Biological diversity of prokaryotic type IV secretion systems.

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

Biological diversity of prokaryotic type IV secretion systems.

Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

Enterococcus faecalis PcfC, a spatially localized substrate receptor for type IV secretion of the pCF10 transfer intermediate.

Upon sensing of peptide pheromone, Enterococcus faecalis efficiently transfers plasmid pCF10 through a type IV secretion (T4S) system to recipient cells. The PcfF accessory factor and PcfG relaxase initiate transfer by catalyzing strand-specific nicking at the pCF10 origin of transfer sequence (oriT). Here, we present evidence that PcfF and PcfG spatially coordinate docking of the pCF10 transfer intermediate with PcfC, a membrane-bound putative ATPase related to the coupling proteins of gram-negative T4S machines. PcfC and PcfG fractionated with the membrane and PcfF with the cytoplasm, yet all three proteins formed several punctate foci at the peripheries of pheromone-induced cells as monitored by immunofluorescence microscopy. A PcfC Walker A nucleoside triphosphate (NTP) binding site mutant (K156T) fractionated with the E. faecalis membrane and also formed foci, whereas PcfC deleted of its N-terminal putative transmembrane domain (PcfCDelta N103) distributed uniformly throughout the cytoplasm. Native PcfC and mutant proteins PcfCK156T and PcfCDelta N103 bound pCF10 but not pcfG or Delta oriT mutant plasmids as shown by transfer DNA immunoprecipitation, indicating that PcfC binds only the processed form of pCF10 in vivo. Finally, purified PcfCDelta N103 bound DNA substrates and interacted with purified PcfF and PcfG in vitro. Our findings support a model in which (i) PcfF recruits PcfG to oriT to catalyze T-strand nicking, (ii) PcfF and PcfG spatially position the relaxosome at the cell membrane to stimulate substrate docking with PcfC, and (iii) PcfC initiates substrate transfer through the pCF10 T4S channel by an NTP-dependent mechanism.

Delayed Thrombus Resolution and Fibroproliferative Vascular Wound Healing From Deficiency of Type III Collagen: a Paradoxical Mechanism for Tissue Fragility

Vascular Ehlers-Danlos syndrome is a heritable disease of connective tissue caused by mutations in COL3A1, conferring a tissue deficiency of type III collagen. Cutaneous wounds heal poorly in these patients, and they are susceptible to spontaneous and catastrophic rupture of expansible hollow organs like the gut, uterus, and medium-sized to large arteries, which leads to premature death. Although the predisposition for organ rupture is often attributed to inherent tissue fragility, investigation of arteries from a haploinsufficient Col3a1 mouse model (Col3a1+/-) demonstrates that mutant arteries withstand even supraphysiologic pressures comparably to wild-type vessels. We hypothesize that injury that elicits occlusive thrombi instead unmasks defective thrombus resolution resulting from impaired production of type III collagen, which causes deranged remodeling of matrix, persistent inflammation, and dysregulated behavior by resident myofibroblasts, culminating in the development of penetrating neovascular channels that disrupt the mechanical integrity of the arterial wall. Vascular injury and thrombus formation following ligation of the carotid artery reveals an abnormal persistence and elevated burden of occlusive thrombi at 21 post-operative days in vessels from Col3a1+/- mice, as opposed to near complete resolution and formation of a patent and mature neointima in wild-type mice. At only 14 days, both groups harbor comparable burdens of resolving thrombi, but wild-type mice increase production of type III collagen in actively resolving tissues, while mutant mice do not. Rather, thrombi in mutant mice contain higher burdens of macrophages and proliferative myofibroblasts, which persist through 21 days while wild-type thrombi, inflammatory cells, and proliferation all regress. At the same time that increased macrophage burdens were observed at 14 and 21 days post ligation, the medial layer of mutant arterial walls concurrently harbored a significantly higher incidence of penetrating neovessels compared with those in wild-type mice. To assess whether limited type III collagen production alters myofibroblast behavior, fibroblasts from vEDS patients with COL3A1 missense mutations were seeded into three-dimensional fibrin gel constructs and stimulated with transforming growth factor-β1 to initiate myofibroblast differentiation. Although early signaling events occur similarly in all cell lines, late extracellular matrix- and mechanically-regulated events like transcriptional upregulation of type I and type III collagen secretion are delayed in mutant cultures, while transcription of genes encoding intracellular contractile machinery is increased. Sophisticated imaging of collagen synthesized de novo by resident myofibroblasts visualizes complex matrix reorganization by control cells but only meager remodeling by COL3A1 mutant cells, concordant with their compensatory contraction to maintain tension in the matrix. Finally, administration of immunosuppressive rapamycin to mice following carotid ligation sufficiently halts the initial inflammatory phase of thrombus resolution and fully prevents both myofibroblast migration into the thrombus and the differential development of neovessels between mutant and wild-type mice, suggesting that pathological defects in mutant arteries develop secondarily to myofibroblast dysfunction and chronic inflammatory stimulation, rather than as a manifestation of tissue fragility. Together these data establish evidence that pathological defects in the vessel wall architecture develop in mutant arteries as sequelae to abnormal healing and remodeling responses activated by arterial injury. Thus, these data support the hypothesis that events threatening the integrity of type III collagen-deficient vessels develop not as a result of inherent tissue weakness and fragility at baseline but instead as an episodic byproduct of abnormally persistent granulation tissue and fibroproliferative intravascular remodeling.

