精神分裂症患者甘露糖结合凝集素基因启动子区多态性及血清浓度的研究

目的:探讨精神分裂症患者甘露糖结合凝集素(MBL)的血清浓度和启动子区基因多态性的相关性及在患者免疫病理机制中的作用。方法:选取2018年6月至2018年12月在我院就诊的精神分裂患者为54例为观察组,另选取同时期本院健康体检中心志愿者56例为正常对照组,采用合酶链反应-序列特异性引物(PCR-SSP)技术分析MBL基因启动子区H/L(-550bp G/C)和X/Y(-221bp C/G)的多态性,酶联免疫吸附法(ELISA)检测血清MBL的浓度。结果:观察组血清MBL浓度(1367.218±1277.429)ng/mL低于正常对照组(1987.781±976.748)ng/mL,差异有统计学意义(P0.05)。观察组HY/HY基因型频率低于对照组(P0.05),HY/LY型频率高于对照组(P0.05)。观察组HY/HY基因型血清MBL浓度明显高于HY/LX、LY/LX型(P0.05);观察组MBL基因启动子区突变型纯合子的血清MBL水平均与对照组差异无统计学意义(P0.05),而MBL基因型杂合子的MBL水平则差异有统计学意义(P0.05)。结论:精神分裂症患者MBL水平低下与患者MBL基因启动子区各基因型的综合调控有关。MBL浓度低下是精神分裂症患者循环免疫复合物(CIC)滞留或清除障碍的分子机制之一。     

Mannose-binding lectin contributes to the severity of Guillain-Barré syndrome

In Guillain-Barré syndrome (GBS), complement activation plays a crucial role in the induction and extent of the postinfectious immune-mediated peripheral nerve damage. Mannose-binding lectin (MBL) activates the complement system via the lectin pathway after recognition of repetitive sugar groups on pathogens. We investigated whether the MBL2 genotype, serum MBL level, and MBL complex activity are associated with the development and severity of GBS. Single nucleotide polymorphisms in the promoter region (-550 H/L and -221 X/Y) and exon 1 (A/O) of the MBL2 gene were determined in 271 GBS patients and 212 healthy controls. The frequencies of the H allele, HY promoter haplotype, and HYA haplotype, which are related to high MBL activity, were all increased in GBS patients compared with healthy controls (p < or = 0.03), particularly in severely affected GBS patients (MRC-sum score < 40) (p < or = 0.02). Severe weakness was also associated with high MBL concentrations and MBL complex activity in sera from GBS patients (p < 0.01). The MBL2 B allele was associated with functional deficiency and relatively mild weakness. These results support the hypothesis that complement activation mediated by MBL contributes to the extent of nerve damage in GBS, which is codetermined by the MBL2 haplotype.     

Association of TLR7 variants with AIDS-like disease and AIDS vaccine efficacy in rhesus macaques

In HIV infection, TLR7-triggered IFN-α production exerts a direct antiviral effect through the inhibition of viral replication, but may also be involved in immune pathogenesis leading to AIDS. TLR7 could also be an important mediator of vaccine efficacy. In this study, we analyzed polymorphisms in the X-linked TLR7 gene in the rhesus macaque model of AIDS. Upon resequencing of the TLR7 gene in 36 rhesus macaques of Indian origin, 12 polymorphic sites were detected. Next, we identified three tightly linked single nucleotide polymorphisms (SNP) as being associated with survival time. Genotyping of 119 untreated, simian immunodeficiency virus (SIV)-infected male rhesus macaques, including an 'MHC adjusted' subset, revealed that the three TLR7 SNPs are also significantly associated with set-point viral load. Surprisingly, this effect was not observed in 72 immunized SIV-infected male monkeys. We hypothesize (i) that SNP c.13G>A in the leader peptide is causative for the observed genotype-phenotype association and that (ii) the underlying mechanism is related to RNA secondary structure formation. Therefore, we investigated a fourth SNP (c.-17C>T), located 17 bp upstream of the ATG translation initiation codon, that is also potentially capable of influencing RNA structure. In c.13A carriers, neither set-point viral load nor survival time were related to the c.-17C>T genotype. In c.13G carriers, by contrast, the c.-17C allele was significantly associated with prolonged survival. Again, no such association was detected among immunized SIV-infected macaques. Our results highlight the dual role of TLR7 in immunodeficiency virus infection and vaccination and imply that it may be important to control human AIDS vaccine trials, not only for MHC genotype, but also for TLR7 genotype.     

