Mouse lysozyme-M knockout mice reveal how the self-determinant hierarchy shapes the T cell repertoire against this circulating self antigen in wild-type mice.

We have studied T cell tolerance to defined determinants within ML-M using wild-type (WT; ML-M(+/+)) and LysMcre (ML-M(-/-)) C3H (H-2(k)) mice to determine the relative contribution of ML-M-derived epitopes vs those from other self Ags in selection of the ML-M-specific T cell repertoire. ML-M was totally nonimmunogenic in WT mice, but was rendered immunogenic in LysMcre mice. Most of the response to ML-M in LysMcre mice was directed to the immunodominant determinant region 105-119.

Analysis of B-cell life-span and homeostasis.

In the past, the life-span of B cells in rodents has been determined by a variety of methods, leading to conflicting results. Among the various techniques employed, labeling of dividing cells with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) has turned out to be a versatile and reliable procedure. Labeling of the cells can be easily performed in vivo by feeding BrdU in the drinking water for extended periods of time or by an ip injection of BrdU for short-term labeling experiments.

Alternative macrophage activation is essential for survival during schistosomiasis and downmodulates T helper 1 responses and immunopathology.

Macrophage/neutrophil-specific IL-4 receptor alpha-deficient mice (LysM(Cre)IL-4Ralpha(-/flox)) were generated to understand the role of IL-4/IL-13 responsive myeloid cells during Type 2 immune responses. LysM(Cre)IL-4Ralpha(-/flox) mice developed protective immunity against Nippostrongylus brasiliensis accompanied by T(H)2 development and goblet cell hyperplasia. In contrast, LysM(Cre)IL-4Ralpha(-/flox) mice were extremely susceptible to Schistosoma mansoni infection with 100% mortality during acute infection.

Renal dimensions measured by ultrasonography in children: variations as a function of the imaging plane and patient position.

The purpose of this study was to determine the effect of patient positioning on sonographic renal measurements and to test if the patient position alters the three-dimensional shape of the kidneys. The maximum longitudinal renal length and transverse renal width and depth were measured in the supine and prone position in 100 children (200 kidneys). Age ranged from 6 months to 16 years (mean age 5 years).

Functional characterization of two naturally occurring mutations in the human sodium-phosphate cotransporter type IIa.

Unlabelled: Mutations in the gene encoding the human sodium-phosphate cotransporter (NPT2), causing reduced phosphate affinity and dominant-negative behavior, were described. We found no evidence of altered kinetics or dominant-negative effects. Thus, the mutations cannot account for the clinical phenotype.

Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor-deficient mice.

Atherosclerosis is now generally accepted as a chronic inflammatory condition. The transcription factor NF-kappaB is a key regulator of inflammation, immune responses, cell survival, and cell proliferation. To investigate the role of NF-kappaB activation in macrophages during atherogenesis, we used LDL receptor-deficient mice with a macrophage-restricted deletion of IkappaB kinase 2 (IKK2), which is essential for NF-kappaB activation by proinflammatory signals.

SOCS3 negatively regulates IL-6 signaling in vivo.

Members of the suppressor of cytokine signaling (SOCS) family are potentially key physiological negative regulators of interleukin-6 (IL-6) signaling. To examine whether SOCS3 is involved in regulating this signaling, we have used conditional gene targeting to generate mice lacking Socs3 in the liver or in macrophages. We show that Socs3 deficiency results in prolonged activation of signal transducer and activator of transcription 1 (STAT1) and STAT3 after IL-6 stimulation but normal activation of STAT1 after stimulation with interferon-gamma (IFN-gamma).

The sodium phosphate cotransporter family SLC34.

This review summarizes the characteristics of the solute carrier family SLC34 that is represented by the type ll Na/P(i)-cotransporters NaPi-lla (SLC34A1), NaPi-llb (SLC34A2) and NaPi-llc (SLC34A3). Other Na/P(i)-cotransporters are described within the SLC17 and SLC20 families. Type ll Na/P(i)-cotransporters are expressed in several tissues and play a major role in the homeostasis of inorganic phosphate.

Essential cysteine residues of the type IIa Na+/Pi cotransporter.

The rat renal Na(+)/P(i) cotransporter (NaP(i)-IIa) contains 12 native cysteines. When individually replaced by a serine, none appears essential for proper expression and function. Nevertheless, the formation of one essential cysteine bridge (C5/C6), together with a postulated second bridge, is necessary.

