Outer membrane protein A renders dendritic cells and macrophages responsive to CCL21 and triggers dendritic cell migration to secondary lymphoid organs

Outer membrane protein A (OmpA) is a class of bacterial cell wall protein that is immunogenic without adjuvant. As specific immune responses are initiated in the lymph nodes (LN, we analyzed the effect of the OmpA from Klebsiella pneumoniae (KpOmpA) onchemokine/ chemokine receptor expression by APC and on cell migration to the LN. Upon contact with KpOmpA, human immature DC and macrophages acquire CCR7 expression and responsiveness to CCL21. In parallel, CCR1 and CCR5 expression is down-regulated and CXCL8, CCL2, CCL3 and CCL5 production is up-regulated. Mice injected subcutaneously with KpOmpA present a transient inflammatory reaction at the site of injection accompanied by an enlargement of the draining LN with a higher proportion of DC and macrophages. Lastly, when exposed to KpOmpA prior injection, DC but not macrophages migrate to the draining LN. In conclusion, KpOmpA confers a migratory phenotype to DC and triggers their migration to the regional LN. This property contributes to explain how innate cells initiate adaptive immune response upon recognition of conserved bacterial components and also why OmpA is immunogenic in the absence of adjuvant.     

Altered regulation of mitogen responsiveness by suppressor cells in multiple sclerosis.

Suppression of the lymphocyte response to concanavalin A (Con A), phytohaemagglutinin (PHA) and protein A from Staphylococcus aureus (SpA) by Con A-induced suppressor cells was measured in twenty-four patients with recently active-recovering multiple sclerosis (MS), twelve with inactive MS and twenty-three healthy controls. Patients with recently active disease displayed significantly greater suppression of the response to Con A. Suppression of the responses to PHA and SpA did not differ among the groups. Lymphocyte stimulation in cultures not showing suppression was similar in all three types of subjects. These results suggest a disturbance of lymphocyte regulation in patients with recently active-recovering MS and illustrate the potential usefulness of measuring the suppression of responsiveness to several mitogens.     

Laboratory strategies for efficient handling of paraffin-embedded tissues for molecular detection of clonality in non-hodgkin lymphomas.

We herein present a technical strategy to optimize DNA isolation from paraffin-embedded tissue (PET). This includes the choice of adequate buffers for proteinase K digestion and multiplex PCR amplifications for assessing the appropriateness of DNA extracts for subsequent PCR assays for detecting clonality. We found that the association of proteinase K digestion in nonionic buffer and subsequent extract dilutions accounted for 79% of successful amplifications. A final efficiency of 88% was achieved by additional organic extractions and/or re-extractions. Comparisons were carried out with control DNA extracts from fresh samples to assess the efficiency of each clonality assay. Immunoglobulin CDRIII rearranged region amplification was more efficient for pregerminal center B-cell lymphomas in contrast to CDRII rearrangement detection, which was more effective for germinal and postgerminal lymphomas. T-cell clonality detection by TCRgamma PCR was less efficient in PET samples than in fresh tissues showing that DNA integrity is more critical for TCR than for IGH amplification. Two inconclusive cases without phenotypic markers and two other atypical lymphoproliferations masked by reactive T cells were diagnosed as plasmablastic lymphomas and as monoclonal B-proliferations, respectively, due to IGH rearrangements.     

Gene therapy of cancer based on interleukin 12

Tumor formation and growth depends mainly on the inability of the organism to elicit a potent immune response, and on the formation of new blood vessels that enable tumor nutrition. Interleukin-12 (IL-12) therapy can target both processes. And IL-12-based gene therapy may restrict IL-12 production to the relevant site in order to obtain enhanced antitumor activity and reduced toxicity. In the clinical setting, IL-12 gene transfer can be used either to improve the pharmacokinetic/pharmacodynamic profile of the cytokine, to transduce dendritic cells or to enhance the efficiency of antitumor vaccination. It can also synergize with other procedures involving the simultaneous transfer of other transgenes or non-gene based strategies. The strong anti-tumoral power shown in many different animal models has not been found in early clinical trials in which cancer patients were treated by peritumoral injections of autologous fibroblasts producing IL-12, intratumoral injections of an adenoviral vector encoding human IL-12 genes, or intratumoral injection of autologous dendritic cells transduced ex vivo with this same adenoviral vector. However, these trials have set the proof-of-concept that local production of IL-12 inside a tumor can stimulate tumor infiltration by effector immune cells and that in some cases it is followed by tumor regression. From the many questions that arise after these disappointing results the most relevant concerns the duration and intensity of transgene expression and the capability to monitor this topics in vivo. New vectors that might achieve regulated, long-term production of this cytokine might have better results and merit clinical testing.     

