Using two monoclonal antibodies, we found subtypes among pneumococcal isolates that are typed as serotype 6A by the quellung reaction. The prevalent subtype bound to both monoclonal antibodies and was labeled here 6A␣, whereas the minor subtype bound to only one monoclonal antibody and was labeled 6A␤. To determine the biochemical nature of the two serologically defined subtypes, we purified capsular polysaccharides (PSs) from the two subtypes and examined their chemical structures with gas-liquid chromatography and mass spectrometry. The study results for 6A␣ PS are consistent with the previously published structure of 6A PS, which is 32) galactose (133) glucose (133) rhamnose (133) Streptococcus pneumoniae is a major human pathogen that is responsible for a large percentage of cases of pneumonia, meningitis, otitis media, and sepsis (6). All pathogenic pneumococci are known to display one of many structurally diverse carbohydrate capsules, which shield pneumococci from host phagocytes and increase their pathogenicity (2). Antisera to a capsule type can be used to treat patients infected with the pneumococci expressing that capsule type (4). Consequently, for the past century, the serological types of pneumococcal capsules have been extensively investigated with quellung reactions. These studies have culminated in identifying 90 different pneumococcal capsules with distinct serological patterns (9) and chemical structures (10).Not all 90 serotypes are equally pathogenic. For instance, serotypes 6A and 6B account for 4.7% and 7%, respectively, of cases of invasive pneumococcal disease in the U.S. population (19,20). Because of their medical importance, the molecular natures of serotype 6A and its related serotype, serotype 6B, have been studied extensively. Biochemical studies found that the capsular polysaccharides (PSs) of serotypes 6A and 6B are linear polymers with a repeating unit containing four monosaccharides/alditols: rhamnose, ribitol-phosphate (P), galactose, and glucose (10). The two PSs are identical except for a difference in the linkage between rhamnose and ribitol (see Fig. 6).Currently available pneumococcal vaccines are designed to elicit antibodies to the capsular PSs of the most common pneumococcal serotypes. Since vaccine-induced immunoprotection is serotype specific, serotyping pneumococcal isolates from patients is an important tool for monitoring the effectiveness of pneumococcal vaccines (3). Because the classical quellung reaction with rabbit antisera is tedious to perform (13), we have developed a new serotyping system based on mouse monoclonal antibodies (mAbs) and a multiplexed immunoassay (27). While validating the new system, we found that a minor fraction of the isolates determined to be serotype 6A by quellung reaction bound to one 6A-specific mAb (Hyp6AG1) but not to the other (Hyp6AM3), whereas the majority of the serotype 6A isolates bound to both mAbs (12). To distinguish between the isolates, we have labeled the isolates reacting with both mAbs as 6A␣ and those reacting wit...

Purpose: Medulloblastoma, a malignant pediatric brain tumor, is incurable in about one third of patients despite multimodal treatments. In addition, current therapies can lead to long-term disabilities. Based on studies of the extensive tropism of neural stem cells (NSC) toward malignant gliomas and the secretion of growth factors common to glioma and medulloblastoma, we hypothesized that NSCs could target medulloblastoma and be used as a cellular therapeutic delivery system. Experimental Design: The migratory ability of HB1.F3 cells (an immortalized, clonal human NSC line) to medulloblastoma was studied both in vitro and in vivo. As proof-of-concept, we used HB1.F3 cells engineered to secrete the prodrug activating enzyme cytosine deaminase. We investigated the potential of human NSCs to deliver a therapeutic gene and reduce tumor growth. Results: The migratory capacity of HB1.F3 cells was confirmed by an in vitro migration assay, and corroborated in vivo by injecting chloromethylbenzamido-Dil^labeled HB1.F3 cells into the hemisphere contralateral to established medulloblastoma in nude mice. In vitro studies showed the therapeutic efficacy of HB1.F3-CD on Daoy cells in coculture experiments. In vitro therapeutic studies were conducted in which animals bearing intracranial medulloblastoma were injected ipsilaterally with HB1.F3-CD cells followed by systemic 5-flourocytosine treatment. Histologic analyses showed that human NSCs migrate to the tumor bed and its boundary, resulting in a 76% reduction of tumor volume in the treatment group (P < 0.01).Conclusion: These studies show for the first time the potential of human NSCs as an effective delivery system to target and disseminate therapeutic agents to medulloblastoma.Medulloblastoma is the most common childhood malignant brain tumor. Although multimodal treatments, including radical surgical resection followed by radiation and chemotherapy, have substantially improved the survival rate for this disease, it remains incurable in about one third of patients.These treatments are also toxic and can lead to long-term disabilities (1, 2). The main cause of death is recurrence associated with tumor dissemination, at which point current therapeutic options have little efficacy (3, 4). Consequently, there is substantial need for novel, effective, low-toxicity therapies for children with medulloblastoma.The discovery of the inherent tumor-tropic properties of neural stem cells (NSC) could serve as a novel adjuvant strategy to current medulloblastoma treatments. Recent studies have shown that NSCs have the capacity to target therapeutic genes to brain tumors, such as malignant glioma (5 -13) and melanoma brain metastasis (14). We have expanded these investigations to determine whether NSCs are capable of targeting medulloblastoma in an orthotopic xenograft animal model. Therapeutic proof-of-concept studies were done using cytosine deaminase (CD) -producing NSCs and systemic 5-fluorocytosine (5-FC) prodrug administration. Our results show for the first time the pote...