Analysis of Agrobacterium tumefaciens VirB6 and VirB9 of the VirB /D4 type IV secretion system

Agrobacterium tumefaciens uses the VirB/D4 type IV secretion system (T4SS) to translocate oncogenic DNA (T-DNA) and protein substrates to plant cells. Independent of VirD4, the eleven VirB proteins are also essential for elaboration of a conjugative pilus termed the T pilus. The focus of this thesis is the characterization and analysis of two VirB proteins, VirB6 and VirB9, with respect to substrate translocation and T pilus biogenesis. Observed stabilizing effects of VirB6 on other VirB subunits and results of protein-protein interaction studies suggest that VirB6 mediates assembly of the secretion machine and T pilus through interactions with VirB7 and VirB9. Topology studies support a model for VirB6 as a polytopic membrane protein with a periplasmic N terminus, a large internal periplasmic loop, five transmembrane segments, and a cytoplasmic C terminus. Topology studies and Transfer DNA immunoprecipitation (TrIP) assays identified several important VirB6 functional domains: (i) the large internal periplasmic loop mediates interaction of VirB6 with the T-DNA, (ii) the membrane spanning region carboxyl-terminal to the large periplasmic loop mediates substrate transfer from VirB6 to VirB8, and (iii) the terminal regions of VirB6 are required for substrate transfer to VirB2 and VirB9. To analyze structure-function relationships of VirB9, the phenotypic consequences of dipeptide insertion mutations were characterized. Substrate discriminating mutations were shown to selectively export the oncogenic T-DNA and VirE2 to plant cells or a mobilizable IncQ plasmid to bacterial cells. Mutations affecting VirB9 interactions with VirB7 and VirB10 were localized to the C- and N- terminal regions respectively. Additionally, “uncoupling” mutations identified in VirB11 and VirB6 that block T pilus assembly, but not substrate transfer to recipient cells, were also identified in VirB9. These results in conjunction with computer analysis establish that VirB9, like VirB6, is also composed of distinct regions or domains that contribute in various ways to secretion channel activity and T pilus assembly. Lastly, in vivo immunofluorescent studies suggest that VirB9 localizes to the outer membrane and may play a role similar to that of secretion/ushers of types II and III secretion systems to facilitate substrate translocation across this final bacterial barrier. ^

Characterization of the Agrobacterium tumefaciens VirB2 pilin of the VirB/D4 Type IV Secretion System

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) delivers oncogenic T-DNA and effector proteins to susceptible plant cells. This leads to the formation of tumors termed Crown Galls. The VirB/D4 T4SS is comprised of 12 subunits (VirB1 to VirB11 and VirD4), which assemble to form two structures, a secretion channel spanning the cell envelope and a T-pilus extending from the cell surface. In A. tumefaciens, the VirB2 pilin subunit is required for assembly of the secretion channel and is the main subunit of the T-pilus. The focus of this thesis is to define key reactions associated with the T4SS biogenesis pathway involving the VirB2 pilin. Topology studies demonstrated that VirB2 integrates into the inner membrane with two transmembrane regions, a small cytoplasmic loop, and a long periplasmic loop comprised of covalently linked N and C termini. VirB2 was shown by the substituted cysteine accessibility method (SCAM) to adopt distinct structural states when integrated into the inner membrane and when assembled as a component of the secretion channel and the T-pilus. The VirB4 and VirB11 ATPases were shown by SCAM to modulate the structural state of membrane-integrated VirB2 pilin, and evidence was also obtained that VirB4 mediates extraction of pilin from the membrane. A model that VirB4 functions as a pilin dislocase by an energy-dependent mechanism was further supported by coimmunoprecipitation and osmotic shock studies. Mutational studies identified two regions of VirB10, an N-terminal transmembrane domain and an outer membrane-associated domain termed the antennae projection, that contribute selectively to T-pilus biogenesis. Lastly, characterization of a VirB10 mutant that confers a ‘leaky’ channel phenotype further highlighted the role of VirB10 in gating substrate translocation across the outer membrane as well as T-pilus biogenesis. Results of my studies support a working model in which the VirB4 ATPase catalyzes dislocation of membrane-integrated pilin, and distinct domains of VirB10 coordinate pilin incorporation into the secretion channel and the extracellular T-pilus.

NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion system.

Type IV secretion (T4S) systems translocate DNA and protein effectors through the double membrane of Gram-negative bacteria. The paradigmatic T4S system in Agrobacterium tumefaciens is assembled from 11 VirB subunits and VirD4. Two subunits, VirB9 and VirB7, form an important stabilizing complex in the outer membrane. We describe here the NMR structure of a complex between the C-terminal domain of the VirB9 homolog TraO (TraO(CT)), bound to VirB7-like TraN from plasmid pKM101. TraO(CT) forms a beta-sandwich around which TraN winds. Structure-based mutations in VirB7 and VirB9 of A. tumefaciens show that the heterodimer interface is conserved. Opposite this interface, the TraO structure shows a protruding three-stranded beta-appendage, and here, we supply evidence that the corresponding region of VirB9 of A. tumefaciens inserts in the membrane and protrudes extracellularly. This complex structure elucidates the molecular basis for the interaction between two essential components of a T4S system.

Evidence for VirB4-mediated dislocation of membrane-integrated VirB2 pilin during biogenesis of the Agrobacterium VirB/VirD4 type IV secretion system.

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.

AGROBACTERIUM VIRB10 CONTRIBUTIONS TO TYPE IV SUBSTRATE SECRETION, T-PILUS BIOGENESIS, AND OUTER MEMBRANE PORE FORMATION

The VirB/D4 type IV secretion system (T4SS) of Agrobacterium tumefaciens functions to transfer substrates to infected plant cells through assembly of a translocation channel and a surface structure termed a T-pilus. This thesis is focused on identifying contributions of VirB10 to substrate transfer and T-pilus formation through a mutational analysis. VirB10 is a bitopic protein with several domains, including a: (i) cytoplasmic N-terminus, (ii) single transmembrane (TM) α-helix, (iii) proline-rich region (PRR), and (iv) large C-terminal modified β-barrel. I introduced cysteine insertion and substitution mutations throughout the length of VirB10 in order to: (i) test a predicted transmembrane topology, (ii) identify residues/domains contributing to VirB10 stability, oligomerization, and function, and (iii) monitor structural changes accompanying energy activation or substrate translocation. These studies were aided by recent structural resolution of a periplasmic domain of a VirB10 homolog and a ‘core’ complex composed of homologs of VirB10 and two outer membrane associated subunits, VirB7 and VirB9. By use of the substituted cysteine accessibility method (SCAM), I confirmed the bitopic topology of VirB10. Through phenotypic studies of Ala-Cys insertion mutations, I identified “uncoupling” mutations in the TM and β-barrel domains that blocked T-pilus assembly but permitted substrate transfer. I showed that cysteine replacements in the C-terminal periplasmic domain yielded a variety of phenotypes in relation to protein accumulation, oligomerization, substrate transfer, and T-pilus formation. By SCAM, I also gained further evidence that VirB10 adopts different structural states during machine biogenesis. Finally, I showed that VirB10 supports substrate transfer even when its TM domain is extensively mutagenized or substituted with heterologous TM domains. By contrast, specific residues most probably involved in oligomerization of the TM domain are required for biogenesis of the T-pilus.

Sensory regulation of network components underlying ciliary locomotion in Hermissenda.