The Role of Serum High Mobility Group Box 1 and Interleukin‐6 Levels in Acute Pancreatitis: A Meta‐Analysis

The purpose of this meta‐analysis was to comprehensively investigate the correlation between high mobility group box 1 (HMGB1) and interleukin‐6 (IL‐6) in relation to acute pancreatitis. A highly regulated exploration of various electronic databases, supplemented by manual searching methods, was performed in an attempt to identify pertinent articles of a useful nature. Subsequently, high‐quality cohort studies that were deemed to comply with the arduous inclusion and exclusion criteria were selected for our meta‐analysis. The extensive data analyses reported in our meta‐analysis were conducted in connection with the Comprehensive Meta‐analysis 2.0 (CMA 2.0). A total of 395 studies (135 Chinese studies and 260 English studies) were initially retrieved. 27 of those studies were selected for our meta‐analysis, comprising of 896 cases of mild acute pancreatitis (MAP), 700 cases of severe acute pancreatitis (SAP) as well as 312 healthy controls. Pooled data suggested that serum HMGB1 and IL‐6 levels of SAP and MAP patients were higher than in healthy controls. Moreover, serum HMGB1 and IL‐6 levels of SAP patients exhibited significantly higher levels than in that of MAP patients. Based on the rigorous investigation of our meta‐analysis, it was concluded that serum HMGB1 and IL‐6 levels might be used as effective indicators for pancreatic lesions as well as the degree of inflammatory response, owing ultimately to the observations and data analyses, suggesting that serum HMGB1 and IL‐6 levels share a close correlation with the severity of pancreatitis. J. Cell. Biochem. 119: 616–624, 2018. © 2017 Wiley Periodicals, Inc.     

胰腺p-STAT3在急性胰腺炎危重演变中的表达变化

目的：检测信号转导与转录激活因子3（STAT3）在不同程度胰腺炎模型小鼠胰腺组织中表达的变化,探讨其在急性胰腺炎危重演变中的作用。方法：48只健康雄性balb/c小鼠随机分为3组（n=16）：对照组（Con）、轻症急性胰腺炎（MAP）组、重症急性胰腺炎（SAP）组。Con组腹腔注射0.9% NaCl;MAP组腹腔注射雨蛙素;SAP组腹腔注射雨蛙素联合脂多糖;分别于造模后2 h、6 h检测血清淀粉酶的活性;分离胰腺、称重,计算胰腺湿重比;检测肺组织髓过氧化物酶（MPO）活性,评估炎细胞浸润肺组织的程度;HE染色切片,光镜下观察胰腺、肺组织病理学改变; Western blot法检测磷酸化STAT3（p-STAT3）的变化。结果：与Con组比较, MAP组和SAP组在各时间点血清淀粉酶活性和胰腺组织湿重比均升高（P<0.05）;肺组织MPO活性显著升高（P<0.05）,且SAP组肺MPO含量明显高于MAP组（P<0.01）。MAP组和SAP组,在造模后2 h,胰腺和肺均可见不同程度的病理学改变; SAP组在造模后2 h胰腺p-STAT3的表达最高,6 h表达有所减弱;MAP组各时间点仅有微量表达;Con组在各时间点为阴性表达。结论：p-STAT3在轻症急性胰腺炎和重症急性胰腺炎模型小鼠胰腺中的表达差异明显,说明重症急性胰腺炎的重症化与STAT3的活化关系密切;抑制STAT3活化将成为阻止急性胰腺炎重症化的靶点之一。     

Study of the effect of DNA polymorphisms in the mannose-binding lectin gene (MBL2) on disease severity in Slovak cystic fibrosis patients

