Epidermal growth factor and trefoil factor family 2 synergistically trigger chemotaxis on BEAS-2B cells via different signaling cascades

Injured areas of the respiratory epithelium are subject to rapid repair by the migration of adjacent epithelial cells, a process termed "restitution". Rapid re-epithelialization is promoted by interactions between migrating cells and the extracellular matrix proteins. Furthermore, epidermal growth factor (EGF) as well as trefoil factor family (TFF) peptides are well known regulators of epithelial restitution due to their motogenic effects. Migration of the human bronchial epithelial cell line BEAS-2B in modified Boyden chambers was used as a model system for airway restitution. EGF or recombinant human TFF2 or TFF3 showed mainly chemotactic activity. The motogenic response was strictly dependent upon a haptotactic substrate, but to different degrees. EGF induced phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, c-Jun-N-terminal kinase, p38, Akt, and p70S6K in BEAS-2B cells. Using specific inhibitors, the signaling cascades responsible for the motogenic response were shown to differ drastically when EGF was compared with TFF2. The motogenic effect of TFF2 was previously demonstrated to depend on ERK1/2 and protein kinase C activation; whereas the EGF-triggered motogenic response was completely independent of ERK1/2 activation but sensitive to the inhibition of phosphoinositide 3-kinase, p38, protein kinase C, or nuclear factor kappaB. However, the motogenic effects of EGF and TFF2 are additive. These data suggest that luminal EGF and TFF peptides can act synergistically in the human respiratory epithelium to enhance rapid repair processes in the course of diseases such as asthma.     

Mice with neonatally induced inactivation of the vascular cell adhesion molecule-1 fail to control the parasite in Toxoplasma encephalitis

Under various inflammatory conditions, cell adhesion molecules are up-regulated in the central nervous system (CNS) and may contribute to the recruitment of leukocytes to the brain. In the present study, the functional role of vascular cell adhesion molecule (VCAM)-1 in Toxoplasma encephalitis (TE) was addressed using VCAM(flox/flox MxCre) mice. Neonatal inactivation of the VCAM-1 gene resulted in a lack of induction of VCAM-1 on cerebral blood vessel endothelial cells, whereas the constitutive expression of VCAM-1 on choroid plexus epithelial cells and the ependyma was unaffected; in these animals, resistance to T. gondii was abolished, and VCAM(flox/flox MxCre) mice died of chronic TE caused by a failure to control parasites in the CNS. Although leukocyte recruitment to the CNS was unimpaired, the B cell response was significantly reduced as evidenced by reduced serum levels of anti-T. gondii-specific IgM and IgG antibodies. Furthermore, the frequency and activation state of intracerebral T. gondii-specific T cells were decreased, and microglial activation was markedly reduced. Taken together, these data demonstrate the crucial requirement of VCAM-1-mediated immune reactions for the control of an intracerebral infectious pathogen, whereas other cell adhesion molecules can efficiently compensate for VCAM-1-mediated homing across cerebral blood vessels.     

Establishment of an HIV cell-cell fusion assay by using two genetically modified HeLa cell lines and reporter gene

Infection of human cells with the human immunodeficiency virus type I (HIV-1) can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 together with the appropriate human chemokine receptors. In this study, a T-tropic HIV cell-cell fusion assay was established that utilized CD4, human CXCR4 and HIV NL4-3 gp160 as fusion components and a T7 polymerase-activated luciferase as a reporter system. The HeLa T4 cells used, expressed CD4 and CXCR4, and the applied HeLa KS386 cells expressed HIV NL4-3 gp160. By combining HeLa T4 cells with HeLa KS386 cells, an approximately about 100- to 300-fold increase in luciferase activity could be elicited relative to the control. The addition of anti-CD4 monoclonal antibody (Mab) (RPA-T4) or anti-CXCR4 Mab (12G5) in the assay significantly inhibited the fusion event; in contrast, an anti-CCR5 Mab (2D7) had no effect, indicating that the fusion assay was CD4 and CXCR4 dependent. In this report, fusion events could be monitored by both the luciferase reporter system and syncytia formation. Fusion events were monitored and compared using these two approaches. The luciferase reporter system was found to be more sensitive than syncytia formation. Moreover, compared with previous HIV fusion models, such as using recombinant vaccinia viruses, this system has several advantages, including simplicity and sensitivity. Finally, the system provides a powerful tool to study fusion mechanisms mediated by T-tropic HIV gp160, as well as to screen for fusion-blocking antibodies and antiviral agents.     

