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During group B streptococcal infection, the alpha C protein (ACP) on the bacterial surface binds to host cell surface heparan sulfate proteoglycans (HSPGs) and facilitates entry of bacteria into human epithelial cells. Previous studies in a Drosophila melanogaster model showed that binding of ACP to the sulfated polysaccharide chains (glycosaminoglycans) of HSPGs promotes host death and is associated with higher bacterial burdens. We hypothesized that ACP-glycosaminoglycan binding might determine infection outcome by altering host responses to infection, such as expression of antimicrobial peptides. As glycosaminoglycans/HSPGs also interact with a number of endogenous secreted signaling molecules in Drosophila , we examined the effects of host and pathogen glycosaminoglycan/HSPG-binding structures in host survival of infection and antimicrobial peptide expression. Strikingly, host survival after infection with wild-type streptococci was enhanced among flies overexpressing the endogenous glycosaminoglycan/HSPG-binding morphogen Decapentaplegic—a transforming growth factor β-like Drosophila homolog of mammalian bone morphogenetic proteins—but not by flies overexpressing a mutant, non-glycosaminoglycan-binding Decapentaplegic, or the other endogenous glycosaminoglycan/HSPG-binding morphogens, Hedgehog and Wingless. While ACP-glycosaminoglycan binding was associated with enhanced transcription of peptidoglycan recognition proteins and antimicrobial peptides, Decapentaplegic overexpression suppressed transcription of these genes during streptococcal infection. Further, the glycosaminoglycan-binding domain of ACP competed with Decapentaplegic for binding to the soluble glycosaminoglycan heparin in an in vitro assay. These data suggest that, in addition to promoting bacterial entry into host cells, ACP competes with Decapentaplegic for binding to glycosaminoglycans/HSPGs during infection and that these bacterial and endogenous glycosaminoglycan-binding structures determine host survival and regulate antimicrobial peptide transcription.

Many pathogens engage host cell surface glycosaminoglycans, but redundancy in pathogen adhesins and host glycosaminoglycan-anchoring proteins (heparan sulfate proteoglycans) has limited the understanding of the importance of glycosaminoglycan binding during infection. The alpha C protein of group B streptococcus, a virulence determinant for this neonatal human pathogen, binds to host glycosaminoglycan and mediates the entry of bacteria into human cells. We studied alpha C protein-glycosaminoglycan binding in Drosophila melanogaster , whose glycosaminoglycan repertoire resembles that of humans but whose genome includes only three characterized membrane heparan sulfate proteoglycan genes. The knockdown of glycosaminoglycan polymerases or of heparan sulfate proteoglycans reduced the cellular binding of alpha C protein. The interruption of alpha C protein-glycosaminoglycan binding was associated with longer host survival and a lower bacterial burden. These data indicate that the glycosaminoglycan-alpha C protein interaction involves multiple heparan sulfate proteoglycans and impairs bacterial killing. Host glycosaminoglycans, anchored by multiple proteoglycans, thereby determine susceptibility to infection. Because there is homology between Drosophila and human glycosaminoglycan/proteoglycan structures and many pathogens express glycosaminoglycan-binding structures, our data suggest that interfering with glycosaminoglycan binding may protect against infections in humans.

The humanBCL6gene on chromosome 3 band q27, which encodes a transcriptional repressor, is implicated in the pathogenesis of human lymphomas, especially the diffuse large B-cell type. We previously identified the humanPDCD2(programmed cell death-2) gene as a target of BCL6 repression.PDCD2encodes a protein that is expressed in many human tissues, including lymphocytes, and is known to interact with corepressor complexes. We now show that BCL6 can bind directly to thePDCD2promoter, repressing its transcription. Knockdown of endogenous BCL6 in a human B cell lymphoma line by introduction of small interfering RNA duplexes increases PDCD2 protein expression. Furthermore, there is an inverse relationship between the expression levels of the BCL6 and PDCD2 proteins in the lymphoid tissues of mice overexpressing human BCL6 (high BCL6 levels, minimal PDCD2) and controls (minimal BCL6, high PDCD2) as well as in tissues examined from some human B and T cell lymphomas. These data confirmPDCD2as a target of BCL6 and support the concept that repression ofPDCD2by BCL6 is likely important in the pathogenesis of certain human lymphomas.

