Development of Immunoglobulin M Memory to Both a T-Cell-Independent and a T-Cell-Dependent Antigen following Infection with Vibrio cholerae O1 in Bangladesh

Vibrio cholerae O1 can cause severe watery diarrhea that can be life-threatening without treatment. Infection results in long-lasting protection against subsequent disease. Development of memory B cells of the immunoglobulin G (IgG) and IgA isotypes to V. cholerae O1 antigens, including serotype-specific lipopolysaccharide (LPS) and the B subunit of cholera toxin (CTB), after cholera infection has been demonstrated. Memory B cells of the IgM isotype may play a role in long-term protection, particularly against T-cell-independent antigens, but IgM memory has not been studied in V. cholerae O1 infection. Therefore, we assayed acute- and convalescent-phase blood samples from cholera patients for the presence of memory B cells that produce cholera antigen-specific IgM antibody upon polyclonal stimulation in in vitro culture. We also examined the development of serological and antibody-secreting cell responses following infection. Subjects developed significant IgM memory responses by day 30 after infection, both to the T-cell-independent antigen LPS and to the T-cell-dependent antigen CTB. No significant corresponding elevations in plasma IgM antibodies or circulating IgM antibody-secreting cells to CTB were detected. In 17 subjects followed to day 90 after infection, significant persistence of elevated IgM memory responses was not observed. The IgM memory response to CTB was negatively correlated with the IgG plasma antibody response to CTB, and there was a trend toward negative correlation between the IgM memory and IgA plasma antibody responses to LPS. We did not observe an association between the IgM memory response to LPS and the vibriocidal titer.Vibrio cholerae continues to be a significant global health burden as a cause of severe secretory diarrhea, resulting in an estimated three to five million annual cases, with more than 100,000 deaths from rapid dehydration (47); cholera has recently become endemic in new regions (44, 45). V. cholerae is a noninvasive pathogen that colonizes the mucosal surface of the small intestine. Strains can be distinguished serologically by the O antigen of the lipopolysaccharide (LPS); V. cholerae O1 is the most common cause of cholera in South Asia as well as globally. The O1 serogroup has two major biotypes, El Tor and classical, and two major serotypes, Inaba and Ogawa (35). Natural infection with V. cholerae O1 El Tor induces protective immunity that lasts for at least 3 to 10 years in both areas where cholera is not endemic and areas where it is endemic (21). It remains unknown, however, what aspects of the adaptive immune response to cholera confer this long-term protection.V. cholerae-infected patients mount immunologic responses to both protein and polysaccharide antigens, including rises in both serum immunoglobulin G (IgG) and IgA antibodies (14). A number of these serological responses have been shown to correlate with protection against reinfection; these include the complement-dependent serum vibriocidal antibody (14) and IgA (but not IgG) responses to LPS, cholera toxin B subunit (CTB), and toxin coregulated pilus A (TcpA) (17). These serological responses, however, are short-lived (4, 32), and the association of the vibriocidal titer with protection is not absolute (36), suggesting that these responses may reflect protection from more recent exposure but that other immunologic mechanisms mediate longer-term protection. In addition to serological responses, development of mucosal immune responses to intestinal antigens can be detected in the blood, when B cells activated by antigen in the gut-associated lymphoid tissues circulate transiently in the blood as antibody-secreting cells (ASCs), before homing back to intestinal mucosal surfaces (11, 26). Circulation of ASCs specific to both LPS and CTB is seen after cholera infection, peaking around the seventh day after infection and declining by day 11 (32).Responses of the IgM isotype to cholera antigens have been less thoroughly investigated than the IgG and IgA responses. However, IgM defenses may be an important component of the overall immunologic response to cholera, since vibriocidal antibodies are principally of the IgM isotype (22) and IgM levels of pooled convalescent-phase serum samples correspond closely with vibriocidal activity (24), which in turn correlates with immunity (14). The pentameric structure of IgM facilitates strong cross-linking of antigens and activation of complement in the defense against other gram-negative enteric bacteria (2).We have recently shown development of memory B cells of both the IgG and IgA isotypes to LPS, CTB, and TcpA; these cells persisted in the circulation beyond 1 year for the protein antigens CTB and TcpA, but were not measurably above baseline levels by 9 to 12 months after infection for the polysaccharide-containing antigen LPS (16, 18). These circulating memory B cells can be detected by ex vivo polyclonal stimulation of peripheral blood mononuclear cells (PBMCs); stimulated memory B cells mature into ASCs detectable by enzyme-linked immunospot (ELISPOT) assay. Alternatively, memory B-cell responses can be detected by measuring antigen-specific antibodies secreted by maturing ASCs during the ex vivo stimulation of PBMCs in the memory B-cell assay (18).Memory B cells relevant for cholera immunity may include IgM⁺ as well as switched-memory (IgA⁺ and IgG⁺) populations. The majority of circulating IgM⁺ cells are naïve B cells, but some IgM⁺ cells bear the memory cell marker CD27⁺, and recent evidence suggests that these IgM⁺CD27⁺ cells are true memory B cells whose immunoglobulin variable region genes have undergone somatic hypermutation in response to antigen in early-stage germinal centers (39). IgM⁺ memory cells can undergo isotype switching to produce IgG, IgA, or IgE antibody, but they also have a role in producing rapid, high-affinity IgM antibody responses to acute infection (19, 37, 46). In this study, we have measured the development of memory B-cell responses of the IgA, IgG, and IgM isotypes to both a protein (CTB) and a nonprotein (LPS) antigen, and we compared these memory responses with other immunologic responses in patients after V. cholerae infection in Bangladesh.     