Proposed quality control guidelines for antimicrobial susceptibility tests using tilmicosin.

Quality control guidelines for tilmicosin, a novel veterinary-use-only macrolide, were developed in a multi-laboratory study according to established National Committee for Clinical Laboratory Standards (NCCLS) procedures (M23-T2). Tilmicosin was incorporated into Sensititre plates for broth microdilution endpoint testing and into two lots of 15-micrograms disks for Kirby-Bauer agar disk diffusion testing. One common lot and five unique lots of Mueller-Hinton media were used. (Broth was cation adjusted, and agar was supplemented with 5% defibrinated sheep blood.) Bacteria used for reference strains included Pasteurella haemolytica 128K, Pasteurella multocida ATCC 43137, and Staphylococcus aureus ATCC 29213 (microdilution) and ATCC 25923 (disk). Replicate tests were conducted. Disk diffusion and broth microdilution quality control ranges are proposed.     

Astroglia—Neuron interactions that promote long-term neuronal survival

Besides intrinsic determinants of cell growth, epigenetic signals have been proposed to regulate development and maintenance of neurons. Here we provide evidence that cerebral astrocytes contribute significantly to the set of environmental influences that are required for long-term survival of neurons derived from the mammalian central nervous system. Cerebral astrocytes in serum-free culture express diffusible and non-diffusible neuron-supporting signals, including cell-adhesive neurite growth-promoting glycoproteins, diffusible neurotrophic factors as well as membrane-bound molecules that mediate cell contact interactions. The combination and synergistic interaction of these environmental signals markedly enhance the survival of brain neurons. While astroglia-derived cell-adhesive substrates that include a high molecular weight complex consisting of laminin β-chains and proteoglycan (Matthiessen et al., 1989) stimulate neurite outgrowth, they fail to enhance long-term neuronal survival when additional neurotrophic and cell-contact interactions are lacking. Astrocytes release a diffusible neurotrophic activity that, when permanently applied, maintains long-term survival of central neurons in culture. The soluble neurotrophic activity seems to interact synergistically with cell-bound signals which are also required for long-term survival and which are expressed by astrocytes and neurons, but not by fibroblasts. Among neurons from different brain areas, such as hippocampus, cerebral cortex and septum, regional differences in their responsiveness to the astroglial neurotrophic activity have been observed.     

20q13.2 Amplification in intraductal hyperplasia adjacent to in situ and invasive ductal carcinoma of the breast

The 20q13 region harboring recently described putative oncogenes is frequently amplified in invasive ductal carcinoma (IDC). The aim of this study was to examine the 20q13 copy number in intraduct hyperplasia (IH), atypical duct hyperplasia (ADH), and ductal carcinoma in situ (DCIS) adjacent to IDC. In 5 patients, comparative genomic hybridization (CGH) after laser microdissection revealed 20q13 amplification in four of five cases of IH, in all of three cases of IH with atypia, all five of DCIS, and all five of IDC. Fluorescence in situ hybridization (FISH) confirmed the amplification at 20q13.2 in IH in the two specimens analyzed. The amplification rate, however, was higher in DCIS and IDC. In phenotypically normal ductal epithelium normal values were found for 20q13 copy number by FISH (n=2) and CGH (n=5). Although the number of cases presented here is small, our results suggest that mutations in the 20q13.2 region in IH may be associated with accelerated proliferation and hyperplasia of the ductal epithelium. Progression to DCIS and ICD is accompanied by a further increase in the 20q13.2 copy number. Received: 17 March 1999 / Accepted: 22 June 1999     

