OBJECTIVE: To investigate the spectrum of antecedent infections in Chinese patients with Guillain-Barré syndrome (GBS) and analyze the infections-related clinical phenotypes locally. METHODS: A prospective case-control study of 150 patients diagnosed with GBS and age- and sex-matched neurological and healthy controls was performed to investigate recent infections of 14 pathogens serologically and collect the clinical data during a follow-up of 12 months. RESULTS: In total, 53% of patients with GBS had a positive serology for recent infection, including Campylobacter jejuni (27%), influenza A (17%) and B (16%), hepatitis A virus (5%), dengue virus (3%), cytomegalovirus (3%), Epstein-Barr virus (3%), Mycoplasma pneumoniae (2%), herpes simplex virus (2%), varicella-zoster virus (1%), and rubella virus (1%). Serology for infections of hepatitis E virus, Haemophilus influenzae, and Zika virus was negative. There was a higher frequency of C. jejuni, influenza A, influenza B, and hepatitis A virus infections in GBS patients than both the neurological and healthy controls. C. jejuni infection was more frequent in younger GBS patients and was associated with antibodies against GM1, GalNAc-GD1a, and GM1:galactocerebroside complex. Influenza B infection was associated with a pure motor form of GBS. INTERPRETATION: C. jejuni, influenza A, influenza B, and hepatitis A virus serve as the most common cause of antecedent infections in GBS locally. Influenza B-related GBS may represent a pure motor phenotype. Differences in the infectious spectrum worldwide may contribute to the geographical clinical heterogeneity of GBS.

Chronic inflammatory demyelinating polyneuropathy with anti-NF155 IgG4 in China.

Patients with chronic inflammatory demyelinating polyneuropathy (CIDP) seropositive for autoantibodies against nodal and paranodal proteins display distinct clinical presentations. We herein tested for autoantibodies against neurofascin (NF) 155, NF186, contactin-associated protein 1 and contactin-1 and investigated the autoantibody-related clinical features in 29 patients with CIDP from China. Six patients with anti-NF155 IgG4 antibodies displayed younger age of onset and poor response to intravenous immunoglobulin than seronegative patients. One patient had anti-NF186 IgG antibody and no patients had anti-contactin-associated protein 1 or anti-contactin-1 antibodies. Clinical features of CIDP patients with anti-NF155 antibodies in China were similar to those reported in other countries.

Intravenous immunoglobulin promotes the proliferation of CD4+CD25+ Foxp3+ regulatory T cells and the cytokines secretion in patients with Guillain-Barré syndrome in vitro.

Intravenous immunoglobulin (IVIg) serves as the first line therapy in Guillain-Barré syndrome (GBS), however, its action mechanism remains unknown. We hereby stimulated peripheral blood mononuclear cells (PBMCs) from patients with GBS and healthy controls using IVIg and an IgG-derived natural Treg epitopes, namely Tregitopes. Our results showed that IVIg significantly promoted both the expansion of CD4+CD25+Foxp3+ regulatory T cells (Tregs) and secretion of IL-10 and TGF-ß1 while Tregitopes promoted secretion of IL-10 and TGF-ß1 only. Further study is necessary to elucidate the molecular mechanism of IVIg and Tregitopes on Tregs and the secretion of IL-10 and TGF-ß1 in GBS.

Memory B cells in Guillain-Barré syndrome.

IgG autoantibodies against gangliosides show the highest titers at the disease onset of axonal Guillain-Barré syndrome (GBS), in which there are no IgM anti-ganglioside antibodies. We hypothesized that memory B cells take part in the development of producing IgG autoantibodies. In this study, we analyzed the memory B cells in patients with GBS using flow cytometry. There was significantly higher percentage of memory B cells in patients with GBS than the healthy controls. The Spearman correlation analysis demonstrated that increased percentage of memory B cells was positively correlated with the clinical severity of the patients with GBS. Our study provides the evidences that memory B cells may be involved in mechanism of GBS.

