Intratracheal administration of liposomal clodronate accelerates alveolar macrophage reconstitution following fetal liver transplantation

To facilitate study of alveolar macrophages in vivo, we developed a method to rapidly and efficiently replace resident alveolar macrophages with macrophages of a different (donor) genotype. Chimeric mice were generated by lethal irradiation followed by fetal liver transplantation (FLT) using green fluorescent protein (GFP) transgenic reporter mice as donors. Kinetics of peripheral blood monocyte (PBM) and alveolar macrophage reconstitution was determined 4 and 10 weeks post-FLT by quantifying the percentage of GFP+ cells. To enhance the recruitment of donor monocytes into the lung after FLT, mice were treated with intratracheal administration of liposomal clodronate to deplete host alveolar macrophages at 6 weeks post-FLT. PBM reconstitution occurred by 4 weeks after FLT (85.7+/-1.6% of CD11b+/Gr-1+ monocytes were GFP+), and minimal alveolar macrophage repopulation was observed (9.5% GFP+). By 10 weeks following FLT, 48% of alveolar macrophages were GFP+ by immunostaining of macrophages on lung tissue sections, and 55.1 +/- 1.6% of lung lavage macrophages were GFP+ by fluorescein-activated cell sorter analysis. Clodronate treatment resulted in a significant increase in GFP+ alveolar macrophages 10 weeks after FLT. By immunostaining, 90% of macrophages were GFP+ on lung tissue sections and 87.5 +/- 1.1% GFP+ in lung lavage (compared with GFP-transgenic controls). The ability of newly recruited alveolar macrophages to clear Pseudomonas aeruginosa and activate nuclear factor-kappaB in response to Eschericia coli lipopolysaccharide demonstrated normal macrophage function. Optimizing this methodology provides an important tool for the study of specific genes and their contribution to alveolar macrophage function in vivo.     

Upregulation of TGF-beta, FOXP3, and CD4+CD25+ regulatory T cells correlates with more rapid parasite growth in human malaria infection

Understanding the regulation of immune responses is central for control of autoimmune and infectious disease. In murine models of autoimmunity and chronic inflammatory disease, potent regulatory T lymphocytes have recently been characterized. Despite an explosion of interest in these cells, their relevance to human disease has been uncertain. In a longitudinal study of malaria sporozoite infection via the natural route, we provide evidence that regulatory T cells have modifying effects on blood-stage infection in vivo in humans. Cells with the characteristics of regulatory T cells are rapidly induced following blood-stage infection and are associated with a burst of TGF-beta production, decreased proinflammatory cytokine production, and decreased antigen-specific immune responses. Both the production of TGF-beta and the presence of CD4+CD25+FOXP3+ regulatory T cells are associated with higher rates of parasite growth in vivo. P. falciparum-mediated induction of regulatory T cells may represent a parasite-specific virulence factor.     

CTLA-4 expression on antigen-specific cells but not IL-10 secretion is required for oral tolerance

CD4(+) T cells play a vital role in mediating the tolerance induced at mucosal sites following exposure to non-pathogenic stimuli, and further understanding of the precise mechanisms by which these cells prevent aberrant responses is required. We have developed a model using transfer of DO11.10 TCR-transgenic bone marrow into irradiated recipients in which it has been possible to track antigen-specific CD4(+) cells in mesenteric lymph nodes (mLN), Peyer's patches (PP) and lamina propria following primary exposure to antigen. Using this model we have demonstrated initial activation in all three gut-associated lymphoid tissue compartments characterized by increases in the frequency of transgenic cells expressing CD69 and CD25. These cells subsequently enter a state of hyporesponsiveness both locally in the mLN and PP and in the periphery following feeding and challenge. Investigating the role of CTLA-4 either using anti-CTLA-4 mAb or by generating chimeras using DO11.10xCTLA-4(-/-) mice as donors we have clearly shown that antigen-specific cells require the expression of this regulatory molecule for oral tolerance. In contrast, oral tolerance was intact in chimeras generated using DO11.10xIL-10(-/-) cells, indicating that secretion of this cytokine by antigen-specific cells is not required.     

Fast network oscillations in the rat dentate gyrus in vitro

The dentate gyrus is a prominent source of gamma frequency activity in the hippocampal formation in vivo. Here we show that transient epochs of gamma frequency network activity (67 +/- 12 Hz) can be generated in the dentate gyrus of rat hippocampal slices, following brief pressure ejections of a high-molarity potassium solution onto the molecular layer. Oscillatory activity remains synchronized over distances >300 microm and is accompanied by a modest rise in [K(+)](o). Gamma frequency oscillations were abolished by a GABA(A) receptor antagonist demonstrating their dependence on rhythmic inhibition. However, in many cases, higher frequency oscillations (>80 Hz) remained in the absence of synaptic transmission, thus demonstrating that nonsynaptic factors may underlie fast oscillatory activity.     

