Use of in vivo-induced antigen technology for identification of Escherichia coli O157:H7 proteins expressed during human infection

Using in vivo-induced antigen technology (IVIAT), a modified immunoscreening technique that circumvents the need for animal models, we directly identified immunogenic Escherichia coli O157:H7 (O157) proteins expressed either specifically during human infection but not during growth under standard laboratory conditions or at significantly higher levels in vivo than in vitro. IVIAT identified 223 O157 proteins expressed during human infection, several of which were unique to this study. These in vivo-induced (ivi) proteins, encoded by ivi genes, mapped to the backbone, O islands (OIs), and pO157. Lack of in vitro expression of O157-specific ivi proteins was confirmed by proteomic analysis of a mid-exponential-phase culture of E. coli O157 grown in LB broth. Because ivi proteins are expressed in response to specific cues during infection and might help pathogens adapt to and counter hostile in vivo environments, those identified in this study are potential targets for drug and vaccine development. Also, such proteins may be exploited as markers of O157 infection in stool specimens.     

The outcome of T-cell costimulation through intercellular adhesion molecule-1 differs from costimulation through leucocyte function-associated antigen-1

Optimal T-cell activation requires both an antigen-specific and a costimulatory signal. The outcome of T-cell activation can be influenced by the nature of the costimulatory signal the T cell receives. We recently demonstrated the ability of stimulation through intercellular adhesion molecule-1 (ICAM-1), resident on the T-cell surface, to provide a second signal for T-cell activation, and have extended that work here to begin an examination of the functional outcome of this set of signals. Costimulation through ICAM-1 resulted in a greater percentage of cells having undergone more than three divisions when compared to costimulation through leucocyte function-associated antigen-1 (LFA-1). Costimulation through ICAM-1 also had an effect similar to costimulation through CD28 in its ability to down-regulate the cyclin dependent kinase inhibitor p27kip1. Costimulation through ICAM-1 provided greater protection from apoptosis than costimulation through LFA-1, especially in cells having divided more than three times. This was supported by the ability of costimulation through ICAM-1 to up-regulate the anti-apoptotic protein Bcl-2. Finally, costimulation through ICAM-1 or CD28 produced a greater number of T cells with a memory phenotype than costimulation through LFA-1.     

In vivo expression of interleukin-8, and regulated on activation, normal, T-cell expressed, and secreted, by human germinal centre B lymphocytes

T-cell homing within germinal centres (GCs) is required for humoral B-cell responses. However, the mechanisms implicated in the recruitment of T cells into the GC are not completely understood. Here we show, by immunohistology, and Northern and Western blots, that in vivo human GC B lymphocytes can express CxC and CC chemokines. Moreover, B-cell subset-specific experiments reveal that interleukin (IL)-8 and regulated on activation, normal, T-cell expressed, and secreted (RANTES) are predominantly expressed by GC centroblast and centrocytes, suggesting that chemokine expression is essential at stages in which B-lymphocytes engage in active antigen-dependent interactions with T lymphocytes. In keeping with this hypothesis, we show that the T cells recruited into the GC correlatively express the receptors for IL-8 and RANTES. We propose that chemokine expression is a key B-cell function that facilitates T-lymphocyte recruitment into the GCs and supports cognate B-cell : T-cell encounters. Moreover, our data are consistent with the impaired homing of T cells to secondary lymphoid organs in mice that are either deficient in CC and CxC chemokines or their receptors.     

Screening microarrays of novel monoclonal antibodies for binding to T-, B- and myeloid leukaemia cells