Sleep Disordered Breathing,Daytime Symptoms,and Functional Performance in Stable Heart Failure

Study Objectives:

To evaluate characteristics of sleep disordered breathing (SDB); clinical and demographic correlates of SDB; and the extent to which SDB explains functional performance and symptoms in stable heart failure patients receiving care in structured HF disease management programs.

Design:

Cross-sectional, observational study.

Setting:

Structured heart failure disease management programs.

Participants:

170 stable chronic heart failure patients (mean age = 60.3 ± 16.8 years; n = 60 [35%] female; n = 50 [29%] African American; left ventricular ejection fraction mean = 32 ± 14.6).

Interventions:

N/A

Measurements and Results:

Full polysomnography was obtained for one night on participants in their homes. Participants completed the 6-minute walk, 3 days of actigraphy, MOS-SF 36, Epworth Sleepiness Scale, Pittsburgh Sleep Quality Index, Multi-Dimensional Assessment of Fatigue Scale, and the Centers for the Epidemiological Studies of Depression Scale. Fifty-one percent had significant SDB; Sixteen (9%) of the total sample had central sleep apnea. Severe SDB was associated with a 4-fold increase in the likelihood of poor self-reported physical function (OR = 4.15, 95%CI = 1.19–14.57) and CSA was associated with low levels of daytime mobility (OR = 4.09, 95%CI = 1.23–13.62) after controlling for clinical and demographic variables. There were no statistically significant relationships between SDB and daytime symptoms or self-reported sleep, despite poorer objective sleep quality in patients with SDB.

Conclusions:

Severe SDB is associated with poor physical function in patients with stable HF but not with daytime symptoms or self-reported sleep, despite poorer objective sleep quality in patients with SDB.

Citation:

Redeker NS; Muench U; Zucker MJ; Walsleben J; Gilbert M; Freudenberger R; Chen M; Campbell D; Blank L; Berkowitz R; Adams L; Rapoport DM. Sleep disordered breathing, daytime symptoms, and functional performance in stable heart failure. SLEEP 2010;33(4):551-560.     

Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation

Flow cytometric detection of antigen-specific CD8+ T cells has previously been limited to MHC-class I tetramer staining or intracellular cytokine production, neither of which measure the cytolytic potential of these cells. Here we present a novel technique to enumerate antigen-specific CD8+ T cells using a marker expressed on the cell surface following activation induced degranulation, a necessary precursor of cytolysis. This assay measures the exposure of CD107a and b, present in the membrane of cytotoxic granules, onto the cell surface as a result of degranulation. Acquisition of cell surface CD107a and b is associated with loss of intracellular perforin and is inhibited by colchicine, indicating that exposure of CD107a and b to the cell surface is dependent on degranulation. CD107a and b are expressed on the cell surface of CD8+ T cells following activation with cognate peptide, concordant with production of intracellular IFNgamma. Finally, CD107-expressing CD8+ T cells are shown to mediate cytolytic activity in an antigen-specific manner. Measurement of CD107a and b expression can also be combined with MHC-class I tetramer labeling and intracellular cytokine staining to provide a more complete assessment of the functionality of CD8+T cells expressing cognate T cell receptors (TCR).     

Haemophilus ducreyi requires an intact flp gene cluster for virulence in humans

An intact Haemophilus ducreyi flp operon is essential for microcolony formation in vitro. tadA is the 9th of 15 genes in the operon and has homology to NTPases of type IV secretion systems. Fifteen human volunteers were experimentally infected with both H. ducreyi 35000HP and the tadA mutant, 35000HP.400. Papules developed at similar rates at sites inoculated with the mutant and parent, while pustules formed at 36.4% of parent sites and at 0% of mutant sites (P = 0.001). Compared to 35000HP, 35000HP.400 had only a modest but significant reduction in lesion scores in the temperature-dependent rabbit model of chancroid. These data suggest that proteins secreted by the flp locus are required for full expression of virulence by H. ducreyi in humans but have less of a role in virulence in an animal model of infection.     

Comparative Analysis of Apoptosis and Inflammation Genes of Mice and Humans

Apoptosis (programmed cell death) plays important roles in many facets of normal mammalian physiology. Host-pathogen interactions have provided evolutionary pressure for apoptosis as a defense mechanism against viruses and microbes, sometimes linking apoptosis mechanisms with inflammatory responses through NFκB induction. Proteins involved in apoptosis and NFκB induction commonly contain evolutionarily conserved domains that can serve as signatures for identification by bioinformatics methods. Using a combination of public (NCBI) and private (RIKEN) databases, we compared the repertoire of apoptosis and NFκB-inducing genes in humans and mice from cDNA/EST/genomic data, focusing on the following domain families: (1) Caspase proteases; (2) Caspase recruitment domains (CARD); (3) Death Domains (DD); (4) Death Effector Domains (DED); (5) BIR domains of Inhibitor of Apoptosis Proteins (IAPs); (6) Bcl-2 homology (BH) domains of Bcl-2 family proteins; (7) Tumor Necrosis Factor (TNF)-family ligands; (8) TNF receptors (TNFR); (9) TIR domains; (10) PAAD (PYRIN; PYD, DAPIN); (11) nucleotide-binding NACHT domains; (12) TRAFs; (13) Hsp70-binding BAG domains; (14) endonuclease-associated CIDE domains; and (15) miscellaneous additional proteins. After excluding redundancy due to alternative splice forms, sequencing errors, and other considerations, we identified cDNAs derived from a total of 227 human genes among these domain families. Orthologous murine genes were found for 219 (96%); in addition, several unique murine genes were found, which appear not to have human orthologs. This mismatch may be due to the still fragmentary information about the mouse genome or genuine differences between mouse and human repertoires of apoptotic genes. With this caveat, we discuss similarities and differences in human and murine genes from these domain families.     