Vinpocetine Inhibited the CpG Oligodeoxynucleotide-induced Immune Response in Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) exert dual roles in immune responses through inducing inflammation and maintaining immune tolerance. A switch of pDC phenotype from pro-inflammation to tolerance has therapeutic promise in the treatment of autoimmune diseases. Vinpocetine, a vasoactive vinca alkaloid extracted from the periwinkle plant, has recently emerged as an immunomodulatory agent. In this study, we evaluated the effect of vinpocetine on phenotype of pDCs isolated from C57BL/6 mice and explored its possible mechanism. Our data showed that vinpocetine significantly downregulated the expression of CD40, CD80, and CD86 on pDCs and increased the expression of translocator protein (TSPO), the specific receptor of vinpocetine, in pDCs. Vinpocetine significantly inhibited the Toll-like receptor 9 signaling pathway and reduced the secretion of related cytokines in pDCs through TSPO. Furthermore, viability of pDCs was significantly promoted by vinpocetine. These findings imply that vinpocetine serves as an immunomodulatory agent for pDCs and may be applied for the treatment of pDCs-related autoimmune diseases.

MicroRNA expression profiling in Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is an acute inflammatory autoimmune disease affecting the peripheral nervous system. MicroRNAs (miRNAs) are a class of small noncoding RNAs that play critical roles in the process of various diseases. The miRNAs in GBS were less studied. In this study, using microarray technology, we found two miRNAs including has-miR-4717-5p and has-miR-642b-5p were upregulated in patients with GBS, which were further confirmed by PCR analysis. Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that the dysregulated miRNAs may be involved in the mechanism of GBS by affecting the cellular differentiation, cell survival and axonal outgrowth.

Increased plasmacytoid dendritic cells in Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is a post-infectious autoimmune disease. Dendritic cells (DCs) can recognize the pathogen and modulate the host immune response. Exploring the role of DCs in GBS will help our understanding of the disease development. In this study, we aimed to analyze plasmacytoid and conventional DCs in peripheral blood of patients with GBS at different stages of the disease: acute phase as well as early and late recovery phases. There was a significant increase of plasmacytoid DCs in the acute phase (p=0.03 vs healthy donors). There was a positive correlation between percentage of plasmacytoid DCs and the clinical severity of patients with GBS (r=0.61, p<0.001). Quantitative polymerase chain reaction and flow cytometry confirmed the aberrant plasmacytoid DCs in GBS. Thus, plasmacytoid DCs may participate in the development of GBS.

Suppressive oligodeoxynucleotides induced tolerogenic plasmacytoid dendritic cells and ameliorated the experimental autoimmune neuritis.

Toll-like receptor (TLR) 9, recognizing different ligands, confers distinct features of plasmacytoid dendritic cells (pDCs). Our previous study demonstrated a role for TLR9 in the mechanism of experimental autoimmune neuritis (EAN). In this study, we explored whether suppressive oligodeoxynucleotides (sODN) could induce tolerogenic pDCs via TLR9 and thus promote the recovery of EAN. Effects of different TLR9 ligands, CpG ODN and sODN on P0 180-199 peptide-stimulated pDCs were measured by detecting the expression of co-stimulatory molecules, indoleamine 2,3-dioxygenase (IDO), secretion of Th1- and Th2-type cytokines and the TLR9 signaling pathway. CpG ODN- or sODN-treated pDCs were intravenously injected into the EAN mice and their effects were compared. Our data showed that P0180-199 peptides significantly promoted mRNA expression of co-stimulatory molecules (CD40, CD80 and CD86) in pDCs and induced secretion of Th1-type cytokines. Treatment of CpG ODN aggravated the effects of P0 180-199 peptides on pDCs; however, sODN had the opposite effects and significantly upregulated the IDO expression in pDCs. Further analysis showed that MYD88 is necessary for sODN to modulate the TLR9/NF-κB signaling in pDCs. Finally, the sODN-treated pDCs significantly promoted recovery of the EAN mice. Taken together, sODN could induce tolerogenic pDCs and thus ameliorate the EAN.