Lung infections are the leading cause of morbidity and mortality in cystic fibrosis (CF). Mannose-binding lectin (MBL) is a key factor in innate immunity. We therefore investigated whether MBL2 gene variants are associated with pulmonary function or susceptibility to Pseudomonas aeruginosa and Burkholderia cepacia infection in Slovak patients affected with CF. DNA polymorphisms in exon 1 and the promoter region were typed by single base primer extension assay in 91 patients and 100 healthy controls. The concentrations of MBL protein were determined in 34 patients by a sandwich enzyme-linked immunosorbent assay, and spirometric and microbiological data were collected from medical records. In this study we found that MBL2 genotypes were associated neither with earlier acquisition of P. aeruginosa or B. cepacia nor with reduced pulmonary function among patients. Although MBL2 genotypes were associated with the MBL2 protein serum level, results were statistically significant only for polymorphisms in exon 1, with p = 0.0008. The role of the MBL2 gene in lung disease severity in CF patients represents a very complex phenomenon where both genetic and environmental factors play an important role in addition to that of the MBL2 gene. Understanding this complexity requires further studies based on a broader scale of genetic factors involving both a whole-genome approach and a larger patient cohort.     

不同评分标准对急性胰腺炎严重程度评估价值的比较

目的:分析和评价Ranson、Glasgow、APACHEⅡ和BISAP 4种临床评分标准对急性胰腺炎严重程度的评估价值。方法:回顾性研究急性胰腺炎患者225例,分别应用APACHEⅡ、Ranson、Glasgow及BISAP评分标准对急性胰腺炎患者进行评分,比较分析不同评分标准对该类患者诊断的敏感性、特异性,以及对合并脏器功能不全的预测情况。结果:225例患者中,轻型胰腺炎188例,重型胰腺炎37例,在轻型和重型胰腺炎患者中,4种评分标准分值差异均有统计学意义(P0.01)。47例患者存在器官功能不全,4种评分标准与患者合并脏器功能不全均显著相关。各评分标准中,APACHEⅡ对急性重症胰腺炎评估的敏感性、特异性最好,分别为76%和72%。结论:4中评分方法各有特点,综合应用可能更准确的评估疾病严重程度及预后。     

Microarray-based detection of mannose-binding lectin 2 (MBL2) polymorphisms in a routine clinical setting

The assessment of allelic variants in the human mannose-binding lectin 2 (MBL2) gene is of great clinical importance in newborns or immune-suppressed patients at high risk for a variety of infections. Here, we present a study on the genotyping accuracy of a DNA microarray-based on-chip PCR method suited for the detection of five different polymorphisms in the MBL2 gene. We tested 153 genomic DNA samples, prepared from archival blood spots on Guthrie cards, for the presence of allelic variants in the human MBL2 gene by the on-chip PCR method and compared the obtained results of three variants to standard DNA capillary sequencing. The genotyping power of the described assay was readily comparable to DNA sequencing (453/459 correct genotype calls in 153 DNA samples; 98.7% accuracy), mainly due to intrinsic technical benefits of microarrays such as high number of test replicates and automated data analysis. This study demonstrates, for the first time, the accuracy and reliability of a microarray-based on-chip PCR genotyping assay for measuring allelic variants in a routine clinical setting.     

Variants of the mannose-binding lectin gene in the Benin population: heterozygosity for the p.G57E allele may confer a selective advantage

Human mannose-binding lectin (MBL) plays an important role in innate immunity. MBL deficiency is associated with mutations in the promoter region and in exon 1 of the MBL2 gene. Such deficiency has been correlated with elevated incidence of infections in infancy and in immunocompromised adults. We determined the distribution profile of the MBL2 gene variants in the general population of Benin (West Africa) and in a vulnerable subset of children with sickle cell disease (SCD) (SS homozygotes). Five hundred forty-two healthy individuals (274 newborns, 268 adults) and 128 patients with SCD (35 newborns, 93 children) were screened for the common variant alleles in the MBL2 secretor haplotype region (exon 1 and promoter). The p.G57E variant allele was the most frequent allele compared to p.G54D (27.5% vs. 1.6%, respectively). The p.R52C allele was not found in this population. There was no difference in allele or genotype frequencies between healthy newborns and newborns with SCD. Alleles associated with MBL deficiency were more frequent in adults than in newborns (69.8% vs. 57.3%, respectively; p = 0.002). This enrichment was exclusively due to an elevated proportion of heterozygotes for the p.G57E allele (47.0% vs. 35.3%, respectively; p = 0.004), supporting a potential selective advantage of this genotype. Our results, compared to those reported in other African countries, support the implication of the MBL2 gene in various major infections in Africa, such as meningitis and tuberculosis in HIV-positive patients.     