We have recently reported a new pneumococcal serotype (6C), which is closely related to serotype 6A (I. H. Park et al., J. Clin. Microbiol. 45:1225-1233, 2007). To investigate the genetic basis for serotype 6C, we studied the capsule gene loci of 14 6C isolates from three different continents, including one isolated in Alabama 27 years ago. The wciN region of all 6C isolates has a 1,029-bp-long sequence that replaces the 1,222-bp-long sequence of the 6A wciN region. This recombination event has created a new 1,125-bp-long open reading frame which encodes a product that is also homologous to glycosyl transferases. Flanking this introduced gene is 300 bp upstream and 100 bp downstream with only about 90% homology with 6A and which is identical in all 6C isolates. Transfer of the wciN region converts 6A to 6C. Determination of the DNA sequence of the entire capsule gene locus of one 6C isolate showed that the 6C capsule gene locus is almost identical (>98% homologous) to that of 6A except for the wciN region. These findings indicate that the 6C capsule type originated more than 27 years ago by a single recombination event in a 6A locus in which 6A wciN was replaced by a gene of unknown origin.

ABSTRACTAlthough virulence ofStreptococcus pneumoniaeis associated with its capsule, some pathogenicS. pneumoniaeisolates lack capsules and are serologically nontypeable (NT). We obtained 64 isolates that were identified as NT “pneumococci” (i.e., bacteria satisfying the conventional definition but without the multilocus sequence typing [MLST]-based definition ofS. pneumoniae) by the traditional criteria. All 64 were optochin sensitive and hadlytA, and 63 hadply. Twelve isolates hadcpsA, suggesting the presence of a conventional but defective capsular polysaccharide synthesis (cps) locus. The 52cpsA-negative isolates could be divided into three null capsule clades (NCC) based onaliC(aliB-like ORF1),aliD(aliB-like ORF2), and our newly discovered gene,pspK, in theircpsloci.pspKencodes a protein with a long alpha-helical region containing an LPxTG motif and a YPT motif known to bind human pIgR. There were nine isolates in NCC1 (pspK+but negative foraliCandaliD), 32 isolates in NCC2 (aliC+aliD+but negative forpspK), and 11 in NCC3 (aliD+but negative foraliCandpspK). Among 52cpsA-negative isolates, 41 were identified asS. pneumoniaeby MLST analysis. All NCC1 and most NCC2 isolates wereS. pneumoniae, whereas all nine NCC3 and two NCC2 isolates were notS. pneumoniae. Several NCC1 and NCC2 isolates from multiple individuals had identical MLST andcpsregions, showing that unencapsulatedS. pneumoniaecan be infectious among humans. Furthermore, NCC1 and NCC2S. pneumoniaeisolates could colonize mice as well as encapsulatedS. pneumoniae, althoughS. pneumoniaewith an artificially disruptedcpslocus did not. Moreover, an NCC1 isolate withpspKdeletion did not colonize mice, suggesting thatpspKis critical for colonization. Thus, PspK may provide pneumococci a means of surviving in the nasopharynx without capsule.IMPORTANCEThe presence of a capsule is critical for many pathogenic bacteria, including pneumococci. Reflecting the pathogenic importance of the pneumococcal capsule, pneumococcal vaccines are designed to elicit anticapsule antibodies. Additional evidence for the pathogenic importance of the pneumococcal capsule is the fact that in pneumococci all the genes necessary for capsule production are together in one genetic locus, which is called thecpslocus. However, there are occasional pathogenic pneumococci without capsules, and how they survive in the host without the capsule is unknown. Here, we show that in these acapsular pneumococci, thecpsloci have been replaced with various novel genes and they can colonize mouse nasopharynges as well as capsulated pneumococci. Since the genes that replace thecpsloci are likely to be important in host survival, they may show new and/or alternative capsule-independent survival mechanisms used by pneumococci.