Mechanism of pellet egestion in great-horned owls (Bubo virginianus).

To study the mechanism of oral pellet egestion in great-horned owls, bipolar electrodes and strain-gauge transducers were chronically implanted in the esophagus, muscular stomach, and duodenum of six owls. Recordings from conscious owls plus simultaneous radiographic observations revealed characteristic gastrointestinal motility patterns associated with egestion. Beginning at about 12 min before egestion, gastric contractions formed the final shape of the pellet and pushed it into the lower esophagus. The pellet was moved out of the esophagus by antiperistalsis during the last 8--10 s before egestion. During pellet egestion, contractions of abdominal muscles were not detected. Pellet egestion appears to be unlike either emesis in mammals with a simple stomach or regurgitation in ruminants.     

Hand involvement in Schmid metaphyseal chondrodysplasia

Schmid metaphyseal chondrodysplasia (Schmid MCD, MIM 156500) is caused by mutations in the COL10A1 gene and is clinically characterized by short stature, bowed legs, and a waddling gait. Radiographic findings include anterior cupping, sclerosis and splaying of the ribs, diffuse metaphyseal flaring, and irregularity that is most pronounced at the knees, coxa vara, and femoral bowing. We reviewed the radiographs of Schmid MCD patients at the International Skeletal Dysplasia Registry in Los Angeles for evidence of hand involvement. We found hand involvement in 47% (7/15) of cases included in our analysis. These changes were subtle and consisted of shortening of the tubular bones and metaphyseal cupping of the proximal phalanges and metacarpals. Mild hand involvement is a common feature of Schmid MCD.     

The Schizosaccharomyces pomberfc3 + gene encodes a homologue of the human hRFC36 and Saccharomyces cerevisiae Rfc3 subunits of replication factor C

In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe replication factor C (RF-C) plays key roles both in chromosomal DNA replication and in DNA replication checkpoint function. At the replication fork, the five-subunit RF-C complex functions to load the trimeric polymerase accessory factor PCNA onto DNA. PCNA then acts as a sliding clamp, tethering Pol δ to the DNA to maximise its processivity. Here we describe the cloning of the S. pomberfc3 ⁺ gene, encoding a homologue of the S. cerevisiae Rfc3 and human hRFC36 proteins. The 1026 bp rfc3 ⁺ ORF is interrupted by five introns, ranging in size from 49 to 165 bp. The spliced ORF is predicted to encode a 342 amino-acid protein that is approximately 50% identical at the amino acid sequence level to the S. cerevisiae Rfc3 and human hRFC36 proteins. As expected, S. pomberfc3 ⁺ is an essential gene, with rfc3Δ cells being defective for DNA replication. Loss of rfc3 ⁺ function can be rescued by heterologous expression of either the S. cerevisiae Rfc3 or human hRFC36 proteins in S. pombe. Received: 15 October 1999     

Signalling during hypoxia in human T lymphocytes – critical role of the src protein tyrosine kinase p56Lck in the O2 sensitivity of Kv1.3 channels

T lymphocytes encounter hypoxia when they migrate to pathological sites such as tumours and wounds. The inability of T cells to provide an efficient defence at these sites can in part be explained by the hypoxic environment. Kv1.3 channels, important components of the T cell activation process are inhibited by hypoxia and their inhibition accounts for a hypoxia-induced decrease in T cell proliferation. Although Kv1.3 channels play a key role in T cell O₂ sensing, the signalling mechanisms mediating their response to hypoxia are still not understood. In this study, we show that the src-protein tyrosine kinase p56Lck (Lck) is required for Kv1.3 channel response to hypoxia. Pre-exposure to the src inhibitor PP2 abolished the hypoxia-induced inhibition of Kv1.3 channels in primary human T lymphocytes. Moreover, Kv1.3 channel sensitivity to hypoxia was lost in Lck-deficient Jurkat T cells. Further studies with recombinant Kv1.3 channels showed that Kv1.3 channels lack intrinsic O₂ sensitivity, but delivery of Lck into the cells and transfection of a constitutively active Lck (Y505FLck) restored their sensitivity to hypoxia. Although Lck is necessary for the Kv1.3 channel response to hypoxia, it does not directly inhibit Kv1.3 channels. Indeed, under normal oxygen tension, delivery of active Lck into L929 cells and overexpression of Y505FLck did not decrease recombinant Kv1.3 currents. On the contrary, activation of endogenous src kinases increased wild-type Kv1.3 currents in T lymphocytes. Our findings indicate that Lck is required for the acute response to hypoxia of human T lymphocytes as it is necessary to confer O₂ sensitivity on Kv1.3 channels.