Mannose-binding lectin 1 haplotypes influence serum MBL-A concentration,complement activity,and milk production traits in Chinese Holstein cattle

Mannose-binding lectin (MBL) is a member of the collectin protein family that binds a broad range of microorganisms and activates the lectin-complement pathway of innate immunity. MBL deficiency is associated with an increased risk for various infections and arises from five polymorphisms in the promoter and first exon of the MBL gene in humans. In this study, three novel single-nucleotide polymorphisms (SNPs) in the promoter region and two previously reported SNPs in exon 2 of the MBL1 gene were detected using PCR single-strand conformation polymorphism, restriction fragment length polymorphism, and DNA sequencing in 537 cattle from three Chinese breeds. Analysis of the genotypes and haplotypes was used to investigate the polymorphisms and their possible implications, especially their association with serum MBL-A levels, complement activity (CH50 and ACH50), and milk production traits was investigated. The g.2651G>A SNP in exon 2 affected the serum MBL-A concentrations and the serum CH50 values, whereas the g.−1330G>A SNP significantly affected CH50 and the somatic cell scores (SCSs). Statistical analysis revealed that cows with the ATGGC/ACAAC combined genotype and those with the AAGGT/ACGGT combined genotype exhibited the lowest and highest SCSs, respectively. Serum antibacterial activities were also conducted to verify the effect of the SNPs on resistance to mastitis pathogens. Results of real-time PCR showed that the liver of cows with clinical mastitis exhibited a higher MBL1 expression compared with healthy ones (P < 0.05). Findings of this study indicate that the MBL1 gene possibly contributes to bacterial infection resistance and can be used as a molecular marker of milk production traits to control mastitis.     

The first report described as an important study: The association of mannose-binding lectin gene 2 polymorphisms in children with Down syndrome

BACKGROUND:

Mannose-binding lectin gene 2 (MBL2) plays a very important role in the first line of host immune response in Down syndrome (DS). The importance of MBL2 gene polymorphisms in children with DS is unclear, and no research has addressed MBL2 gene polymorphisms in patients with DS. This is the first report describing an important association between MBL2 gene polymorphisms and infections in children with DS.

MATERIALS AND METHODS:

We compared the frequency of single-nucleotide polymorphisms (SNPs) at two codons of the MBL2 gene in a cross sectional cohort of 166 children with DS and 229 controls. Polymorphisms at codons 54 (GGC→GAC) and 57 (GGA→GAA) in exon 1 of the MBL2 gene were typed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique using the restriction enzymes BshN1 (derivated from Bacillus sphaericus) and MboII (derivated from Moraxella bovis), respectively.

RESULTS:

MBL2 codon 54 GA genotype frequency was found to be lower in patients with DS (22.9%) than those of healthy controls (35.8%), differences were statistically significant (OR = 0.532, 95% CI = 0.339-0.836, P = 0.008). On the other hand, codon 57 polymorphism in the MBL2 gene was detected in none of the DS patients, but only one person in the control group showed codon 57 GA genotype (OR = 1.004, 95% CI = 0.996-1.013, P = 1.000).

CONCLUSION:

Our data provides an evidence for the first time that a homozygote or heterozygote for the variant, MBL2 alleles, is not associated with infections in patients with DS, and do not influence the incidence of infections.     

MBL在结肠直肠癌中的基因分型

目的：MBL是补体激活凝集素途径的关键因素。MBL基因多态性影响MBL血清水平。结肠直肠癌患者血清MBL水平升高。低水平的MBL则预示着术后肺炎的发生,目前还不清楚此相关性是否与遗传相关。本实验分析调查了结肠直肠癌患者和健康对照者的MBL基因分型,评估基因分析和复发率、生存率之间潜在的相关性。方法：使用TaqMan基因分型分析法和实时定量PCR分析MBL基因的4个SNP、启动子区2个SNP、非编码区1个SNP;ELISA测定标本血清MBL含量。结果：所有标本中发现了8种不同的MBL单体型,它们在患者和健康者中出现频率几乎是完全一样的;YA/YA基因型与高水平的MBL相关,YO／YO与低水平的MBL水平相关,6种不同基因型CRC患者的MBL水平存在着明显不同。结论：MBL基因型与血清MBL浓度显薯相关（P〈0．0001）;突变型B,C,D和启动子单体型Y,X对MBL含量的影响起主要作用;MBL基因型和术后感染并发症没有明显相关性（P=0．33）,与复发癌和存活时间也没有明显相关性（P=0．74）。因此,从基因水平还不能解释为何结肠直肠癌患者血清MBL水平增加。对比已经检测出的血清MBL水平,其基因型还不能预测结肠直肠癌患者的疾病进程。     

