Coxsackievirus B3-Induced Myocarditis : Perforin Exacerbates Disease, But Plays No Detectable Role in Virus Clearance

Viral myocarditis is remarkably common, being detected in approximately 1% of unselected asymptomatic individuals. Many cases are attributable to enteroviral infection, and in particular to coxsackievirus B3. The underlying pathogenesis is controversial, but most studies admit the important immunopathological role of infiltrating CD8⁺ (cytotoxic) T lymphocytes (CTLs). We have previously shown that CTLs play conflicting roles in coxsackievirus B (CVB) myocarditis; they assist in controlling virus replication, but also are instrumental in causing the extensive inflammatory disease, which often results in severe myocardial scarring. A role for perforin, the major CTL cytolytic protein, in CVB myocarditis has been suggested, but never proven. In the present study we use perforin knockout (PKO) mice to show that perforin plays a major role in CVB infection; in broad terms, perforin is important in immunopathology, but not in CVB clearance. For example, PKO mice are better able to withstand a normally lethal dose of CVB (100% survival of PKO mice compared with 90% death in +/+ littermates). In addition, PKO mice given a nonlethal dose of CVB develop only a mild myocarditis, whereas their perforin⁺ littermates have extensive myocardial lesions. The myocarditis in PKO mice resolves more quickly, and these mice show minimal histological sequelae; in contrast, late in disease the perforin⁺ mice develop severe myocardial fibrosis. PKO mice, despite lacking this major CTL effector function, can control the infection and eradicate the virus; growth kinetics and peak CVB titers are indistinguishable in PKO and perforin⁺ mice. Therefore, the immunopathological and antiviral effects of CTLs can be uncoupled by ablation of perforin; this offers a promising target for therapy of myocarditis. Furthermore, we evaluate the possible roles of apoptosis, and of chemokine expression, in CVB infection. In perforin⁺ mice, apoptotic cells are detected within the inflammatory infiltrate, whereas in their PKO counterparts, apoptotic myocyte nuclei are seen. Chemokine expression in both PKO and perforin⁺ mice precedes and parallels the course of myocarditis. Several chemokines are detectable earlier in PKO mice than in perforin⁺ mice, but PKO mice show reduced peak levels, and chemokine expression decays sooner. In particular, MIP-1α expression is barely detectable at any time point in PKO mice, but it is readily identified in perforin⁺ animals, peaking just before the time of maximal myocarditis; this is particularly interesting, given that MIP-1α knockout mice are resistant to CVB myocarditis, but remain able to control viral infection. Thus, the chemokine pathway offers a second route of intervention to diminish myocarditis and its sequelae, while permitting the host to eradicate the virus.     

A conserved protein network controls assembly of the outer kinetochore and its ability to sustain tension

Kinetochores play an essential role in chromosome segregation by forming dynamic connections with spindle microtubules. Here, we identify a set of 10 copurifying kinetochore proteins from Caenorhabditis elegans, seven of which were previously uncharacterized. Using in vivo assays to monitor chromosome segregation, kinetochore assembly, and the mechanical stability of chromosome-microtubule attachments, we show that this copurifying protein network plays a central role at the kinetochore-microtubule interface. In addition, our analysis suggests that the network is comprised of three groups of proteins that contribute in distinct ways to this interface: KNL proteins act after the assembly of centromeric chromatin to generate the core of the microtubule-binding interface, MIS proteins control the rate and extent of formation of this interface, and NDC proteins are necessary to sustain tension during interactions with spindle microtubules. We also purify a similar set of associated proteins from human cells that includes four novel proteins and has recognizable homologs from each functional class. Thus, this protein network is a conserved constituent of the outer kinetochore, and the functions defined by our analysis in C. elegans are likely to be widely relevant.     

Haplotype structure,LD blocks,and uneven recombination within the LRP5 gene

Patterns of linkage disequilibrium (LD) in the human genome are beginning to be characterized, with a paucity of haplotype diversity in "LD blocks," interspersed by apparent "hot spots" of recombination. Previously, we cloned and physically characterized the low-density lipoprotein-receptor-related protein 5 (LRP5) gene. Here, we have extensively analysed both LRP5 and its flanking three genes, spanning 269 kb, for single nucleotide polymorphisms (SNPs), and we present a comprehensive SNP map comprising 95 polymorphisms. Analysis revealed high levels of recombination across LRP5, including a hot-spot region from intron 1 to intron 7 of LRP5, where there are 109 recombinants/Mb (4882 meioses), in contrast to flanking regions of 14.6 recombinants/Mb. This region of high recombination could be delineated into three to four hot spots, one within a 601-bp interval. For LRP5, three haplotype blocks were identified, flanked by the hot spots. Each LD block comprised over 80% common haplotypes, concurring with a previous study of 14 genes that showed that common haplotypes account for at least 80% of all haplotypes. The identification of hot spots in between these LD blocks provides additional evidence that LD blocks are separated by areas of higher recombination.     