Effects of a brisk walk on lipoprotein lipase activity and plasma triglyceride concentrations in the fasted and postprandial states

This study aimed to determine whether changes in plasma heparin-releasable lipoprotein lipase (LPL) activity following a brisk walk were associated with decreases in fasting and/or postprandial triglyceride (TG) concentrations. Two groups of pre-menopausal women participated. In one group (fasting study group, n=10), TG concentrations and post-heparin plasma LPL activity were measured in the fasted state on two occasions: ~18 h after a 2-h treadmill walk at 50% maximal oxygen uptake (exercise trial); and after a day of no exercise (control trial). The other group (postprandial study group, n=9) undertook two oral fat tolerance tests (blood samples taken fasting and for 6 h after a high-fat meal), with plasma LPL activity measured 6 h after meal ingestion. Pre-conditions were the same as for the fasting study group (i.e. control and prior exercise). Prior exercise reduced fasting TG concentrations by 23 (7)% (fasting study group) [mean (SEM)] and by 18 (9)% (postprandial study group) (both P<0.05), and the postprandial TG response by 23 (6)% (postprandial study group) (P<0.01). Plasma LPL activity was not significantly increased by exercise in either the fasting or postprandial study groups. However, exercise-induced changes in both fasting and postprandial LPL activity were significantly correlated with the respective exercise-induced changes in fasting TG concentration and the postprandial TG response (r=−0.70 and −0.77 respectively, P<0.05 for both). These data suggest that increased LPL activity may contribute to the hypotriglyceridaemic effect of moderate exercise, although other mechanisms are also likely to be involved. Electronic Publication     

Increasing access to medical education for students from medically underserved communities: one program's success.

The Premedical Honors College (PHC) is an eight-year, BS-MD program created in 1994 by Baylor College of Medicine (BCM) and The University of Texas-Pan American (UT-PA) to increase the number of physicians addressing the health care needs of underserved populations in Texas. The PHC targets South Texas, a 13-county, medically underserved area with a population that is 82% Hispanic. To date, the PHC has had 159 matriculants and 71 graduates, of whom 60 (84.5%) have matriculated into medical school. These results are significant considering that in 1996, only four students from all five South Texas colleges (combined enrollment of 30000 students) were accepted to medical school. An outcomes study comparing PHC matriculants with students of similar academic ability, ethnicity, and interest in medicine revealed that the odds of medical school matriculation were seven times higher for PHC students than for non-PHC students. The PHC's initial success has been acknowledged by the Texas legislature, which recently passed a bill to promote the PHC's replication. In addition, the number of PHC students-of whom 95% are Mexican American-who matriculate into medical school annually is significant nationally. In 2001, only 386 U.S. medical school matriculants (2.3% of all matriculants) were Mexican American; 17 of these students (4.4%) were PHC graduates. If current trends continue, the PHC could significantly expand the number of physicians serving minority and medically underserved populations in Texas and the nation. Also, the PHC provides an opportunity for research on programs designed to create pathways from high school to medical school.     

VEGFR1/CXCR4-positive progenitor cells modulate local inflammation and augment tissue perfusion by a SDF-1-dependent mechanism

Recruitment and retention of circulating progenitor cells at the site of injured or ischemic tissues facilitates adult neo-vascularization. We hypothesized that cell therapy could modulate local neo-vascularization through the vascular endothelial growth factor (VEGF)/stromal cell-derived factor-1 (SDF-1) axis and by paracrine effects on local endothelial cells. We isolated from rat bone marrow a subset of multipotent adult progenitor cell-derived progenitor cells (MDPC). In vitro, MDPCs secreted multiple cytokines related to inflammation and angiogenesis, including monocyte chemotactic protein-1, SDF-1, basic fibroblast growth factor, and VEGF, and expressed the chemokine receptors CXCR4 and VEGFR1. To investigate in vivo properties, we transplanted MDPCs into the ischemic hind limbs of rats. Elevated levels of the chemokine SDF-1 and colocalization of CD11b⁺ cells marked the initial phase of tissue remodeling after cell transplantation. Prolonged engraftment was observed in the adventitial–medial border region of arterioles of ischemic muscles. However, engrafted cells did not differentiate into endothelial or smooth muscle cells. Limb perfusion normalized 4 weeks after cell injection. Inhibition of SDF-1 reduced the engraftment of transplanted cells and decreased endothelial cell proliferation. These findings suggest a two-stage model whereby transplanted MDPCs modulate wound repair through recruitment of inflammatory cells to ischemic tissue. This is an important potential mechanism for cell transplantation, in addition to the direct modulation of local vascular cells through paracrine mechanisms.