急性胰腺炎患者胃肠动力紊乱与胃肠激素的关系分析

目的:探讨急性胰腺炎(AP)患者的胃肠动力紊乱与胃肠激素的关系。方法:收集2014年3月~2015年4月我院消化科收治的90例AP患者为病例组,按病情严重程度分为轻症急性胰腺炎组(MAP组,39例)、中重症急性胰腺炎组(MSAP组,33例)及重症急性胰腺炎组(SAP组,18例),并于同期抽取30例健康体检者作为对照组,通过采用双抗体夹心酶联免疫法(ELASA)测量入院第1、3、7天的血清胃动素(MTL)、血管活性肠肽(VIP)在各组中的水平,同时测量口盲传递时间(OCTT)来衡量AP的严重程度。结果:MAP组、MSAP组及SAP组血清MTL水平均较对照组明显降低,且随着入院治疗时间的延长逐渐升高,差异均有统计学意义(P0.05)。MAP组、MSAP组及SAP组血清VIP水平均较对照组明显升高,且随着入院治疗时间的延长逐渐降低,差异均有统计学意义(P0.05)。血清MTL水平与OCTT呈负相关性(rs=-0.534、-0.629、-0.468,P0.05);血清VIP水平与OCTT呈正相关性(rs=0.464、0.326、0.671,P0.05)。结论:胃肠动力紊乱可能与胃肠激素异常有关,表现为MTL水平降低、VIP水平升高,且其改变幅度与病情的严重程度相一致。     

急性胰腺炎1796例病因及治疗效果分析

目的：分析我院急性胰腺炎（AP）的主要病因及治疗效果,为临床诊疗工作提供参考。方法：回顾性分析2008年1月至2012年12月于我院住院治疗的1796例AP患者的临床资料,统计其病因及治疗效果,对其病因及预后进行分析总结。结果：在1796例患者中,重症急性胰腺炎（SAP）522例,轻症急性胰腺炎（MAP）1274例。胆源性AP1025例,高脂血症性AP209例,酒精性AP200例。高脂血疰性AP中,SAP占49．8％,高于胆道疾病及酒精因素引起的SAP的比例（P〈0．05）。33例患者死亡,总病死率1．8％。结论：胆道疾病、酒精、高脂血症是我国AP主要病因,随着医疗水平的提高,AP的病死率有所下降。     

Nasopharyngeal bacterial colonization and gene polymorphisms of mannose-binding lectin and toll-like receptors 2 and 4 in infants

Background

Human nasopharynx is often colonized by potentially pathogenic bacteria. Gene polymorphisms in mannose-binding lectin (MBL), toll-like receptor (TLR) 2 and TLR4 have been reported. The present study aimed to investigate possible association between nasopharyngeal bacterial colonization and gene polymorphisms of MBL, TLR2 and TLR4 in healthy infants.

Methodology/Principal Findings

From August 2008 to June 2010, 489 nasopharyngeal swabs and 412 blood samples were taken from 3-month-old healthy Finnish infants. Semi-quantitative culture was performed and pyrosequencing was used for detection of polymorphisms in MBL structural gene at codons 52, 54, and 57, TLR2 Arg753Gln and TLR4 Asp299Gly. Fifty-nine percent of subjects were culture positive for at least one of the four species: 11% for Streptococcus pneumoniae, 23% for Moraxella catarrhalis, 1% for Haemophilus influenzae and 25% for Staphylococcus aureus. Thirty-two percent of subjects had variant types in MBL, 5% had polymorphism of TLR2, and 18% had polymorphism of TLR4. Colonization rates of S. pneumoniae and S. aureus were significantly higher in infants with variant types of MBL than those with wild type (p = .011 and p = .024). Colonization rates of S. aureus and M. catarrhalis were significantly higher in infants with polymorphisms of TLR2 and of TLR4 than those without (p = .027 and p = .002).

