Granulocyte turnover in the feline intestine

The objective of this study was to determine the turnover rate of the extravascular pool of granulocytes in different regions of the feline gastrointestinal tract. Leukocyte emigration from the vasculature was prevented over a 48-h period by repeated intravenous injections of a monoclonal antibody (MAb IB₄) directed against the leukocyte adhesion glycoprotein complex CD11/CD18. Tissue-associated myeloperoxidase (MPO) activity was used to monitor the total tissue granulocyte pool at 0.5, 12, 24, and 48 h after MAb IB₄ administration. The mucosal layer of the duodenum, jejunum, ileum, and colon exhibited different kinetics of granulocyte clearance, with average life-spans (t1/2) ranging between 6.9 (colon) and 10.4 h (duodenum). Granulocyte clearance rates of 0.5 × 10⁶ and 2.4 x 10⁶ cells/h/g tissue were estimated (from measured values oft1/2 and tissue granulocyte pool) for the small bowel and colonie mucosae, respectively. The submucosal layer of the intestine exhibited a biphasic reduction in tissue MPO activity following immunoneutralization of CD11/CD18, with an initialt1/2 0.5 h followed by at1/2 of 36–60 h. The initial rapid decline in tissue MPO suggests that a significant fraction of granulocytes in the submucosa is localized in a readily exchangeable pool (e.g., marginated cells within the vasculature). The results of this study indicate that the average life-span of resident granulocytes varies significantly between different regions of the gastrointestinal tract, with the intestinal mucosa exhibiting at1/2 comparable to that previously reported for circulating feline neutrophils (R 8 h).     

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.     

Functional consequences of ROMK mutants linked to antenatal Bartter's syndrome and implications for treatment

The antenatal variant of Bartter's syndrome is an autosomal recessive kidney disease characterized by polyhydramnios, premature delivery, hypokalemic alkalosis and hypercalciuria. It is genetically heterogeneous, having been linked recently to mutations in an ATP- sensitive, renal outer medullary K+channel, ROMK, and earlier to mutations in the Na-K-2Cl co-transporter, NKCC2. We characterized four of the mutations reported in three heterozygous ROMK variants of antenatal Bartter's and found that each expressed a distinct phenotype in Sf9 cells. One mutation expressed normal function and appears to be an allelic polymorphism. The other three mutations produced channels with significantly reduced K+fluxes. However, the mechanisms in each case were different and reflected abnormalities in phosphorylation, proteolytic processing or protein trafficking. The different mechanisms may be important in the design of appropriate therapy for patients with this disease.      

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.