Group B Streptococcus (GBS) frequently colonizes the human gastrointestinal and gynecological tracts and less frequently causes deep tissue infections. The transition between colonization and infection depends upon the ability of the organism to cross epithelial barriers. The alpha C protein (ACP) on the surface of GBS contributes to this process. A virulence factor in mouse models of infection, and prototype for a family of Gram-positive bacterial surface proteins, ACP facilitates GBS entry into human cervical epithelial cells and movement across cell layers. ACP binds to host cell surface glycosaminoglycan (GAG). From crystallography, we have identified a cluster of basic residues (BR2) that is a putative GAG binding area in Domain 2, near the junction of the N-terminal domain of ACP and the first of a series of tandem amino acid repeats. D2-R, a protein construct including this region, binds to cells similarly to full-length ACP. We now demonstrate that the predicted charged BR2 residues confer GAG binding; site-directed mutagenesis of these residues (Arg(172), Arg(185), or Lys(196)) eliminates cell-binding activity of construct D2-R. In addition, we have constructed a GBS strain expressing a variant ACP with a charge-neutralizing substitution at residue 185. This strain enters host cells less effectively than does the wild-type strain and similarly to an ACP null mutant strain. The point mutant strain transcytoses similarly to the wild-type strain. These data indicate that GAG-binding activity underlies ACP-mediated cellular entry of GBS. GBS entry into host cells and transcytosis of host cells may occur by distinct mechanisms.

Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-A resolution crystal structure of NtACP comprising residues Ser(52) through Leu(225) of the full-length ACP. NtACP has two domains, an N-terminal beta-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the beta-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp(146), Arg(110), and Asp(118). A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.

Summary Group B Streptococcus (GBS) is the leading cause of bacterial chorioamnionitis and neonatal pneumonia, sepsis, and meningitis. Deletion of the alpha C protein gene (bca) attenuates the virulence of GBS in an animal model; significant survival differences in the first 24 h of infection suggest a pathogenic role for the alpha C protein early in the infection process. We examined the role of alpha C protein in the association between GBS and mucosal surfaces using a human cervical epithelial cell line, ME180. Fluorescent and confocal microscopy and flow cytometry demonstrated that 9-repeat alpha C protein binds to the surface of ME180 cells. Isolated N-terminal region of this protein also binds to these cells and competitively inhibits binding of the full protein. Wild-type GBS strain A909 and the bca-null isogenic mutant JL2053 bound similarly to the surface of ME180 cells. However, A909 entered these cells threefold more. Internalization of A909 was inhibited with 2- and 9-repeat alpha C and with N-terminal region alone but not by repeat region-specific peptide. Translocation across polarized ME180 membranes was fivefold greater for A909 than for JL2053. These findings suggest a role for the alpha C protein in interaction with epithelial surfaces and initiation of infection.

Objective.Substantial research has associated exposure to entertainment media with increased levels of interpersonal violence, risky sexual behavior, body image distortion, substance abuse, and obesity. The objective of this study was to determine what pediatric residency programs are teaching trainees about media and the influence of media on the physical and mental health of children and adolescents.Design.Survey of residency curricula, consisting of 17 items about children's exposure to media, including television, movies, popular music, computer/video games and the Internet, the effects of this exposure on specific health risks, and associations between program characteristics and media education in the residency curriculum.Participants.Directors of the 209 accredited pediatric residency programs in the United States.Results.Two hundred four programs (97.6%) responded. Fifty-eight programs (28.4%) offered formal education on 1 or more types of media; 60 programs (29.4%) discussed the influences of media when teaching about specific health conditions. Residents in 96 programs (47.1%) were encouraged to discuss media use with patients and parents; 13 programs (6.4%) taught media literacy as an intervention. Among program characteristics, only media training received by program directors was significantly associated with inclusion of media in residency curricula.Conclusions.Despite increasing awareness of media influence on child health, less than one-third of US pediatric residency programs teach about media exposure. Developing a pediatric media curriculum and training pediatric residency directors or designated faculty may be a resource-effective means of improving health for children growing up in a media-saturated environment.