We have developed a microarray (DotScan) that enables rapid immunophenotyping and classification of leukaemias and lymphomas by measuring the capture of cells by immobilized dots of 82 CD antibodies [Belov, L., de la Vega, O., dos Remedios, C.G., Mulligan, S.P., 2001. Immunophenotyping of leukemia using a cluster of differentiation antibody microarray. Cancer Res. 61, 4483; Belov, L., Huang, P., Barber, N., Mulligan, S.P., Christopherson, R.I., 2003. Identification of repertoires of surface antigens on leukemias using an antibody microarray. Proteomics 3, 2147]. The DotScan technology has been used to investigate the properties of 498 new antibodies submitted to the HLDA8 Workshop. These antibodies have been applied as 10 nl dots to a film of nitrocellulose on a microscope slide to make an HLDA8 microarray. After blocking the remaining nitrocellulose surface, individual arrays were incubated with each of 7 cell types from a human leukaemia cell panel consisting of three cell lines, CCRF-CEM (a T-cell acute lymphocytic leukaemia), MEC-1 (derived from B-cell chronic lymphocytic leukaemia) and HL-60 (a promyelocytic leukaemia), and four leukaemias from patients: a T-cell prolymphocytic leukaemia, a B-cell chronic lymphocytic leukaemia, and two acute myeloid leukaemias. Leukaemia cells were captured by those immobilized antibodies for which they expressed the corresponding surface molecule. Unbound cells were gently washed off, bound cells were fixed to the arrays and dot patterns were recorded using a DotScan array reader and quantified using DotScan data analysis software. The data obtained show the unique expression profiles of the 7 cell types in the leukaemia cell panel obtained with the DotScan microarray, and the differential capture patterns for these 7 cell types screened against the 498 antibodies in the HLDA8 microarray constructed for this study.     

Detection of Vi-negative Salmonella enterica serovar typhi in the peripheral blood of patients with typhoid fever in the Faisalabad region of Pakistan

The synthesis and transportation proteins of the Vi capsular polysaccharide of Salmonella enterica serovar Typhi (serovar Typhi) are encoded by the viaB operon, which resides on a 134-kb pathogenicity island known as SPI-7. In recent years, Vi-negative strains of serovar Typhi have been reported in regions where typhoid fever is endemic. However, because Vi negativity can arise during in vitro passage, the clinical significance of Vi-negative serovar Typhi is not clear. To investigate the loss of Vi expression at the genetic level, 60 stored strains of serovar Typhi from the Faisalabad region of Pakistan were analyzed by PCR for the presence of SPI-7 and two genes essential for Vi production: tviA and tviB. Nine of the sixty strains analyzed (15%) tested negative for both tviA and tviB; only two of these strains lacked SPI-7. In order to investigate whether this phenomenon occurred in vivo, blood samples from patients with the clinical symptoms of typhoid fever were also investigated. Of 48 blood samples tested, 42 tested positive by fliC PCR for serovar Typhi; 4 of these were negative for tviA and tviB. Three of these samples tested positive for SPI-7. These results demonstrate that viaB-negative, SPI-7-positive serovar Typhi is naturally occurring and can be detected by PCR in the peripheral blood of typhoid patients in this region. The method described here can be used to monitor the incidence of Vi-negative serovar Typhi in regions where the Vi vaccine is used.     

Regenerative medicine and developmental biology: the role of the extracellular matrix

The principles and ultimate goals of regenerative medicine and developmental biology involve a complex sequence of events, culminating in the formation of structurally and functionally normal tissues and organs. The molecular composition of the extracellular matrix (ECM) plays a critical role in cellular migration and differentiation events. Mammalian ECM, derived from various tissues and organs, has been used as a biologic scaffold for therapeutic regenerative applications. Hundreds of thousands of human patients have benefited from the use of biologic scaffolds composed of naturally occurring ECM. The mechanisms by which ECM induces constructive remodeling instead of scar tissue formation are only beginning to be understood. This article reviews composition of mammalian ECM, its poorly understood role in developmental biology, and the clinical applications that have resulted from the use of this naturally occurring scaffold.     

Loss of cell polarity causes severe brain dysplasia in Lgl1 knockout mice

Disruption of cell polarity is seen in many cancers; however, it is generally considered a late event in tumor progression. Lethal giant larvae (Lgl) has been implicated in maintenance of cell polarity in Drosophila and cultured mammalian cells. We now show that loss of Lgl1 in mice results in formation of neuroepithelial rosette-like structures, similar to the neuroblastic rosettes in human primitive neuroectodermal tumors. The newborn Lgl1(-/-) pups develop severe hydrocephalus and die neonatally. A large proportion of Lgl1(-/-) neural progenitor cells fail to exit the cell cycle and differentiate, and, instead, continue to proliferate and die by apoptosis. Dividing Lgl1(-/-) cells are unable to asymmetrically localize the Notch inhibitor Numb, and the resulting failure of asymmetric cell divisions may be responsible for the hyperproliferation and the lack of differentiation. These results reveal a critical role for mammalian Lgl1 in regulating of proliferation, differentiation, and tissue organization and demonstrate a potential causative role of disruption of cell polarity in neoplastic transformation of neuroepithelial cells.     

