All solid organs contain resident monocyte-derived cells that appear early in organogenesis and persist throughout life. These cells are critical for normal development in some organs. Here we report the use of a previously described transgenic line, with EGFP driven by the macrophage-restricted Csf1r (c-fms) promoter, to image macrophage production and infiltration accompanying organogenesis in many tissues. Using microarray analysis of FACS-isolated EGFP-positive cells, we show that fetal kidney, lung and brain macrophages show similar gene expression profiles irrespective of their tissue of origin. EGFP-positive cells appeared in the renal interstitium from 12 days post coitum, prior to nephrogenesis, and maintain a close apposition to renal tubules postnatally. CSF-1 added to embryonic kidney explants increased overall renal growth and ureteric bud branching. Expression profiling of tissue macrophages and of CSF-1-treated explants showed evidence of the alternate, pro-proliferative (M2) activation profile, including expression of macrophage mannose receptor (CD206), macrophage scavenger receptor 2 (Msr2), C1q, CD163, selenoprotein P, CCL24 and TREM2. This response has been associated with the trophic role of tumour-associated macrophages. These findings suggest a trophic role of macrophages in embryonic kidney development, which may continue to play a similar role in postnatal repair.

The end point of immune and nonimmune renal injury typically involves glomerular and tubulointerstitial fibrosis. Although numerous studies have focused on the events that lead to renal fibrosis, less is known about the mechanisms that promote cellular repair and tissue remodeling. Described is a model of renal injury and repair after the reversal of unilateral ureteral obstruction (UUO) in male C57bl/6J mice. Male mice (20 to 25 g) underwent 10 d of UUO with or without 1, 2, 4, or 6 wk of reversal of UUO (R-UUO). UUO resulted in cortical tubular cell atrophy and tubular dilation in conjunction with an almost complete ablation of the outer medulla. This was associated with interstitial macrophage infiltration; increased hydroxyproline content; and upregulated type I, III, IV, and V collagen expression. The volume density of kidney occupied by renal tubules that exhibited a brush border was measured as an assessment of the degree of repair after R-UUO. After 6 wk of R-UUO, there was an increase in the area of kidney occupied by repaired tubules (83.7 ؎ 5.9%), compared with 10 d UUO kidneys (32.6 ؎ 7.3%). This coincided with reduced macrophage numbers, decreased hydroxyproline content, and reduced collagen accumulation and interstitial matrix expansion, compared with obstructed kidneys from UUO mice. GFR in the 6-wk R-UUO kidneys was restored to 43 to 88% of the GFR in the contralateral unobstructed kidneys. This study describes the regenerative potential of the kidney after the established interstitial matrix expansion and medullary ablation associated with UUO in the adult mouse. R enal injury and repair comprises a delicate balance between cell loss and proliferation and extracellular matrix (ECM) accumulation and remodeling. Although many studies have focused on the cellular and molecular events that lead to the development of renal fibrosis, less is understood about the process of endogenous renal repair.We have characterized a model of reversal of unilateral ureteral obstruction (R-UUO) in the mouse to assess the regenerative potential of the kidney after interstitial matrix expansion and medullary ablation. UUO initiates a marked increase in numbers of interstitial macrophages that contribute to the cascade of events that lead to the development of fibrosis and renal cell loss. The facilitation and recruitment of macrophages into the renal interstitium is coupled to the upregulated expression of an array of chemokines and adhesion molecules (1,2). After R-UUO in the rat, both cortical (3) and medullary (4) interstitial macrophages are found to decrease gradually in number toward control values by 4 wk after release.A marked decline in GFR and renal plasma flow is observed by 24 h after UUO in dogs, rabbits, and rats (5-7). The duration of UUO and the extent of cell loss play a pivotal role in the recovery of GFR and subsequent renal remodeling and repair. Whereas short-term UUO in rats seems to be completely reversible (8), UUO for Ͼ72 h may lead to renal fibrotic and apoptotic changes culminating in a perm...

We describe an approach for the rapid mapping of epitopes within a malaria antigen using a combination of phage display techniques. Phage display of antigen fragments identifies the location of the epitopes, then random peptide libraries displayed on phage are employed to identify accurately amino acids involved in the epitope. Finally, phage display of mutant fragments confirms the role of each residue in the epitope. This approach was applied to the apical membrane antigen-1 (AMA1), which is a leading candidate for inclusion in a vaccine directed against the asexual blood stages of Plasmodium falciparum. As part of the effort both to understand the function of AMA1 in the parasite life cycle and to define the specificity of protective immune responses, a panel of monoclonal antibodies (MAbs) was generated to obtain binding reagents to the various domains within the molecule. There is a pressing need to determine rapidly the regions recognized by these antibodies and the structural requirements required within AMA1 for high affinity binding of the MAbs. Using phage displaying random AMA1 fragments, it was shown that MAb5G8 recognizes a short linear epitope within the pro-domain of AMA1 whereas the epitope recognized by MAb 1F9 is reduction sensitive and resides within a disulphide-bonded 57 amino acid sub-domain of domain-1. Phage displaying random peptide libraries and mutant AMA1 fragments were employed for fine mapping of the MAb5G8 core epitope to a three-residue sequence in the AMA1 prodomain.

STEM CELLS 2007;25:697-706

The recent introduction of technologies capable of reprogramming human somatic cells into induced pluripotent stem (iPS) cells offers a unique opportunity to study many aspects of neurodegenerative diseases in vitro that could ultimately lead to novel drug development and testing. Here, we report for the first time that human dermal fibroblasts from a patient with relapsing-remitting Multiple Sclerosis (MS) were reprogrammed to pluripotency by retroviral transduction using defined factors (OCT4, SOX2, KLF4, and c-MYC). The MSiPS cell lines resembled human embryonic stem (hES) cell-like colonies in morphology and gene expression and exhibited silencing of the retroviral transgenes after four passages. MSiPS cells formed embryoid bodies that expressed markers of all three germ layers by immunostaining and Reverse Transcriptase (RT)-PCR. The injection of undifferentiated iPS cell colonies into immunodeficient mice formed teratomas, thereby demonstrating pluripotency. The MSiPS cells were successfully differentiated into mature astrocytes, oligodendrocytes and neurons with normal karyotypes. Although MSiPS-derived neurons displayed some differences in their electrophysiological characteristics as compared to the control cell line, they exhibit properties of functional neurons, with robust resting membrane potentials, large fast tetrodotoxin-sensitive action potentials and voltage-gated sodium currents. This study provides for the first time proof of concept that disease cell lines derived from skin cells obtained from an MS patient can be generated and successfully differentiated into mature neural lineages. This represents an important step in a novel approach for the study of MS pathophysiology and potential drug discovery.