Identification, recombinant expression, immunolocalization in macrophages, and T-cell responsiveness of the major extracellular proteins of Francisella tularensis

A safer and more effective vaccine than the previously developed live attenuated vaccine is needed for combating Francisella tularensis, a highly infectious bacterial pathogen. To search for potential candidates for inclusion in a new vaccine, we characterized the proteins present in the culture filtrates of a virulent recent clinical isolate and the attenuated live vaccine strain of F. tularensis using a proteomic approach. We identified a total of 12 proteins; among these, catalase-peroxidase was much more abundant in the culture filtrate of the virulent clinical isolate, whereas bacterioferritin was more abundant in the culture filtrate of the live vaccine strain. Streptolysin O treatment of infected human macrophages indicated that catalase-peroxidase and the heat shock protein GroEL are released intracellularly by actively growing F. tularensis. Mice immunized with F. tularensis developed significant cell-mediated immune responses to catalase-peroxidase, the heat shock protein GroEL, and bacterioferritin as measured by splenic lymphocyte proliferation and gamma interferon production. Finally, we expressed the major culture filtrate proteins that are promising vaccine candidates in Escherichia coli at high levels in soluble form to facilitate study of their immunobiology and potential role in vaccines.     

Abnormal cervicovaginal smears due to endometriosis: a continuing problem.

Endometriosis may be challenging when identified on cervicovaginal smears (CVS), leading to an incorrect interpretation of high-grade squamous intraepithelial lesion (HSIL), or atypical glandular cells of undetermined significance (AGUS) including adenocarcinoma in situ (AIS). Awareness of cervical endometriosis, particularly in predisposed patients, is crucial for a correct diagnosis. While cervical endometriosis has been reported to be a diagnostic pitfall of glandular abnormalities, its characteristic features are still not well-established. This may partially be attributed to the varied cytomorphologic features endometriosis shows, depending on menstrual cycle hormonal changes. We describe our experience with three examples where CVS were interpreted as either AGUS or HSIL, which led to a hysterectomy in 2 of 3 patients. Cervical endometriosis needs to be considered with other well-known benign conditions that mimic glandular abnormalities, including cervicitis, tubal metaplasia, lower uterine segment sampling, and microglandular hyperplasia. Published series and our own experience lead us to suggest that these smears will continue to present diagnostic difficulties.     

Hemolytic uremic syndrome after bone marrow transplantation: clinical characteristics and outcome in children.

Hemolytic uremic syndrome (HUS) is an uncommon but potentially life-threatening complication of hematopoietic stem cell transplantation. We retrospectively studied the medical records of 293 children who underwent allogeneic bone marrow transplantation at St. Jude Children's Research Hospital between 1992 and 1999 to describe the clinical course of and to identify risk factors for transplant-associated HUS. Conditioning regimens included cyclophosphamide, cytarabine, and total body irradiation for patients with hematologic malignancies (n = 244); patients with nonmalignant diseases (n = 49) received disease-specific regimens. Grafts from unrelated or mismatched related donors were depleted of T lymphocytes, whereas matched sibling grafts were unmanipulated. All patients received cyclosporine as prophylaxis for graft-versus-host disease. Recipients of grafts from matched siblings also received pentoxifylline or short-course methotrexate. HUS developed in 28 (9.6%) patients at a median of 171 days after transplantation. We identified older donor age (P = .029), use of antithymocyte globulin in the conditioning regimen (P = .008), and recipient CMV seronegativity (P = .011) as being associated with an increased risk of HUS. With a multiple regression analysis, the use of antithymocyte globulin (beta = .86; P = .04) and recipient cytomegalovirus seronegativity (beta = .93; P = .035) remained significant risk factors for the development of HUS.     