Ciliary locomotion in the nudibranch mollusk Hermissenda is modulated by the visual and graviceptive systems. Components of the neural network mediating ciliary locomotion have been identified including aggregates of polysensory interneurons that receive monosynaptic input from identified photoreceptors and efferent neurons that activate cilia. Illumination produces an inhibition of type I(i) (off-cell) spike activity, excitation of type I(e) (on-cell) spike activity, decreased spike activity in type III(i) inhibitory interneurons, and increased spike activity of ciliary efferent neurons. Here we show that pairs of type I(i) interneurons and pairs of type I(e) interneurons are electrically coupled. Neither electrical coupling or synaptic connections were observed between I(e) and I(i) interneurons. Coupling is effective in synchronizing dark-adapted spontaneous firing between pairs of I(e) and pairs of I(i) interneurons. Out-of-phase burst activity, occasionally observed in dark-adapted and light-adapted pairs of I(e) and I(i) interneurons, suggests that they receive synaptic input from a common presynaptic source or sources. Rhythmic activity is typically not a characteristic of dark-adapted, light-adapted, or light-evoked firing of type I interneurons. However, burst activity in I(e) and I(i) interneurons may be elicited by electrical stimulation of pedal nerves or generated at the offset of light. Our results indicate that type I interneurons can support the generation of both rhythmic activity and changes in tonic firing depending on sensory input. This suggests that the neural network supporting ciliary locomotion may be multifunctional. However, consistent with the nonmuscular and nonrhythmic characteristics of visually modulated ciliary locomotion, type I interneurons exhibit changes in tonic activity evoked by illumination.

Agrobacterium ParA/MinD-like VirC1 spatially coordinates early conjugative DNA transfer reactions.

Agrobacterium tumefaciens translocates T-DNA through a polar VirB/D4 type IV secretion (T4S) system. VirC1, a factor required for efficient T-DNA transfer, bears a deviant Walker A and other sequence motifs characteristic of ParA and MinD ATPases. Here, we show that VirC1 promotes conjugative T-DNA transfer by stimulating generation of multiple copies per cell of the T-DNA substrate (T-complex) through pairwise interactions with the processing factors VirD2 relaxase, VirC2, and VirD1. VirC1 also associates with the polar membrane and recruits T-complexes to cell poles, the site of VirB/D4 T4S machine assembly. VirC1 Walker A mutations abrogate T-complex generation and polar recruitment, whereas the native protein recruits T-complexes to cell poles independently of other polar processing factors (VirC2, VirD1) or T4S components (VirD4 substrate receptor, VirB channel subunits). We propose that A. tumefaciens has appropriated a progenitor ParA/MinD-like ATPase to promote conjugative DNA transfer by: (i) nucleating relaxosome assembly at oriT-like T-DNA border sequences and (ii) spatially positioning the transfer intermediate at the cell pole to coordinate substrate-T4S channel docking.

Alpha C protein of group B Streptococcus as a potential vaccine candidate

Group B Streptococcus (GBS) is a leading cause of life-threatening infection in neonates and young infants, pregnant women, and non-pregnant adults with underlying medical conditions. Immunization has theoretical potential to prevent significant morbidity and mortality from GBS disease. Alpha C protein (α C), found in 70% of non-type III capsule polysaccharide group B Streptococcus, elicits antibodies protective against α C-expressing strains in experimental animals and is an appealing carrier for a GBS conjugate vaccine. We determined whether natural exposure to α C elicits antibodies in women and if high maternal α C-specific serum antibody at delivery is associated with protection against neonatal disease. An ELISA was designed to measure α C-specific IgM and IgG in human sera. A case-control design (1:3 ratio) was used to match α C-expressing GBS colonized and non-colonized women by age and compare quantified serum α C-specific IgM and IgG. Sera also were analyzed from bacteremic neonates and their mothers and from women with invasive GBS disease. Antibody concentrations were compared using t-tests on log-transformed data. Geometric mean concentrations of α C-specific IgM and IgG were similar in sera from 58 α C strain colonized and 174 age-matched non-colonized women (IgG 245 and 313 ng/ml; IgM 257 and 229 ng/ml, respectively). Delivery sera from mothers of 42 neonates with GBS α C sepsis had similar concentrations of α C-specific IgM (245 ng/ml) and IgG (371 ng/ml), but acute sera from 13 women with invasive α C-expressing GBS infection had significantly higher concentrations (IgM 383 and IgG 476 ng/ml [p=0.036 and 0.038, respectively]). Convalescent sera from 5 of these women 16-49 days later had high α C-specific IgM and IgG concentrations (1355 and 4173 ng/ml, respectively). In vitro killing of α C-expressing GBS correlated with total α C-specific antibody concentration. Invasive disease but not colonization elicits α C-specific IgM and IgG in adults. Whether α C-specific IgG induced by vaccine would protect against disease in neonates merits further investigation. ^