Central nervous system (CNS) aspergillosis is a relatively uncommon complication of human immunodeficiency virus (HIV) infection. We describe 6 patients with the acquired immunodeficiency syndrome (AIDS) who developed CNS aspergillosis, and we review a total of 33 cases of CNS aspergillosis among HIV-infected individuals that were diagnosed by histology and/or culture. All patients were diagnosed with advanced HIV infection. Major risk factors for the disease included neutropenia and corticosteroid use. The most common presenting symptoms were nonspecific neurologic manifestations including headache, cranial or somatic nerve weakness or paresthesia, altered mental status, and seizures. The most common sites of additional Aspergillus involvement were the lungs, sinuses, ears, and orbits, while in one-fourth of the cases CNS was the only site of Aspergillus infection. The final diagnosis of CNS aspergillosis was made on autopsy in more than half the cases, and medical treatment of CNS aspergillosis was unsuccessful in all cases. CNS aspergillosis should be included in the differential diagnosis of HIV-infected patients who present with nonspecific neurologic symptoms and signs. If we take into account the much higher prevalence of invasive aspergillosis of the lungs, the findings in the present report suggest that CNS aspergillosis in HIV-infected individuals occurs more often as a result of direct extension from the sinuses, orbits, and ears than through hematogenous spread from the lungs. Physicians should be aware that the CNS might be the only site of Aspergillus involvement and include CNS aspergillosis in the differential diagnosis of HIV-infected patients presenting with focal neurologic signs and symptoms, especially when the head CT reveals hypodense lesions.

Objective. To characterize the clinical features of hepatitis C virus (HCV) infection associated with the administration of intravenous immunoglobulin (IVIG) in patients with varied immunodeficiencies. Design. Prospective collection of clinical and virologic logic data in patients determined to have HCV exposure associated with Gammagard. Setting. Outpatient department of children's Hospital tal, Boston. Patients. Twenty-one patients with evidence of HCV infection were identified during a screening program initiated to detect infection in exposed individuals. They ranged from 5 to 53 years of age; 14 were children under age 18. Results. Six patients presented with severe clinical hepatitis before detection by screening, 13 were detected by screening only, and 2 were first detected by screening and subsequently developed symptomatic hepatitis. Follow-up is available on 20 patients; 4 without viremia at identification have remained clinically well. Hepatitis and viremia have resolved in 2, 2 additional subjects have developed normal alanine aminotransferase (ALT) values with persistent viremia, and 13 have biochemical and/or virologic evidence of chronic hepatitis. Eight patients (7 children) have undergone liver biopsies; 7 have histologic findings of chronic hepatitis, 5 have mild fibrosis, and 2 have moderate fibrosis. HCV genotypes 1a and 1b were observed with equal frequency in this group. Conclusions. Some HCV infections associated with IVIG had a more severe, acute course than is ordinarily described. This may be attributable to host factors, such as immunodeficiencies, or virologic factors, such as inoculum or genotype. Although a large percentage (87.5%) of these individuals developed chronic infection, the natural history is not as yet completely defined.

Background Some US residents travel abroad to undergo cosmetic surgery for fat removal, a practice referred to as "lipotourism." Mycobacterium abscessus can cause postsurgical wound infection. Methods US residents who developed M. abscessus wound infection after undergoing cosmetic surgery in the Dominican Republic in 2003 and 2004 were identified using the Emerging Infections Network listserv. Results Twenty returning US travelers with M. abscessus infection were detected. Eight patients had matching isolates, as determined by pulsed-field gel electrophoresis and repetitive element polymerase chain reaction. All 8 patients, who had previously been healthy Hispanic women, underwent abdominoplasties at the same clinic in the Dominican Republic. Symptoms first developed 2-18 weeks after the procedure (median interval, 7 weeks). Only 2 of the 8 patients received a correct diagnosis at the initial presentation. Most patients presented with painful, erythematous, draining subcutaneous abdominal nodules. Seven patients underwent drainage procedures. Six patients received a combination of antibiotics that included a macrolide plus cefoxitin, imipenem, amikacin, and/or linezolid; 2 received clarithromycin monotherapy. All patients but 1 were cured after a median of 9 months of therapy (range, 2-12 months). Because of a lack of access to the surgical clinic, the cause of the outbreak of infection was not identified. The patients who were infected with nonmatching isolates underwent surgeries in different facilities but otherwise had demographic characteristics and clinical presentations similar to those of the 8 patients infected with matching isolates. Conclusions This case series of M. abscessus infection in US "lipotourists" highlights the risks of traveling abroad for surgery and the potential role of the Internet in identifying and investigating outbreaks.