Purpose: Brainstem gliomas are usually inoperable and have a dismal prognosis.Based on the robust tropisms of neural stem cells (NSC) and mesenchymal stem cells (MSC) to brain tumors, we compared the tumor-tropic migratory capacities of these stem cells and evaluated the therapeutic potential of genetically engineered human NSCs encoding cytosine deaminase (CD) and IFNβ against brainstem gliomas. Experimental Design: The directed migratory capacities of NSCs and MSCs to brainstem glioma (F98) were evaluated both in vitro and in vivo. The human NSCs (HB1. F3) and various human MSCs, such as bone marrow-derived MSCs (HM3.B10), adipose tissue-derived MSCs, and umbilical cord blood-derived MSCs, were tested. Human fibroblast cells (HFF-1) were used as the negative control. As a proof of concept, the bioactivity of HB1.F3-CD-IFNβ was analyzed with a cell viability assay, and animals with brainstem gliomas were injected with HB1.F3-CD-IFNβ cells followed by systemic 5-fluorocytosine treatment.Results: In an in vitro modified Transwell migration assay and in vivo stem cell injection into established brainstem gliomas in rats, all the stem cells showed a significant migratory capacity compared with that of the control (P < 0.01). Histologic analysis showed a 59% reduction in tumor volume in the HB1.F3-CD-IFNβ-treated group (P < 0.05). Apoptotic cells were increased 2.33-fold in animals treated with HB1.F3-CD-IFNβ compared with the respective control groups (P < 0.01). Conclusion: The brainstem glioma-tropic migratory capacities of MSCs from various sources were similar to those of NSCs. Genetically engineered NSCs show therapeutic efficacy against brainstem gliomas.

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Nuclear protein HMGB1 is secreted in response to various stimuli and functions as a danger-associated molecular pattern. Extracellular HMGB1 induces inflammation, cytokine production, and immune cell recruitment via activation of various receptors. As HMGB1 does not contain an endoplasmic reticulumtargeting signal peptide, HMGB1 is secreted via the endoplasmic reticulum-Golgi independently via an unconventional secretion pathway. However, the mechanism underlying HMGB1 secretion remains largely unknown. Here, we investigated the role of secretory autophagy machinery and vesicular trafficking in HMGB1 secretion. We observed that HSP90AA1 (heat shock protein 90 alpha family class A member 1), a stress-inducible protein, regulates the translocation of HMGB1 from the nucleus to the cytoplasm and its secretion through direct interaction. Additionally, geldanamycin, an HSP90AA1 inhibitor, reduced HMGB1 secretion. GORASP2/GRASP55 (golgi reassembly stacking protein 2), ARF1 Q71L (ADP ribosylation factor 1), and SAR1A T39N (secretion associated Ras related GTPase 1A), which promoted unconventional protein secretion, increased HMGB1 secretion. HMGB1 secretion was inhibited by an early autophagy inhibitor and diminished in ATG5-deficient cells even when GORASP2 was overexpressed. In contrast, a late autophagy inhibitor increased HMGB1 secretion under the same conditions. The multivesicular body formation inhibitor GW4869 dramatically decreased HMGB1 secretion under HMGB1 secretion-inducing conditions. Thus, we demonstrated that secretory autophagy and multivesicular body formation mediate HMGB1 secretion.

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