Protocol for ultrarapid immunostaining of frozen sections

Rapid immunostaining of frozen sections within a tolerable time span would be very helpful for intraoperative diagnosis. A protocol was therefore established using the enhanced polymer one-step staining (EPOS) system (Dako) with antibodies against leucocyte common antigen (LCA), cytokeratin (CK), and anti-melanoma (MEL). Best results with reliable and specific immunostaining and a labelling intensity comparable to standard immunostaining protocols were achieved with fixation of samples in 100% acetone for 20 seconds (CK, LCA) or two minutes (MEL), followed by incubation of the primary antibody and development of the chromogen reaction with 3,3'diaminobenzidine (DAB) for three and five minutes at 37 degrees C, respectively. The total procedure takes only 12 minutes, thus enabling rapid immunostaining on intraoperative frozen sections. Apart from its use in tumour classification, this method is especially useful in detecting tumour cells in sentinel lymph nodes.     

A staphylococcal radioimmunoassay for detection of antibodies toMycoplasma pneumoniae

A radioimmunoassay (RIA) which depends on the property of protein A ofStaphylococcus aureus to combine with the Fc-fragment of immunoglobulins was developed.This technique was employed to measure antibodies in human and various animal sera. It could be demonstrated that the staphylococcal RIA was at least as sensitive as the previously described radioimmunoprecipitation technique in detecting antibodies toM.pneumoniae in human sera. In addition, antibodies toM.pneumoniae could be demonstrated in sera of hamsters intranasally inoculated with the organisms. Antibodies could also be demonstrated in rabbit sera after immunization withM.pneumoniae. The test proved to be considerably more sensitive than conventional tests for detection of antibodies to the organisms. The test requires only small amounts of reagents and is relatively inexpensive.The results were presented in a preliminary form at the annual meeting of the Local Branch of the American Society for Microbiology, Frankfurt, March 1976 and the 77th ordinary meeting of the Society for General Microbiology, Glasgow, September 1976     

Rush Hymenoptera venom immunotherapy: a safe and practical protocol for high-risk patients

BACKGROUND: Hymenoptera venom immunotherapy in allergic patients is a well-established treatment modality for the prevention of systemic anaphylactic reactions caused by insect stings. A variety of therapy regimens exists, from conventional to rush and ultrarush modalities that operate on continuous or intermittent schedules. OBJECTIVE: The aim of this study was to report the 8-year experience with our rush venom immunotherapy regimen in predominantly high-risk patients and to compare data on safety and convenience with the results of 26 studies published from 1978 to 2001. METHODS: One hundred one patients allergic to bee, yellow jacket, or hornet venom were treated with rush Hymenoptera venom immunotherapy. Diagnosis and selection of patients for venom immunotherapy were carried out according to the recommendations of the European Academy of Allergology and Clinical Immunology. We used a 4-day regimen, and the incidence and nature of systemic reactions (SRs) were documented. Fifty-two patients were treated with honeybee venom, and 49 were treated with yellow jacket venom. RESULTS: One hundred (99%) patients reached the maintenance dose. We observed 8 injection-related SRs (0.47% of all injections given) in 7 (6.9%) patients. The number of SRs was higher in patients treated with bee venom extract (12%) compared with in patients receiving yellow jacket venom extract (2%). There was no significant difference in the risk of SRs between female and male patients. The incidence of SRs was considerably lower than the average of 17.8% reported in the literature. CONCLUSION: With a rush immunotherapy regimen over a time period of 8 years in predominantly high-risk patients, the incidence of SRs was low, despite the high number of patients with bee venom allergy, who are more likely to have side effects. Epinephrine as rescue medication was never necessary, and the regimen proved to be safe and convenient for both the patients and the medical staff.