O-antigen-deficient Francisella tularensis Live Vaccine Strain mutants are ingested via an aberrant form of looping phagocytosis and show altered kinetics of intracellular trafficking in human macrophages

We examined the uptake and intracellular trafficking of F. tularensis Live Vaccine Strain (LVS) and LVS with disruptions of wbtDEF and wbtI genes essential for synthesis of the O antigen of lipopolysaccharide. Unlike parental bacteria, O-antigen-deficient LVS is efficiently killed by serum with intact complement but not by serum lacking terminal complement components. Opsonization of O-antigen-deficient LVS in serum lacking terminal complement components allows efficient uptake of these live bacteria by macrophages. In the presence of complement, whereas parental F. tularensis LVS is internalized within spacious pseudopod loops, mutant LVS is internalized within tightly juxtaposed multiple onion-like layers of pseudopodia. Without complement, both parental and mutant LVSs are internalized within spacious pseudopod loops. Thus, molecules other than O antigen are important in triggering dramatic pseudopod extensions and uptake by spacious pseudopod loops. Following uptake, both parental and mutant LVSs enter compartments that show limited staining for the lysosomal membrane glycoprotein CD63 and little fusion with secondary lysosomes. Subsequently, both parental and mutant LVSs lose their CD63 staining. Whereas the majority of parental LVS escapes into the cytosol by 6 h after uptake, mutant LVS shows a marked lag but does escape by 1 day after uptake. Despite the altered kinetics of phagosome escape, both mutant and parental strains grow to high levels within human macrophages. Thus, the O antigen plays a role in the morphology of uptake in the presence of complement and the kinetics of intracellular growth but is not essential for escape, survival, altered membrane trafficking, or intramacrophage growth.     

Contaminating cells alter gene signatures in whole organ versus laser capture microdissected tumors: a comparison of experimental breast cancers and their lymph node metastases

Genome-wide expression profiling has expedited our molecular understanding of the different subtypes of breast cancers, as well as defined the differences among genes expressed in primary tumors and their metastases. Laser-capture microdissection (LCM) coupled to gene expression analysis allows us to understand how specific cell types contribute to the total cancer gene expression signature. Expression profiling was used to define genes that contribute to breast cancer spread into and/or growth within draining lymph nodes (LN). Whole tumor xenografts and their matched whole LN metastases were compared to LCM captured cancer cells from the same tumors and matched LN metastases. One-thousand nine-hundred thirty genes were identified by the whole organ method alone, and 1,281 genes by the LCM method alone. However, less than 1% (30 genes) of genes that changed between tumors and LN metastases were common to both methods. Several of these genes have previously been implicated in cancer aggressiveness. Our data show that whole-organ and LCM based gene expression profiling yield distinctly different lists of metastasis-promoting genes. Contamination of the tumor cells, and cross reactivity of mouse RNA to human-specific chips may explain these differences, and suggests that LCM-derived data may be more accurate.     

Identification of a novel mycobacterial histone H1 homologue (HupB) as an antigenic target of pANCA monoclonal antibody and serum immunoglobulin A from patients with Crohn's disease

pANCA is a marker antibody associated with inflammatory bowel disease (IBD), including most patients with ulcerative colitis and a subset with Crohn's disease. This study addressed the hypothesis that pANCA reacts with an antigen(s) of microbial agents potentially relevant to IBD pathogenesis. Using a pANCA monoclonal antibody, we have previously identified the C-terminal basic random-coil domain of histone H1 as a pANCA autoantigen. BLAST analysis of the peptide databases revealed H1 epitope homologues in open reading frames of the Mycobacterium tuberculosis genome. Western analysis of extracts from six mycobacterial species directly demonstrated reactivity to a single, conserved approximately 32-kDa protein. Direct protein sequencing, followed by gene cloning, revealed a novel 214-amino-acid protein, an iron-regulated protein recently termed HupB. Sequence analysis demonstrated its homology with the mammalian histone H1 gene family, and recombinant protein expression confirmed its reactivity with the 5-3 pANCA monoclonal antibody. Binding activity of patient serum immunoglobulin G (IgG) to HupB did not correlate with reactivity to histone H1 or pANCA, indicating the complex character of the pANCA antigen. However, anti-HupB IgA was strongly associated with Crohn's disease (P < 0.001). These findings indicate that the 5-3 pANCA monoclonal antibody detects a structural domain recurrent among mycobacteria and cross-reactive with a DNA-binding domain of histone H1. The association of HupB-binding serum IgA with IBD provides new evidence for the association of a mycobacterial species with Crohn's disease.     

