Antibodies to PAI-1 alter the invasive and migratory properties of human tumour cells in vitro

Recent reports suggest that elevated levels of plasminogen activator inhibitor-1 (PAI-1) may contribute to tumour progression. The studies reported here were designed to help elucidate PAI-1's contribution to the invasive and migratory phenotype. Antibodies to PAI-1 dose-dependently, and significantly, inhibited the invasive and migratory potential of human HT1080 fibrosarcoma cells, as did an antibody to uPA and the plasmin inhibitor aprotinin. Invasion of the human melanoma cell line, BLM, was also attenuated by the anti-PAI-1 monoclonal antibody MAI-12. The non-invasive human melanoma cell line, IF6, which does not express uPA, provided further confirmation of PAI-1 and uPA's role as, upon transfection with uPA, this cell line attained an invasive phenotype, which was again attenuated by MAI- 12. Although antibodies to PAI-1 did not affect the adhesion of HT1080 cells to vitronectin, the antibody to uPA reduced their attachment. Addition of exogenous PAI-1, however, prevented HT1080 cell adhesion (IC₅₀ 180nM) and promoted cell detachment from vitronectin. Furthermore melanoma cells transfected with a uPA variant, which had an impaired interaction with PAI-1, were not invasive and had impaired binding to vitronectin. These data highlight the importance of a balanced proteolysis and suggest an additional role for PAI-1 distinct from its role in proteolysis. These data also suggest that uPA and PAI-1 may co-operate in the migratory process by respectively facilitating the attachment to, and subsequent detachment from, vitronectin in the extracellular matrix. These results support the clinical findings and indicate that modulation of PAI-1 activity may be of therapeutic benefit for the treatment of cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Rapid differentiation of the major causative agents of bacterial meningitis by use of frequency-pulsed electron capture gas-liquid chromatograph: analysis of acids.

The major causative agents of bacterial meningitis, Haemophilus influenzae serogroup B, Neisseria meningitidis serogroups B and C, Klebsiella pneumoniae, Streptococcus pneumoniae, and two types of Escherichia coli, were cultured in a modified chemically defined Catlin medium and in a commercial version of the unmodified Catlin medium. The spent media were extracted under acidic conditions, and electron-capturing derivatives were prepared by derivatization with trichloroethanol or haptafluorobutyric anhydride. The derivatives were analyzed on a gas chromatograph equipped with a frequency-pulsed electron capture detector and a PEP-2 computer. The data obtained from the study show that these organisms can be easily distinguished from each other on the basis of metabolic products detected in either type of medium. Three different metabolic groups were detected within two serogroups of N. meningitidis. The methods are practical, and the new technique should offer clinical laboratories and hospitals a better method for rapid identification of this important group of pathogens.     

Analysis of the effect of DNA purification on detection of human papillomavirus in oral rinse samples by PCR

Human papillomavirus (HPV) has recently been associated with oral cancers. To prepare for a study of the natural history of oral HPV infection, the effect of the DNA purification method on HPV genomic DNA detection in Scope mouthwash oral rinse samples and the reproducibility of HPV detection in rinse samples collected 7 days apart were investigated. The study was conducted with a population at high risk for oral HPV infection: human immunodeficiency virus-infected men with CD4-cell counts <200. Five DNA purification methods were compared among equal aliquots of oral rinse samples collected from a subset of individuals. The purification methods included (i) proteinase K digestion (PKD) and heat inactivation; (ii) PKD and ethanol precipitation (EP); (iii) PKD, phenol-chloroform extraction, and EP; (iv) use of the Puregene DNA purification kit; and (v) use of the QIAamp DNA Blood Midi kit. HPV was detected by PCR amplification with PGMY09 and PGMY11 L1 primer pools and by use of a Roche linear array. Puregene-purified samples had higher human DNA yields and purities, and Puregene purification detected the greatest number of HPV-positive subjects and total HPV infections in comparison to the numbers detected by all other methods. The total number of HPV infections and HPV prevalence estimates were also higher for Puregene-processed oral rinse samples when a fixed volume (10 mul) rather than a fixed cell number ( approximately 50,000 cells) was used for PCR amplification. A good concordance was observed for oral HPV infection status (agreement, 80%; kappa value, = 0.60) and type-specific infection (agreement, 98%; kappa value, 0.57) in matched oral rinse samples. The method of DNA purification significantly affects the detection of HPV genomic DNA from oral rinse samples and may result in exposure misclassification that could contribute to the inconsistent associations reported in the literature.     

How patients stress, con, and intimidate physicians to file dubious disability reports.

This study was undertaken to determine the association between health providers'' stress and performances, and patients who presented with disability forms that the providers did not feel were justified. Two questionnaires were given to health-care providers at the Family Practice Clinic, Martin Luther King/Drew Medical Center, Los Angeles, California. One questionnaire assessed the level of stress they suffered from, and a second questionnaire assessed how these types of patients may affect their general performance. The data suggested that the performance of providers was definitely affected by hostile, demanding, threatening, and malingering patients. The students, residents, nurses, and clinic administrative staff were all affected. Third-year medical students and foreign-trained doctors appeared not to be affected as much, probably because of a lack of knowledge about the "system" or because they preferred not to "rock the boat".     