Conclusions

Our study suggests that there is an association between nasopharyngeal bacterial colonization and genetic variation of MBL, TLR2 and TLR4 in young infants. This finding supports a role for these genetic variations in susceptibility of children to respiratory infections.     

Association analysis of monocyte chemotactic protein-3 (MCP3) polymorphisms with asthmatic phenotypes

The monocyte chemotactic protein-3 (MCP3), on chromosome 17q11.2-q12, is a secreted chemokine, which attracts macrophages during inflammation and metastasis. In an effort to discover additional polymorphism(s) in genes whose variant(s) have been implicated in asthma, we scrutinized the genetic polymorphisms in MCP3 to evaluate it as a potential candidate gene for asthma host genetic study. By direct DNA sequencing in twenty-four individuals, we identified four sequence variants within the 3 kb full genome including 1,000bp promoter region of MCP3; one in promoter region (-420T>C), three in intron (+136C>G, +563C>T, +984G>A) respectively. The frequencies of those four SNPs were 0.020 (-420T>C), 0.038 (+136C>G), 0.080 (+563C>T), 0.035 (+984G>A), respectively, in Korean population (n = 598). Haplotypes, their frequencies and linkage disequilibrium coefficients (|D'|) between SNP pairs were estimated. The associations with the risk of asthma, skin-test reactivity and total serum IgE levels were analyzed. Using statistical analyses for association of MCP3 polymorphisms with asthma development and asthma-related phenotypes, no significant signals were detected. In conclusion, we identified four genetic polymorphisms in the important MCP3 gene, but no significant associations of MCP3 variants with asthma phenotypes were detected. MCP3 variation/haplotype information identified in this study will provide valuable information for future association studies of other allergic diseases.     

Association of EGFR c.2073A>T polymorphism with decreased risk of diffusely infiltrating astrocytoma in a Brazilian case-control study

Epidermal growth factor receptor (EGFR) gene overexpression has been implicated in the development of many types of tumors, including glioblastomas, the most frequent diffusely infiltrating astrocytomas. However, little is known about the influence of the polymorphisms of EGFR on EGFR production and/or activity, possibly modulating the susceptibility to astrocytomas. This study aimed to examine the association of two EGFR promoter polymorphisms (c.-191C>A and c.-216G>T) and the c.2073A>T polymorphism located in exon 16 with susceptibility to astrocytomas, EGFR gene expression and survival in a case-control study of 193 astrocytoma patients and 200 cancer-free controls. We found that the variant TT genotype of the EGFR c.2073A>T polymorphism was associated with a significantly decreased risk of astrocytoma when compared with the AA genotype [sex- and age-adjusted odds ratio 0.51, 95% confidence interval 0.26-0.98]. No association of the two promoter EGFR polymorphisms (or combinations of these polymorphisms) and risk of astrocytomas, EGFR expression or survival was found. Our findings suggest that modulation of the EGFR c.2073A>T polymorphism could play a role in future therapeutic approaches to astrocytoma.     

Toll-like receptor 2 and Toll-like receptor 4 polymorphisms in invasive pneumococcal disease

BACKGROUND: Toll-like receptors (TLRs) recognize distinct pathogen-associated molecular patterns and trigger anti-microbial host defense responses. Several in vitro and in vivo studies in mice indicate that TLR2 and TLR4 are involved in the defense against Streptococcus pneumoniae. Studies have revealed associations between polymorphisms in TLRs and human diseases. The effect of polymorphisms in TLR2 and TLR4 in the human defense to S. pneumoniae has not been studied. METHODS: We genotyped 99 Caucasian patients with invasive pneumococcal disease and 178 Caucasian controls for the known R579H, P631H and R753Q polymorphisms in TLR2 and the D299G polymorphism in TLR4 with PCR-RFLP methods. RESULTS: The distribution of the TLR2 R579H, P631H and R753Q and TLR4 D299G variants was not significantly different between the patients and the controls. After stratification of the patient population by age, sex, diagnosis, and mortality no significant differences for the TLR2 R753Q genotype and TLR4 D299G genotype were found between various patient subgroups and between patient subgroups and the control population. It should be mentioned that for the TLR2 polymorphisms neither the control group nor the patient group contains homozygous mutant individuals. CONCLUSION: We found no association between TLR2 and TLR4 polymorphisms and invasive pneumococcal infection.