Ectopic Tissue Formation Research Articles

Leukocytes and the kidney contribute to interstitial inflammation in lupus nephritis

Interstitial leucocyte infiltration, a prominent feature of lupus nephritis, predicts deterioration of renal function. We used two models of lupus nephritis in mice, one with chronic spontaneous disease and the other with acute interferon-alpha (IFN alpha)-mediated disease. The latter is characterized by the virtual absence of interstitial infiltration. In vivo migration assays showed that splenic leukocytes from spontaneously nephritic mice tended to migrate into non-inflamed syngeneic kidneys. This was enhanced if the recipient kidneys were already inflamed. Kidneys from both chronically and acutely nephritic mice showed similar ability to recruit splenic leukocytes from chronically diseased mice. Leukocytes from acutely diseased mice, however, failed to migrate into chronically inflamed kidney. Compared with those with chronic nephritis, the kidneys of acute nephritic mice expressed less of the inflammatory chemokine CXCL13/BLC. Moreover, leukocytes from acute nephritic mice displayed impaired migration, in vitro, to T-cell chemokine attractants. This study links leukocyte infiltration to both kidney chemokine expression, and leukocyte chemotaxis to kidney-expressed chemokines.

Bone tissue formation in sheep muscles induced by a biphasic calcium phosphate ceramic and fibrin glue composite

Some biomaterials are able to induce ectopic bone formation in muscles of large animals. The osteoinductive potential of macro- micro-porous biphasic calcium phosphate (MBCP) ceramic granules with fibrin glue was evaluated by intramuscular implantation for 6 months in six adult female sheep. The MBCP granules were 1-2 mm in size and were composed of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) in a 60/40 ratio. The fibrin glue was composed of fibrinogen, thrombin and other biological factors. After 6 months of implantation in the dorsal muscles of sheep, the explants were rigid. Histology, back-scattered electron microscopy and micro-computed tomography of the implants indicated that approximately 12% of mineralized bone had formed in between the MBCP granules. The ectopic bone appeared well-mineralized with mature osteocytes and Haversian structures. In addition, the number and thickness of bone trabeculae formed in between the MBCP particles were similar to those measured in trabecular bone in sheep. The overall results therefore confirmed the formation of well-mineralized ectopic bone tissue after intramuscular implantation of MBCP/fibrin glue composites. These bone substitutes exhibiting osteoinductive properties could be used for the reconstruction of large bone defects.

Pulmonary expression of CXC chemokine ligand 13, CC chemokine ligand 19, and CC chemokine ligand 21 is essential for local immunity to influenza

CXC chemokine ligand 13 (CXCL13), CC chemokine ligand 21 (CCL21), and CCL19 are constitutively expressed in secondary lymphoid organs, where they control the placement of lymphocytes and dendritic cells. However, these chemokines are also inducibly expressed in the lung after influenza infection. Here we show that, in the absence of spleen and lymph nodes, the expression of homeostatic chemokines in the lung is essential for local B and T cell responses to influenza and for the development and organization of inducible bronchus-associated lymphoid tissue (iBALT). Surprisingly, despite the association between local CXCL13 expression and the formation of ectopic lymphoid tissues, the loss of CXCL13 in the lung had minimal impact on either the development or function of iBALT. In contrast, the loss of CCL19 and CCL21 impaired iBALT formation as well as B and T cell responses. These results demonstrate that the local expression of homeostatic chemokines in nonlymphoid organs, such as the lung, plays an important role in protective immune responses.

New concepts on progressive multiple sclerosis

Multiple sclerosis is generally regarded as a putative autoimmune disease of the central nervous system in which a chronic T-cell-mediated inflammation leads to focal plaques of demyelination in the white matter of the central nervous system. This plaque-centered view of the disease, however, fails to explain clinical deterioration of the patients when they have reached the progressive stage of the disease. It was thus postulated during the past few years that besides inflammation there is a neurodegenerative component of the disease that leads to progressive and global brain damage. This article reviews recent findings that suggest a different explanation. It describes that in the early stage of acute and relapsing multiple sclerosis, focal plaques in the white matter are formed by relapsing waves of inflammation. With chronicity, however, the inflammatory response becomes trapped behind the blood-brain barrier, giving rise to slowly progressive inflammatory damage that affects the brain and spinal cord in a global sense. This is mainly reflected by extensive cortical demyelination and diffuse axonal injury within the normal-appearing white matter. This process seems to be driven by the aberrant formation of ectopic lymphatic tissue within the brain compartment.

Phase II Trial of Prochymal™ (Ex-Vivo Cultured Adult Human Mesenchymal Stem Cells) and Corticosteroids as Primary Treatment for Acute Graft-Vs-Host Disease (aGVHD).