Squash Procedure for Protein Immunolocalization in Meiotic Cells

Several techniques have been developed for protein immunolocalization in meiotic cells. However, most of them include treatments that lead to cell disruption and are only suitable for prophase-I cells. We describe a novel squash procedure of cell preparation for protein immunolabelling of different meiotic stages. This procedure is an alternative to both cryosectioning and whole spreading procedures. We present results obtained in mouse spermatocytes with three different antibodies: the MPM-2 mAb against mitotic phosphoepitopes, an anticentromere serum and a polyclonal serum against the SCP3 protein of the axial elements and lateral elements of the synaptonemal complex. The procedure was tested for single and double immunolabelling. With this technique a large number of cells at different meiotic stages can be analysed. Cell stages are easily identified and cell and chromosome structures are preserved. Thus, it allows the study of chromosome behaviour and the relations hips between the different structural elements of the cell throughout meiotic divisions. Our procedure is also suitable for three-dimensional (3D) analyses and proved to be reliable in a wide range of systems including insects and mammals. In addition, the procedure may be interesting to obtain a rapid immunological diagnosis.     

Targeted retroviral gene transfer into the rat biliary tract

The ability to induce proliferation by temporary duct ligation suggested an hypothesis that retrovirus-mediated gene transfer into cells of the biliary tract could be accomplished. The time course of histologic changes, incorporation of³H-thymidine and immunofluorescent staining with a monoclonal antibody to cytokeratin-19 (a marker for differentiated bile ducts) was studied in male Fischer F344 rats. A recombinant Gibbon ape leukemia virus (GALV), containing a gene encodingEscherichia coli β-galactosidase was next introduced into 24 hr obstructed bile ducts. Gene transfer was maximal when virus was exposed to the obstructed duct for 12 hr (∼0.1%). The majority of X-gal positive cells were in cytokeratin-19 negative peribiliary tissues, which had the appearance of newly forming bile ducts. The data suggest that cells targeted by retroviral infection of the obstructed rat bile duct may be a precursor of mature, fully differentiated biliary epithelium.     

Novel KChIP2 isoforms increase functional diversity of transient outward potassium currents

Kv4.3 channels conduct transient outward K⁺ currents in the human heart and brain where they mediate the early phase of action potential repolarization. KChIP2 proteins are members of a new class of calcium sensors that modulate the surface expression and biophysical properties of Kv4 K⁺ channels. Here we describe three novel isoforms of KChIP2 with an alternatively spliced C-terminus (KChIP2e, KChIP2f) or N-terminus (KChIP2g). KChIP2e and KChIP2f are expressed in the human atrium, whereas KChIP2g is predominantly expressed in the brain. The KChIP2 isoforms were coexpressed with Kv4.3 channels in Xenopus oocytes and currents recorded with two-microelectrode voltage-clamp techniques. KChIP2e caused a reduction in current amplitude, an acceleration of inactivation and a slowing of the recovery from inactivation of Kv4.3 currents. KChIP2f increased the current amplitude and slowed the rate of inactivation, but did not alter the recovery from inactivation or the voltage of half-maximal inactivation of Kv4.3 channels. KChIP2g increased current amplitudes, slowed the rate of inactivation and shifted the voltage of half-maximal inactivation to more negative potentials. The biophysical changes induced by these alternatively spliced KChIP2 proteins differ markedly from previously described KChIP2 proteins and would be expected to increase the diversity of native transient outward K⁺ currents.     

Color modulation of Doppler spectra with the use of a personal computer.

This paper describes a method for the use of a personal computer in developing color modulated spectral maps of blood flow echoes from a Doppler flowmeter. The acoustical signal from a Doppler flowmeter is digitized and transformed into the frequency domain with a fast Fourier technique with the use of an Apple IIe microcomputer in conjunction with a Motorola 68000 co-processor. The resultant transform is displayed as a pseudo three-dimensional, color modulated spectral map by way of a color monitor, or a television projector onto the meeting room screen. We have found this technique to be useful in appreciating differences in blood flow from different hydraulic conditions, as well as in helping the medical students appreciate these differences in pulsatile flow in various arteries.     

Psychiatric morbidity in three primary medical care clinics in the city of Sao Paulo

Summary The aim of this study was to assess the prevalence of psychiatric disturbance in three primary medical care settings (Brasilandia, Servidor and Barra Funda) in a large city of a developing country and the effect of relevant sociodemographic factors (sex, age, marital status, occupation, migration, colour of skin and housing) on minor psychiatric morbidity.The prevalence rates of psychiatric morbidity were estimated at two levels: for minor and for severe psychiatric disturbance. The extent of minor psychiatric morbidity was found to be very high in the three primary medical care clinics (Brasilandia 56%, Servidor 50% and Barra Funda 47%) and about one-quarter of persons attending each clinic presented a severe psychiatric disturbance.Two factors were found to be associated with minor psychiatric morbidity (sex and family income per capita) when the joint effects of the sociodemographic variables were investigated. Women were more likely than men to present a minor psychiatric disturbance and the lower the family income the higher the risk of minor psychiatric morbidity. These findings are discussed in the light of the mental health of the urban poor living in the large cities of developing countries.