HIF-1alpha is essential for myeloid cell-mediated inflammation.

Granulocytes and monocytes/macrophages of the myeloid lineage are the chief cellular agents of innate immunity. Here, we have examined the inflammatory response in mice with conditional knockouts of the hypoxia responsive transcription factor HIF-1alpha, its negative regulator VHL, and a known downstream target, VEGF. We find that activation of HIF-1alpha is essential for myeloid cell infiltration and activation in vivo through a mechanism independent of VEGF.

Compartmentalized production of CCL17 in vivo: strong inducibility in peripheral dendritic cells contrasts selective absence from the spleen.

Dendritic cells (DCs)(*) fulfill an important regulatory function at the interface of the innate and adaptive immune system. The thymus and activation-regulated chemokine (TARC/CCL17) is produced by DCs and facilitates the attraction of activated T cells. Using a fluorescence-based in vivo reporter system, we show that CCL17 expression in mice is found in activated Langerhans cells and mature DCs located in various lymphoid and nonlymphoid organs, and is up-regulated after stimulation with Toll-like receptor ligands.

Anti-interleukin-18 therapy in murine models of inflammatory bowel disease.

Interleukin (IL)-18 is a cytokine with a broad array of effector functions, the most prominent of which is to act synergistically with IL-12 in interferon-gamma production and the induction of a strong T-helper-1-mediated immune response. In addition, IL-18 also upregulates the production of proinflammatory cytokines such as IL-1 and tumor necrosis factor-alpha. Analysis of IL-18-deficient mice revealed an important role of IL-18 in the activation of macrophages and natural killer cells in the context of infection with intracellular bacteria or parasites.

Regulation of Na/Pi transporter in the proximal tubule.

The physiological tuning and pathophysiological alterations of renal proximal reabsorption of inorganic phosphate can be ascribed to the net amount of the Na/Pi-cotransporter NaPi-IIa localized in the brush border membrane. The net amount of NaPi-IIa appears to be the result of an endocytotic rate regulated by a complex network of different protein kinases. New approaches demonstrated that NaPi-IIa is part of heteromeric protein complexes, organized by PDZ (postsynaptic protein PSD95, Drosophila junction protein Disc-large, tight junction protein ZO-1) proteins.

Interleukin-10 is crucial for maintenance but not for developmental induction of peripheral T cell tolerance.

To asses the requirement of interleukin (IL)-10 for peripheral CD4 T cell tolerance, the IL-10 knockout (KO) was introduced into a T cell receptor-transgenic mouse model (TCR1) specific for SV40 T antigen (Tag). IL-10-deficient TCR1-transgenic mice failed to establish antigen-specific T cell tolerance following sequential injections with Tag peptide. Nevertheless, IL-10 was not required for the establishment of CD4 T cell tolerance in double transgenic RT2/TCR1 mice in which Tag is expressed endogenously under control of the insulin promoter.

Genetic dissection of the cellular pathways and signaling mechanisms in modeled tumor necrosis factor-induced Crohn's-like inflammatory bowel disease.

Recent clinical evidence demonstrated the importance of tumor necrosis factor (TNF) in the development of Crohn's disease. A mouse model for this pathology has previously been established by engineering defects in the translational control of TNF mRNA (Tnf(Delta)(ARE) mouse). Here, we show that development of intestinal pathology in this model depends on Th1-like cytokines such as interleukin 12 and interferon gamma and requires the function of CD8(+) T lymphocytes.

Transport function of the renal type IIa Na+/P(i) cotransporter is codetermined by residues in two opposing linker regions.

Two highly similar regions in the predicted first intracellular (ICL-1) and third extracellular loop (ECL-3) of the type IIa Na+/P(i) cotransporter (NaPi-IIa) have been shown previously to contain functionally important sites by applying the substituted cysteine accessibility method (SCAM). Incubation in methanethiosulfonate (MTS) reagents of mutants that contain novel cysteines in both loops led to full inhibition of cotransport activity. To elucidate further the role these regions play in defining the transport mechanism, a double mutant (A203C-S460C) was constructed with novel cysteines in each region.

Forging the link between structure and function of electrogenic cotransporters: the renal type IIa Na+/Pi cotransporter as a case study.