A novel and functional variant within the ATG5 gene promoter in sporadic Parkinson's disease.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Majority of PD are sporadic, for which genetic causes remain largely unknown. Alpha-synuclein, the main component of Lewy bodies, plays a central role in the PD pathogenesis. Macroautophagy is a highly conserved cellular process that digests dysfunctional macromolecules and damaged organelles. Accumulating evidence indicates that macroautophagy (hereafter referred to as autophagy) is involved in alpha-synuclein degradation. Dysregulation of autophagy has been observed in the brain tissues from PD patients and animal models. We hypothesized that change expression levels of autophagy-related genes (ATG), including ATG5, may contribute to PD. In this study, we genetically and functionally analyzed the ATG5 gene promoter in groups of sporadic PD patients and ethnic-matched healthy controls. A novel heterozygous variant, 106774459T>A, was identified in one female patient, but in none of controls, which significantly enhanced transcriptional activities of the ATG5 gene promoter. Furthermore, ATG5 gene expression level in the PD patient was significantly elevated than that in controls. Four novel heterozygous variants, 106774423C>A, 106774418C>A, 106774382C>A and 106774206G>A, were only found in controls. The variant, 106774464C>T, and SNP-106774030A>G (rs510432) were found in PD patients and controls with similar frequencies. Collectively, the variant identified in PD patient may change ATG5 protein levels and alter autophagy activities, contributing to PD onset as a risk factor.

Genetic analysis of the ATG7 gene promoter in sporadic Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease. The majority of PD cases are sporadic, for which genetic causes and underlying molecular mechanisms remain largely unclear. Autophagy, a highly conserved cellular process that governs the breakdown of long-lived proteins and organelles, has been involved in the degradation of α-synuclein (α-Syn), the main component of Lewy bodies. Accumulating evidence implicates deregulation of autophagy in the development and progression of sporadic PD. Altered autophagic gene expression has been observed in the brain tissues from PD patients and animal models. We hypothesized that changes in expression levels of autophagy-related genes (ATGs), rather than mutations associated with amino acid changes, may contribute to PD onset. In this study, the ATG7 gene promoter was sequenced bi-directionally in groups of sporadic PD patients and ethnic-matched healthy controls. As predicted, four novel heterozygous variants, 11313449G>A, 11313811T>C, 11313913G>A and 11314041G>A, were identified in five PD patients, but in none of the controls, which significantly decreased transcriptional activities of the ATG7 gene promoter. Two novel heterozygous variants, 11312947G>A and 11313006C>G, were only found in controls, which did not affect transcriptional activities of the ATG7 gene promoter. The other five novel variants were found in PD patients and controls with similar frequencies. Taken together, the sequence variants within the ATG7 gene promoter identified in PD patients may change ATG7 protein levels, which in turn would influence autophagic activity, contributing to PD onset as a risk factor.

Silencing of miR155 promotes the production of inflammatory mediators in Guillain-Barré syndrome in vitro.

MicroRNA-155 (miR155) has been demonstrated as a central regulator of immune responses induced by inflammatory mediators. Previous studies suggest that miR155 may play adverse effects in various diseases. We hereby explored the roles of miR155 in the pathogenesis of Guillain-Barré syndrome (GBS). Peripheral blood mononuclear cells (PBMCs) were separated from GBS patients and healthy controls. Expression of miR155 in PBMCs was detected by quantitative PCR. An inhibitor of miR155 was transfected into the cultured PBMCs and the GBS-related cytokines were detected. Significantly, our study demonstrated that miR155 was downregulated in PBMCs from GBS patients and silencing of miR155 profoundly promoted the production of Th1-type cytokines in vitro. Our data effectively demonstrate a protective role of miR155 in GBS, which suggests that miR155 may be a promising target for the therapy of the disease.

Decreased expression of lysosomal alpha-galactosiase A gene in sporadic Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease. To date, the causal genes and variants associated with sporadic PD are largely unknown. Accumulating evidence demonstrates that autophagy delivers alpha-syncuclein proteins to lysosome for degradation and dysfunctional autophagy is involved in the PD pathogenesis. We have previously screened a group of lysosomal hydrolases and found that alpha-galactosidase A (GLA) activity is significantly decreased in the peripheral leukocytes of sporadic PD patients. In this study, GLA transcript and protein levels were semi-quantitatively examined. The GLA transcript (P = 0.020) and protein (P = 0.027) levels in the peripheral leukocytes of sporadic PD patients were significantly decreased, compared to age- and sex-matched healthy controls. Furthermore, decreased GLA gene expression levels were strongly associated with sporadic PD (OR 3.33, 95%CI 1.17-9.52, P = 0.024). Therefore, our data suggest that insufficient GLA activity may contribute to the pathogenesis of sporadic PD. The underlying molecular mechanisms remain to be determined.