AGVHD is a major cause of morbidity and mortality after allogeneic stem cell transplantation (SCT). Primary treatment of aGVHD with corticosteroids achieves a complete response (CR) rate of 20–40%. Mesenchymal stem cells (MSC) derived from adult bone marrow have immunomodulatory effects. Preclinical and clinical data suggest that MSC inhibit T-cell alloreactivity. Thus, we initiated a trial to investigate the use of Prochymal, ex-vivo cultured adult human MSC derived from unrelated donors, added to conventional steroid therapy for the primary treatment of aGVHD. Patients (pts) and Methods: Pts must have had newly clinically diagnosed aGVHD, grades II-IV using standard grading criteria, after undergoing a related or unrelated allogeneic SCT, or donor lymphocyte infusion (DLI). Endpoints of the study included safety of administration of Prochymal and response rates of aGVHD by day 28 after Prochymal infusion. Treatment with steroids (2mg/kg) and Prochymal, randomized to 2 (low) or 8 million (high) cells/kg, was initiated at the time of GVHD diagnosis. Pts were kept at therapeutic levels of tacrolimus, cyclosporine, or MMF for GVHD prophylaxis. 2 doses of Prochymal were given 3–5 days apart, with the first given within 72 hrs of steroid initiation. Corticosteroids were maintained at 2 mg/kg for at least 1 week. Pts were monitored for response and toxicity weekly for 4 weeks, and then followed for safety on a 2 year long-term follow-up study. Results: 32 pts were enrolled with interim data available for 30 pts (21 males, 9 females) with median age 52 (range 34–67). AGVHD developed following matched sibling (n=15), matched unrelated (n=11), or DLI (n=4) infusions. Overall aGVHD was noted to be grade II (n=20), grade III (n=7), and grade IV (n=3), and involved GI (n=13), skin (n=11), GI and skin (n=4), and GI and liver (n=2). Prochymal dose was low in 17 pts and high in 13 pts. All pts received both Prochymal infusions. 26 of 29 evaluable pts (90%) initially responded to treatment of their aGVHD: 19 achieved CR with no evidence for GVHD, and 7 achieved PR as documented by a reduction in 1 organ stage. Nine pts (31%) eventually required a second line agent to control aGVHD. No infusional toxicities were noted. One pt developed atrial fibrillation 1 day following the second Prochymal infusion. No unexpected ectopic tissue formation was noted in any pt by CT scans at day 28. Currently, 8 pts have died from progression of aGVHD (n=2), intracranial bleed following fall (n=1), relapse (n=1), and infection (n=4) at a median of 44 days (range 13–58) following Prochymal infusion. Conclusions: The addition of Prochymal to corticosteroids resulted in a high response rate with minimal added toxicity when used for the primary treatment of aGVHD, and suggests that MSC have unique biological properties that may be effective for treatment of GVHD. Longer follow-up is necessary to determine if Prochymal has any impact on survival. Phase III studies are warranted, including studies in steroid refractory GVHD.

Odd-skipped genes encode repressors that control kidney development

Odd-skipped family of proteins (Odd in Drosophila and Osr in vertebrates) are evolutionarily conserved zinc finger transcription factors. Two Osr genes are present in mammalian genomes, and it was recently reported that Osr1, but not Osr2, is required for murine kidney development. Here, we show that in Xenopus and zebrafish both Osr1 and Osr2 are necessary and sufficient for the development of the pronephros. Osr genes are expressed in early prospective pronephric territories, and morphants for either of the two genes show severely impaired kidney development. Conversely, overexpression of Osr genes promotes formation of ectopic kidney tissue. Molecularly, Osr proteins function as transcriptional repressors during kidney formation. We also show that Drosophila Odd induces kidney tissue in Xenopus. This might be accomplished through recruitment of Groucho-like co-repressors. Odd genes may also be required for proper development of the Malpighian tubules, the Drosophila renal organs. Our results highlight the evolutionary conserved involvement of Odd-skipped transcription factors in the development of kidneys.

Effector memory T cells as driving force of granuloma formation and autoimmunity in Wegener's granulomatosis

Effector memory T cells as driving force of granuloma formation and autoimmunity in Wegener's granulomatosis

Type I Interferon Production by Tertiary Lymphoid Tissue Developing in Response to 2,6,10,14-Tetramethyl-Pentadecane (Pristane)

Lymphoid neogenesis is associated with antibody-mediated autoimmune diseases such as Sjogren's syndrome and rheumatoid arthritis. Although systemic lupus erythematosus is the prototypical B-cell-mediated autoimmune disease, the role of lymphoid neogenesis in its pathogenesis is unknown. Intraperitoneal injection of 2,6,10,14-tetramethyl-pentadecane (TMPD, pristane) or mineral oil causes lipogranuloma formation in mice, but only TMPD-treated mice develop lupus. We report that lipogranulomas are a form of lymphoid neogenesis. Immunoperoxidase staining of lipogranulomas revealed B cells, CD4(+) T cells, and dendritic cells and in some cases organization into T- and B-cell zones. Lipogranulomas also expressed the lymphoid chemokines CCL21, CCL19, CXCL13, CXCL12, and CCL22. Expression of the type I interferon (IFN-I)-inducible genes Mx1, IRF7, IP-10, and ISG-15 was greatly increased in TMPD- versus mineral oil-induced lipogranulomas. Dendritic cells from TMPD lipogranulomas underwent activation/maturation with high CD86 and interleukin-12 expression. Magnetic bead depletion of dendritic cells markedly diminished IFN-inducible gene (Mx1) expression. We conclude that TMPD-induced lupus is associated with the formation of ectopic lymphoid tissue containing activated dendritic cells producing IFN-I and interleukin-12. In view of the increased IFN-I production in systemic lupus erythematosus, these studies suggest that IFN-I from ectopic lymphoid tissue could play a role in the pathogenesis of experimental lupus in mice.