Performance of a commercial, type-specific enzyme-linked immunosorbent assay for detection of herpes simplex virus type 2-specific antibodies in Ugandans

Two hundred forty-eight human immunodeficiency virus (HIV)-positive and 496 HIV-negative subjects in Uganda were tested by HerpeSelect herpes simplex virus type 2 enzyme-linked immunosorbent assay (ELISA) and Western blotting to optimize the ELISA for use in this population. A higher index cutoff value was required for optimal sensitivity and specificity, and overall performance of the assay was not affected by HIV status.     

Antipneumococcal effects of C-reactive protein and monoclonal antibodies to pneumococcal cell wall and capsular antigens.

Antibodies to pneumococcal capsular polysaccharides are well known for their ability to protect against pneumococcal infection. Recent studies indicate that antibodies to cell wall antigens, including pneumococcal surface protein A and the phosphocholine (PC) determinant of teichoic acids as well as human C-reactive protein (which also binds to PC), can protect mice against pneumococcal infection. In the present study we compared the protective effects of these agents as measured by mouse protection, the blood bactericidal assay, and clearance of pneumococci from the blood and peritoneal cavity. Our findings extend previous results indicating that human C-reactive protein and antibodies to noncapsular antigens are generally less protective than anticapsular antibodies. The new results obtained indicate the following: (i) mouse protection studies with intraperitoneal and intravenous infections provide very similar results; (ii) monoclonal immunoglobulin G2a (IgG2a) antibodies to PC, like IgG1, IgG2b, and IgG3 antibodies to PC, are highly protective against pneumococcal infection in mice; (iii) human antibody to PC is able to protect against pneumococcal infection in mice; (iv) antibodies to PspA are effective at mediating blood and peritoneal clearance of pneumococci; (v) complement is required for the in vivo protective effects of both IgG and IgM antibodies to PC; (vi) IgG1, IgG2b, and IgG3 anti-PC antibodies all mediate complement-dependent lysis of PC-conjugated erythrocytes; and (vii) antibodies and human C-reactive proteins that are reactive with capsular antigens but not cell wall antigens are able to mediate significant antibacterial activity in the blood bactericidal assay.     

Murine neurotropic retrovirus spongiform polioencephalomyelopathy: acceleration of disease by virus inoculum concentration.

A 10-fold reduction in the incubation period of murine neurotropic retrovirus spongiform polioencephalomyelopathy was effected by a 1,000-fold concentration of the cloned virus inoculum.     

Impaired hepatocyte survival and liver regeneration in Atm-deficient mice

Atm is a stress-induced DNA damage checkpoint protein kinase with multiple roles in cell-cycle progression. Recent evidence indicates that Atm also plays a role in stem cell maintenance and self-renewal. It is not known whether Atm has a role during tissue regeneration. Using liver regeneration as a model system, we examined the role of Atm in this process. Here, we show that the expression levels of Atm protein were gradually increased during liver regeneration and this was correlated with the onset of DNA replication. The induction of Stat3 and JNK signaling, which are essential processes in normal regeneration response, was attenuated during the early phases of liver regeneration in Atm-deficient mice. P53 was transiently phosphorylated at serine 23 during liver regeneration in an Atm-dependent manner. In addition, we found that cyclin A induction was delayed and p21 was over-expressed, both of these processes were correlated with reduced and delayed DNA replication in Atm(-/-) mice during liver regeneration. Finally, we show that increased apoptosis was observed in Atm(-/-) mice in response to partial hepatectomy, indicating that Atm is required for the survival of hepatocytes. Collectively, these data indicate that liver regeneration is impaired in Atm-deficient mice. Given that liver is the first line of defense against environmental toxins, the elucidation of the function of Atm and Atm-mediated signaling pathways in liver metabolism and in response to environmental toxins is of fundamental interest.     

Longitudinal study of viruses associated with canine infectious respiratory disease

In this investigation a population of dogs at a rehoming center was monitored over a period of 2 years. Despite regular vaccination of incoming dogs against distemper, canine adenovirus type 2 (CAV-2), and canine parainfluenza virus (CPIV), respiratory disease was endemic. Tissue samples from the respiratory tract as well as paired serum samples were collected for analysis. The development of PCR assays for the detection of CPIV, canine adenovirus types 1 and 2, and canine herpesvirus (CHV) is described. Surprisingly, canine adenovirus was not detected in samples from this population, whereas 19.4% of tracheal and 10.4% of lung samples were positive for CPIV and 12.8% of tracheal and 9.6% of lung samples were positive for CHV. As reported previously, a novel canine respiratory coronavirus (CRCoV) was detected in this population (K. Erles, C. Toomey, H. W. Brooks, and J. Brownlie, Virology 310:216-223, 2003). Infections with CRCoV occurred mostly during the first week of a dog's stay at the kennel, whereas CPIV and CHV were detected at later time points. Furthermore, the evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to CPIV and an immunofluorescence assay for detection of antibodies to CHV is described. This study shows that CPIV is present at kennels despite vaccination. In addition, other agents such as CHV and CRCoV may play a role in the pathogenesis of canine respiratory disease, whereas CAV-2 and canine distemper virus were not present in this population, indicating that their prevalence in the United Kingdom is low due to widespread vaccination of dogs.