Immunoperoxidase stains of ganglion cells and abnormal mucosal nerve proliferations in Hirschsprung's disease

Hirschsprung's disease is congenital aganglionosis of the distal colon. The affected bowel shows an abnormal proliferation of mucosal nerve fibers by acetylcholinesterase stains. We retrospectively reviewed biopsy specimens from patients with suspected and proven Hirschsprung's disease, performed immunoperoxidase stains for S-100 protein and neuron-specific enolase (NSE), and compared these results to routine histologic findings and acetylcholinesterase stains. Ganglion cells were demonstrated by immunoperoxidase in 63 of 69 specimens containing ganglion cells and in 1 specimen interpreted as aganglionic by hematoxylin-eosin staining. Increased numbers of nerve fibers in the muscularis mucosae and deep lamina propria by S-100 staining were detected in 8 of 8 specimens diagnostic for Hirschsprung's disease by hematoxylin-eosin and acetylcholinesterase stains and in 1 specimen diagnostic for colonic neuronal dysplasia (a disorder related to Hirschsprung's disease). Whereas 45 of 67 specimens from unaffected bowel showed a normal number and distribution of nerve fibers by S-100 staining, in 22 the pattern resembled that of Hirschsprung's disease. Specimens from affected colon also showed hypertrophied submucosal nerve trunks by S-100 stain (average nerve trunk thickness, 29.8 micron in affected bowel, 16.1 micron in unaffected segments--p less than 0.03). We conclude that NSE and S-100 stains are of value in demonstrating ganglion cells in suspected cases of Hirschsprung's disease and colonic neuronal dysplasia. The acetylcholinesterase stain is preferred over S-100 stain for detecting mucosal nerve proliferations in affected bowel. Submucosal nerve trunk thickness, although significantly different in affected and unaffected colon, is not of diagnostic value because of the wide variation in the measurements in the two groups.     

Gene discovery in the Entamoeba invadens genome

Entamoeba invadens, a parasite of reptiles, is a model for the study of encystation by the human enteric pathogen Entamoeba histolytica, because E. invadens form cysts in axenic culture. With approximately 0.5-fold sequence coverage of the genome, we were able to get insights into E. invadens gene and genome features. Overall, the E. invadens genome displays many of the features that are emerging from ongoing genome sequencing efforts in E. histolytica. At the nucleotide level the E. invadens genome has on average 60% sequence identity with that of E. histolytica. The presence of introns in E. invadens was predicted with similar consensus (GTTTGT em leader A/TAG) sequences to those identified in E. histolytica and Entamoeba dispar. Sequences highly repeated in the genome of E. histolytica (rRNAs, tRNAs, CXXC-rich proteins, and Leu-rich repeat proteins) were found to be highly repeated in the E. invadens genome. Numerous proteins homologous to those implicated in amoebic virulence, (Gal/GalNAc lectins, amoebapores, and cysteine proteinases) and drug resistance (p-glycoproteins) were identified. Homologs of proteins involved in cell cycle, vesicular trafficking and signal transduction were identified, which may be involved in en/excystation and cell growth of E. invadens. Finally, multiple copies of a number of E. invadens genes coding for predicted enzymes involved in core metabolism and the targets of anti-amoebic drugs were identified.     

Evidence for linkage of nonsyndromic cleft lip with or without cleft palate to a region on chromosome 2

Results from a genome-wide screen of 10 multiplex families ascertained through probands with nonsyndromic cleft lip with or without cleft palate (CL/P) in Mexico, Argentina, and the United States yielded suggestive evidence of linkage to chromosomes 2, 6, 17 and 18. Fine mapping excluded all regions except chromosome 2. Subsequent analysis was performed on the original 10 families plus an additional 16 families using 31 markers on chromosome 2. This analysis showed intriguing evidence of linkage to 2q (Zlr=2.26, empirical P-value=0.028 in a chromosome-wide analysis). Transmission disequilibrium tests also revealed evidence of linkage and disequilibrium for two markers in this region (D2S168 and D2S1400 with P-values=0.022 and 0.006, respectively). A subset of these 26 families provided additional evidence for a susceptibility gene for CL/P on 2q, suggesting that further studies of genes in this region are warranted.     

Data-Driven Dietary Patterns,Nutrient Intake and Body Weight Status in a Cross-Section of Singaporean Children Aged 6–12 Years

Pattern analysis of children’s diet may provide insights into chronic disease risk in adolescence and adulthood. This study aimed to assess dietary patterns of young Singaporean children using cluster analysis. An existing dataset included 15,820 items consumed by 561 participants (aged 6–12 years) over 2 days of dietary recall. Thirty-seven food groups were defined and expressed as a percentage contribution of total energy. Dietary patterns were identified using k-means cluster analysis. Three clusters were identified, “Western”, “Convenience” and “Local/hawker”, none of which were defined by more prudent dietary choices. The “Convenience” cluster group had the lowest total energy intake (mean 85.8 ± SD 25.3% of Average Requirement for Energy) compared to the other groups (95.4 ± 25.9% for “Western” and 93.4 ± 25.3% for “Local/hawker”, p < 0.001) but also had the lowest calcium intake (66.3 ± 34.7% of Recommended Dietary Allowance), similar to intake in the “Local/hawker” group (69.5 ± 38.9%) but less than the “Western” group (82.8 ± 36.1%, p < 0.001). These findings highlight the need for longitudinal analysis of dietary habit in younger Singaporeans in order to better define public health messaging targeted at reducing risk of major noncommunicable disease.