Electrogenic cotransporters are membrane proteins that use the electrochemical gradient across the cell membrane of a cosubstrate ion, for example Na(+) or H(+), to mediate uphill cotransport of a substrate specific to the transport protein. The cotransport process involves recognition of both cosubstrate and substrate and translocation of each species according to a defined stoichiometry. Electrogenicity implies net movement of charges across the membrane in response to the transmembrane voltage and therefore, in addition to isotope flux assays, the cotransport kinetics can be studied in real-time using electrophysiological methods.

Potassium-selective atomic force microscopy on ion-releasing substrates and living cells.

A new method for simultaneous mapping of cell topography and ion fluxes was developed. A highly sensitive ion sensor system was generated by coating atomic force microscopy tips with a PVC layer containing valinomycin, an ionophore for potassium. The activity of specific ions was traced on artificial ion-releasing PVC substrates.

Functional studies on a split type II Na/P(i)-cotransporter.

Analysis of rat and mouse proximal tubular brush-border membrane expression of the type IIa Na/P(i)-cotransporter provides evidence for its cleavage in the large extracellular loop (ECL-2). To study functional properties and membrane distribution of this split NaP(i)-IIa transporter we followed two strategies. In one strategy we expressed the transporter as two complementary parts (p40 and p45) in Xenopus laevis oocytes and as another strategy we cleaved the WT protein with trypsin.

The renal type IIa Na/Pi cotransporter: structure-function relationships.

The type IIa Na/Pi cotransporter mediates proximal tubular brush-border membrane secondary active phosphate (Pi) flux. It is rate limiting in tubular Pi reabsorption and, thus, a final target in many physiological and pathophysiological situations of altered renal Pi handling. In the present short review, we will briefly summarize our current knowledge about the transport mechanism (cycle) as well as particular regions of the transporter protein ("molecular domains") that potentially determine transport characteristics.

Failure of HY-specific thymocytes to escape negative selection by receptor editing.

Editing of autoreactive antigen receptors by secondary V(D)J recombination efficiently rescues B lymphocyte precursors from apoptosis induced by negative selection, but its role has not been rigorously assessed in T cell development. We therefore generated a transgenic mouse model in which self-reactive thymocytes could edit their TCR by secondary recombination at the TCR alpha locus. For this purpose, the V alpha J alpha exon of a male-specific TCR was inserted into the TCR alpha locus followed by Cre-loxP-mediated deletion of the TCR delta locus.

Modulation of renal type IIa Na+/Pi cotransporter kinetics by the arginine modifier phenylglyoxal.

The effects of the arginine-modifying reagent phenylglyoxal on the kinetics of the type IIa Na + /Pi cotransporter expressed in Xenopus, oocytes were studied by means of 32Pi uptake and electrophysiology. Phenylglyoxal incubation induced up to 60% loss of cotransport function but only marginally altered the Na+-leak. Substrate activation and pH dependency remained essentially unaltered, whereas the voltage dependency of Pi-induced change in electrogenic response was significantly reduced.

Identification of functionally important sites in the first intracellular loop of the NaPi-IIa cotransporter.

Intrasequence comparison of the type IIa Na(+)-P(i) cotransport protein revealed two regions with high similarity in the first intracellular (ICL-1) and third extracellular (ECL-3) loops. Because the ECL-3 loop contains functionally important sites that have been identified by cysteine scanning, we applied this method to corresponding sites in the ICL-1 loop. The accessibility of novel cysteines by methanethiosulfonate reagents was assayed electrophysiologically.

High tibial osteotomy in knee instability: the rationale of treatment and early results.

We treated 14 patients having knee instability and varus alignment with tibial osteotomy with or without ligament reconstruction. Five patients with varus angulated anterior cruciate deficiency (double varus) were treated with single-stage closed-wedge tibial osteotomy and anterior cruciate ligament reconstruction. The remaining nine patients had varying amount of posterior cruciate and postero-lateral corner ligament injuries with varus angulation (triple varus); six of these patients had a ligament reconstruction using the Ligament Advanced Reconstruction System ligament with tibial osteotomy (intra-articular--posterior cruciate ligament/extra-articular--postero-lateral corner reconstruction), while the remaining three had a tibial osteotomy without a ligament reconstruction.