Fine-needle aspiration biopsy of salivary gland mycoses

This report details the fine-needle aspiration biopsy (FNAB) cytomorphologic features of two cases of salivary gland mycosis. Both patients had acquired immunodeficiency syndrome (AIDS) and presented with parotid gland masses. The first patient had Histoplasmosis with secondary infection by Candida. Cytopathologically, the FNAB smears showed classic features of a deep-seated mycosis characterized by necrosis and scattered fungal forms. The second patient had a colonizing sialadenitis caused by either Asperigillus or Fusarium. Cytopathologically, the findings were similar to those seen in aspergillomas of the lung orparanasal sinuses with numerous hyphal forms and an absence of an inflammatory response. Because mycotic disease can induce a wide spectrum of pathogenic change, other benign or malignant, solid or cystic lesions enter into the differential diagnosis. Diagn Cytopathol 1994; 11:286–290. © 1994 Wiley-Liss, Inc.     

Eccentric and concentric torque-velocity characteristics of the quadriceps femoris in man

Summary The primary purpose of this investigation was to study the eccentric and concentric torque-velocity characteristics of the quadriceps femoris in man using a recently developed combined isometric, concentric and eccentric controlled velocity dynamometer (the SPARK System). A secondary purpose was to compare the method error associated with maximal voluntary concentric and eccentric torque output over a range of testing velocities. 21 males (21–32 years) performed on two separate days maximal voluntary isometric, concentric and eccentric contractions of the quadriceps femoris at 4 isokinetic lever arm velocities of 0° · s^–1 (isometric), 30° · s^–1 120° · s^–1 and 270° · s^–1. Eccentric peak torque and angle-specific torques (measured every 10° from 30° to 70°) did not significantly change from 0° · s^–1 to 270° · s^–1 (p>0.05) (with the exception of angle-specific 40° torque, which significantly increased;p<0.05). The mean method error was significantly higher for the eccentric tests (10.6%±1.6%) than for the concentric tests (8.1%±1.7%) (p<0.05). The mean method error decreased slightly with increasing concentric velocity (p>0.05), and increased slightly with increasing eccentric velocity (p>0.05). A tension restricting neural mechanism, if active during maximal eccentric contractions, could possibly account for the large difference seen between the present eccentric torque-velocity results and the classic results obtained from isolated animal muscle.     

Genotypic analysis of Mycobacterium tuberculosis in Bangladesh and prevalence of the Beijing strain

Genotypic analysis was performed on 48 Mycobacterium tuberculosis complex strains collected from a hospital in Dhaka city. Deletion analysis showed that the isolates were all M. tuberculosis; 13 of them were found to be of the "ancestral" type, while 35 were of the "modern" type, indicating that both endemic (ancestral type) and epidemic (modern type) strains cause tuberculosis in Bangladesh. Genotyping based on the spoligotype and variable-number tandem repeats (VNTR) of mycobacterial interspersed repetitive units (MIRU) was also done. A total of 34 strains (71%) were grouped by spoligotyping into nine different clusters; the largest comprised 15 isolates of the Beijing genotype, whereas the remaining eight clusters consisted of two to five isolates. MIRU-VNTR typing detected 32 different patterns among 44 tested strains, and the 15 Beijing strains were further discriminated by MIRU-VNTR typing (7 distinct patterns for the 15 isolates). These results indicate that MIRU-VNTR typing, along with spoligotyping and deletion analysis, can be used effectively for molecular epidemiological studies to determine ongoing transmission clusters; to our knowledge, this is the first report about the type of strains prevailing in Bangladesh.