Group B streptococci (GBSs) are the leading cause of neonatal meningitis. GBSs enter the CNS by penetrating the blood-brain barrier (BBB), which consists of specialized human brain microvascular endothelial cells (hBMECs). To identify GBS factors required for BBB penetration, we generated random mutant libraries of a virulent strain and screened for loss of hBMEC invasion in vitro. Two independent hypo-invasive mutants possessed disruptions in the same gene, invasion associated gene (iagA), which encodes a glycosyltransferase homolog. Allelic replacement of iagA in the GBS chromosome produced a 4-fold decrease in hBMEC invasiveness. Mice challenged with the GBS DeltaiagA mutant developed bacteremia comparably to WT mice, yet mortality was significantly lower (20% vs. 90%), as was the incidence of meningitis. The glycolipid diglucosyldiacylglycerol, a cell membrane anchor for lipoteichoic acid (LTA) and predicted product of the IagA glycosyltransferase, was absent in the DeltaiagA mutant, which consequently shed LTA into the media. Attenuation of virulence of the DeltaiagA mutant was found to be independent of TLR2-mediated signaling, but bacterial supernatants from the DeltaiagA mutant containing released LTA inhibited hBMEC invasion by WT GBS. Our data suggest that LTA expression on the GBS surface plays a role in bacterial interaction with BBB endothelium and the pathogenesis of neonatal meningitis.

Group B Streptococcus (GBS) colonizes mucosal surfaces of the human gastrointestinal and gynecological tracts and causes disease in a wide range of patients. Invasive illness occurs after organisms traverse an epithelial boundary and enter deeper tissues. Previously we have reported that the alpha C protein (ACP) on the surface of GBS mediates GBS entry into ME180 cervical epithelial cells and GBS translocation across layers of these cells. We now demonstrate that ACP interacts with host cell glycosaminoglycan (GAG); the interaction of ACP with ME180 cells is inhibited if cells are pretreated with sodium chlorate, an inhibitor of sulfate incorporation, or with heparitinases. The interaction is also inhibited in the presence of soluble heparin or heparan sulfate or host cell-derived GAG. In addition, ACP binds soluble heparin specifically in inhibition and dot blot assays. After interaction with host GAG, soluble ACP enters ME180 cells and fractionates to the eukaryotic cell cytosol. These events are inhibited in cells pretreated with cytochalasin D or with Clostridium difficile toxin B. These data indicate that full-length ACP interacts with ME180 cell GAG and enters the eukaryotic cell cytosol by a mechanism that involves Rho GTPase-dependent actin rearrangements. We suggest that these molecular interactions drive ACP-mediated translocation of GBS across epithelial barriers, thereby facilitating invasive GBS infection.

The beta C protein of group B streptococci (GBS) elicits antibody that is protective against GBS challenge in animals and is considered to be a potential component of a GBS conjugate vaccine. We developed a quantitative enzyme-linked immunosorbent assay to measure beta-specific serum immunoglobulin G (IgG) levels and used it to compare beta-specific IgG in a group of mothers of neonates with invasive type Ib/beta GBS disease and a group of mothers colonized with Ib/beta strains whose neonates remained well. beta-Specific IgG concentrations from these 2 groups were similar. To investigate differences in beta-specific antibody in animals and humans, protein fragments were generated that corresponded to major regions within the beta C protein. A single major region was predominantly recognized in human and rabbit serum samples. Thus, in contrast to immunized animals, no relationship was seen between levels of naturally acquired human beta-specific IgG and protection from neonatal disease. This difference was not explained by a major difference in epitope specificity.

Objective. —To determine the risk of and risk factors for hepatitis C virus (HCV) infection among persons with immune deficiencies who had received intravenous immune globulin (IGIV) between March 1993 and February 1994. Design. —Retrospective cohort study. Setting. —An immunology program in a tertiary care hospital. Patients. —Of 341 persons who had received IGIV between March 1,1993, and February 22,1994, 278 (82%) were enrolled. The mean age for the enrolled persons was 9 years, and 99% had primary immune deficiencies. Main Outcome Measures. —Evidence of HCV infection by detection in sera of antibody to HCV and/or HCV RNA by reverse transcriptase polymerase chain reaction. Results. —Twenty-three (11%) of 210 persons who received the IGIV Gammagard (Baxter Healthcare Corporation, Deerfield, III) became infected compared with none of 52 persons who received exclusively other IGIV products (P=.01). In a multivariate analysis, HCV infection was associated only with Gammagard produced from plasma screened by second-generation (multiantigen) anti-HCV tests (P=.03). Hepatitis C virus RNA was detected in Gammagard, and the risk of transmission to recipients increased with increasing quantity of HCV RNA infused, from 0 for those who received no HCV RNA-positive lots to 29% for the quartile of patients receiving the greatest amount (P Conclusion. —Gammagard was the only IGIV product implicated in the transmission of HCV. Infection was associated with higher quantities of HCV RNA in Gammagard produced from second-generation anti-HCV-screened plasma. Further studies are needed to determine reasons for the infectivity of Gammagard, and viral inactivation and removal steps are needed to ensure the safety of IGIV products.