T-cell epitope mapping of the three most abundant extracellular proteins of Mycobacterium tuberculosis in outbred guinea pigs

The three most abundant extracellular proteins of Mycobacterium tuberculosis, the 30-, 32-, and 16-kDa major extracellular proteins, are particularly promising vaccine candidates. We have mapped T-cell epitopes of these three proteins in outbred guinea pigs by immunizing the animals with each protein and assaying splenic lymphocyte proliferation against a series of overlapping synthetic peptides covering the entire length of the mature proteins. The 30-kDa protein contained nine immunodominant epitopes, the 32-kDa protein contained two immunodominant epitopes, and the 16-kDa protein contained a highly immunodominant region at its N terminus. The immunodominant epitopes of the 30- and 32-kDa proteins in outbred guinea pigs were frequently identified in healthy purified-protein-derivative-positive or BCG-vaccinated individuals in previous studies. The immunodominant epitopes of these major extracellular proteins have potential utility in an epitope-based vaccine against tuberculosis.     

Caregiving and cardiovascular disease risk factors in male and female Luo elders from Kenya

Background: The HIV/AIDS pandemic has created over 11 million orphans, who are primarily being cared for by grandparents. It has been suggested that this renewed parenting responsibility presents elders with added stressors. Few studies have systematically examined the impact of caregiving on health outcomes.

Aim: The aim of this study was to examine the impact of caregiving on cardiovascular risk. It was hypothesized that caregiving would increase cardiovascular disease risk as measured by Framingham risk scores.

Subjects and method: 386 Luo elders (age = 73±8), divided into caregiving and non-caregiving groups, were recruited from the Nyanza Province, Kenya. Data were obtained from the participants including: Total cholesterol, HDL cholesterol, glucose, blood pressure, age, sex and smoking status.

Results: No significant difference was found between the Framingham risk scores of caregivers and non-caregivers. Among women increased BMI was positively associated with Framingham score (p=0.017), and among men increased waist circumference was positively associated with the score (p<0.001). Among women, the number of orphans under one's care lowered the risk of falling into the top quartile of the Framingham score while being a caregiver increased the risk of falling into the top quartile.

Conclusion: This study demonstrates that there is not a simple relationship between caregiving and cardiovascular risk.     

Cellular localization of complement C3 and C4 transcripts in intestinal specimens from patients with Crohn's disease

It has been suggested that the increase in C3 and C4 levels in jejunal perfusates of patients with Crohn's disease (CD) results from local intestinal synthesis of complement. The present study evaluated the expression of these complement genes in inflamed tissues from patients with CD. Surgically resected specimens from patients with CD and control tissue obtained from subjects with adenocarcinoma of the colon were evaluated for C3 and C4 gene expression by the use of 35S-labelled anti-sense RNA probes. All tissue samples, diseased and normal tissue, expressed C4 mRNA throughout in the intestinal epithelium. C3 mRNA was not detected in epithelial cells in histologically normal tissue, but in diseased specimens there was a focal distribution of C3 mRNA in epithelial cells of the crypts, but not in villous epithelium. Focal C3 gene expression correlated with crypt abscess formation and the presence of polymorphonuclear leucocytes in the lumen of the crypts. In addition, C3 mRNA was also found in macrophages of the submucosa. These macrophages were CD68+, fusiform with faint cytoplasm and morphologically different from the large rounded lamina propria macrophages, which do not express C3 mRNA. Multinucleated giant cells did not express either C3 or C4 genes. In addition to its presence in intestinal epithelium, C4 mRNA was also expressed in mast cells, which however did not express C3 mRNA. These observations identify cells in the intestinal wall expressing complement genes and support the hypothesis that there is local regulated production of complement in the intestine of patients with CD, and subsequent complement activation may contribute to the inflammatory process.