Genetic analysis of lysosomal alpha-galactosidase A gene in sporadic Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease. Majority of PD cases are sporadic, resulting from interaction of genetic and environmental factors. Accumulating evidence indicates that autophagy, which delivers alpha-synuclein to lysosomes for degradation, is involved in the PD pathogenesis. Some lysosomal hydrolases, such as glucocerebrosidase gene and ATP13A2, a lysosomal ATPase gene, have been implicated in PD. We have previously screened the activities of a group of lysosomal hydrolases in sporadic PD patients and found that alpha-galactosidase A (GLA) activities are significantly decreased. In this study, we analyzed GLA gene in sporadic PD patients by sequencing its promoter and exon regions. One single-nucleotide polymorphism (SNP) in the promoter region, rs3027580 (NG_007119.1:g.4292G>C), and two SNPs in the GLA 5'-untranslated region, rs2071225 (NM_000169.2:c.-10C>T) and rs3027585 (NM_000169.2:c.-12G>A), were identified with similar frequencies in sporadic PD patients and healthy controls. A novel variant (NG_007119.1:g.4488C>G) within the promoter region, at the -573 site upstream of the translation start codon (ATG), was found in one male PD patient, but not in female PD patients or healthy controls. Our data suggest that the sequence variant may affect GLA gene expression by altering transcription factor binding sites, contributing to the pathogenesis of sporadic PD.

Altered expression of autophagic genes in the peripheral leukocytes of patients with sporadic Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease caused by interaction of genetic and environmental factors. To date, genetic genes and variants causing PD remain largely unknown. Autophagy is a conserved cellular process including three subtypes, macroautophagy (hereafter referred to as autophagy), microautophagy and chaperone-mediated autophagy (CMA). Although reduced CMA and induced autophagy are observed in human PD brain samples, cell and animal PD models, CMA and autophagy have not been systemically studied in sporadic PD patients. In the peripheral leukocytes of sporadic PD patients, we examined gene expression levels of lysosome-associated membrane 2 (LAMP-2), a CMA receptor and a limiting step, and microtubule-associated protein 1 light chain 3 (LC3), product of which is sequentially cleaved and lipidated to form LC3-II as an autophagosome marker. Compared to age- and sex-matched healthy controls, LAMP-2 gene expression and protein levels in sporadic PD patients were significantly decreased, which may lead to reduced CMA activity and impaired fusion of autophagosome and lysosome. LC3 gene expression and LC3-II protein levels were significantly increased in sporadic PD patients, suggesting that autophagosomes are accumulated. Our findings, decreased LAMP-2 gene expression and increased LC3 gene expression, are consistent to the previous studies with dopaminergic neuronal cells in vitro and in vivo, which may contribute to the pathogenesis of sporadic PD by altering CMA and autophagy activities. The genetic causes leading to decreased LAMP-2 gene expression need further investigation and genetic or pharmacological restoration of LAMP-2 might be a novel strategy for treating PD patients.

Decreased activities of lysosomal acid alpha-D-galactosidase A in the leukocytes of sporadic Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease that affects aged people. Although a number of genes have been linked to familial PD, the genetic causes of sporadic PD that accounts for 90% of all PD cases remain unclear. Accumulating evidence has demonstrated that alpha-synuclein aggregation is essential to the pathogenesis of PD. Recent studies suggest that autophagic-lysosomal system play major roles in the process of alpha-synuclein aggregation. We hypothesized that lysosomal acid hydrolases may be involved in the alpha-synuclein degradation and aggregation. In this study, we examined the activities of 11 lysosomal acid hydrolases in peripheral blood leukocytes of 38 sporadic PD patients and 258 age- and sex-matched healthy controls. The activities of alpha-D-galactosidase A were significantly decreased in sporadic PD patients, compared to age- and sex-matched controls. In contrast, no significant differences of the activities of other 10 lysosomal acid hydrolases was observed. This initial study suggests that decreased activities of lysosomal alpha-D-galactosidase A in the central nervous system may be involved in the degradation and aggregation of alpha-synuclein protein and contribute to the pathogenesis of sporadic PD as a risk factor.