The burden of parainfluenza virus infection in patients with hematological malignancy and hematopoietic stem cell transplant (HSCT) recipients in the absence of active immunization and approved therapy: The role of infection control

Background. Community respiratory viruses, mainly RSV and influenza, are significant causes of morbidity and mortality in patients with leukemia and HSCT recipients. The data on impact of PIV infections in these patients is lacking. Methods. We reviewed the records of patients with leukemia and HSCT recipients who developed PIV infection from Oct'02–Nov'07 to determine the outcome of such infections. Results. We identified 200 patients with PIV infections including 80(40%) patients with leukemia and 120 (60%) recipients of HSCT. Median age was 55 y (17-84 y). As compared to HSCT recipients, patients with leukemia had higher APACHE II score (14 vs. 10, p<0.0001); were more likely to have ANC<500 (48% vs. 10%, p<0.0001) and ALC<200 (45% vs. 23.5%, p=0.02). PIV type III was the commonest isolate (172/200, 86%). Most patients 141/200 (70%) had upper respiratory infection (URI), and 59/200 (30%) had pneumonia at presentation. Patients in leukemia group were more likely to require hospitalization due to PIV infection (77% vs. 36% p=0.0001) and were more likely to progress to pneumonia (61% vs. 39%, p=0.002). Fifty five patients received aerosolized ribavirin and/or IVIG. There were no significant differences in the duration of symptoms, length of hospitalization, progression to pneumonia or mortality between the treated verses untreated group. The clinical outcome was unknown in 13 (6%) patients. Complete resolution of symptoms was noted in 91% (171/187) patients and 9% (16/187) patients died. Mortality rate was 17% (16/95) among patients who had PIV pneumonia, with no significant difference between leukemia and HSCT group (16% vs. 17%). The cause of death was acute respiratory failure and/or multi-organ failure in (13, 81%) patients. Conclusions. Patients with leukemia and HSCT could be at high risk for serious PIV infections including PIV pneumonia. Treatment with aerosolized ribavirin and/or IVIG may not have significant effect on the outcome of PIV infection.^

Type I hypersensitivity and its pharmacological modulation in {\it Trichinella\/}-induced intestinal fluid secretion and rapid rejection of the parasite in immunized rats

Studies were performed to test the hypothesis that type I hypersensitivity underlies worm induced intestinal fluid secretion and the rapid rejection of Trichinella spiralis from immunized rats, and the two events may be related in a cause-effect manner.^ Two approaches were taken. One was to determine whether inhibition of anaphylaxis-mediated Cl$\sp{-}$ and fluid secretion accompanying a secondary infection impedes worm rejection from immune hosts. The other was to determine whether induction of intestinal fluid secretion in nonimmune hosts interfered with worm establishment. In both studies, fluid secretion was measured volumetrically 30 min after a challenge infection and worms were counted.^ In immunized rats indomethacin did not affect the worm-induced fluid secretion when used alone, despite inhibiting mucosal prostaglandin synthesis. Fluid secretion was reduced by treatment with diphenhydramine and further reduced by the combination of diphenhydramine and indomethacin. The paradoxical effects of indomethacin when used alone compared with its coadministration with diphenhydramine is explained by the enhancing effect of indomethacin on histamine release. Abolishing net fluid secretion in these studies had no effect on rapid worm rejection in immune hosts.^ Worm establishment was reduced in recipients of immune serum containing IgE antibodies. Net intestinal fluid secretion induced in normal rats by PGE$\sb2$, cholera toxin, or hypertonic mannitol solution had no effect on worm establishment compared with untreated controls.^ In a related experiment, worm-induced intestinal fluid secretion and worm rejection in immune rats were partially blocked by concurrent injection with 5-HT$\sb2$ and 5-HT$\sb3$ blockers (Ketanserin and MDL-72222), suggesting that 5-HT is involved. This possible involvement was supported in that treatment of nonimmune rats with 5-HT significantly inhibited worm establishment in the intestine.^ Results indicate that anaphylaxis is the basis for both worm-induced intestinal fluid secretion and rapid rejection of T. spiralis in immune rats, but these events are independent of one another. 5-HT is a possible mediator of worm rejection, however, its mechanism of action is related to something other than fluid secretion. ^

A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells.

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.