Co-localization of Chondromodulin-I (ChM-I) and Bone Morphogenetic Protein-6 (BMP-6) in Myoepithelial Cells of Canine Mammary Tumors

To compare the roles of chondromodulin-I (ChM-I) and bone morphogenetic protein-6 (BMP-6) in ectopic mesenchymal tissue formation in canine mammary gland tumors, 33 tumors and 2 normal mammary glands were examined. Immunohistochemical analysis revealed co-expression of ChM-I and BMP-6 in canine mammary tumors. In mixed tumors, newly formed woven bone with ossified cartilage matrix was observed in 4/9 cases. The osteoblasts lining the woven bone showed moderate immunoreactivity to ChM-I and BMP-6. Almost all chondrocytes and proliferative myoepithelial cells within the basement membrane showed intense immunoreactivity to both, and the myoepithelial cells adjacent to the mature cartilage showed the most intense immunoreactivity. The immunoreactivity to ChM-I and BMP-6 of the interstitial myoepithelial cells in the myxomatous stroma varied in each focus of mixed tumors. Similar findings were found in complex adenomas. In simple adenomas, hyperplasic myoepithelial cells within the basement membrane showed moderate immunoreactivity to both markers. Western blot analysis detected a 25 kDa band of ChM-I in fresh tissue samples from three mixed tumors. Our results support the hypothesis that proliferating myoepithelial cells with ChM-I and BMP-6 expression play important roles in mesenchymal metaplasia in canine mammary tumors.

Search for the precursor of ectopic follicular dendritic cells

Chronic inflammation is often accompanied by the formation of ectopic lymphoid tissue, including infiltrations of T lymphocytes and B lymphocytes, and formation of follicular structures around a network of follicular dendritic cells (FDCs). Ectopic follicular structures may serve as survival niches for undesirable B-cell clones, and FDCs provide powerful survival signals to B lymphocytes [1]. There is broad consensus that FDCs show phenotypic overlap with fibroblasts and are of mesenchymal origin, but the formation of FDCs is poorly understood and a FDC-precursor cell has not been identified [2]. It has been shown in mice that the expression of members of the tumor necrosis factor receptor family, especially of the receptor for lymphotoxin-beta (LTβR), on FDC precursors is a condition for FDC maturation.

Regulation of eye formation by the Rx and pax6 homeobox genes.

The Rx and pax6 homeobox genes are among the earliest genes expressed in the eye primordia and play crucial roles in the specification of ocular fate. These genes exhibit strong conservation of sequence and expression patterns among vertebrates. As transcription factors, Rx and Pax6 exert their effects through the activation and repression of downstream targets. Overexpression of each protein induces ectopic retinal tissue formation, as well as induction of the other. pax6 gene mutations have been correlated with an array of human diseases, and a similar array of mutations potentially exists for the human Rx gene. Based on functional studies, the vertebrate Rx and pax6 genes are likely to regulate cell proliferation and are required for the initial commitment to retinal and lens cell fate, respectively, while pax6 appears to play additional roles in the formation of the retina and cornea.

Cell differentiation in vascular calcification.

Ectopic tissue formation is commonly found in calcified atherosclerotic plaques. This suggests that cell differentiation plays an important role in vascular calcification, even though the origin of the cells involved is unclear. Calcifying vascular cells (CVCs), derived from bovine aortic media, have been used as an in vitro model for vascular calcification. CVCs have many characteristics in common with bone cells, but there are also differences suggesting mechanisms that may be applicable to the problem of osteoporosis in the setting of vascular calcification. Matrix GLA protein (MGP) deficient mice develop severe vascular calcification and die prematurely from heart failure and/or aortic rupture. The molecular mechanism of MGP is unknown. It has been hypothesized that MGP acts as a calcification inhibitor by binding calcium, preventing mineral deposition in extracellular fluids near the saturation point for calcium and phosphate. Alternatively, MGP expression may be an attempt to regulate cell differentiation in the vascular wall, possibly by acting as an inhibitor to a factor able to induce cartilage and bone such as bone morphogenetic proteins (BMPs).

Anterior patterning by synergistic activity of the early gastrula organizer and the anterior germ layer tissues of the mouse embryo.

Fragments of the germ layer tissues isolated from the early-primitive-streak (early-streak) stage mouse embryos were tested for axis induction activity by transplantation to late-gastrula (late-streak to early-bud) stage host embryos. The posterior epiblast fragment that contains the early gastrula organizer was able to recruit the host tissues to form an ectopic axis. However, the most anterior neural gene that was expressed in the ectopic axis was Krox20 that marks parts of the hindbrain, but markers of the mid- and forebrain (Otx2 and En1) were not expressed. Anterior visceral endoderm or the anterior epiblast alone did not induce any ectopic neural tissue. However, when these two anterior germ layer tissues were transplanted together, they can induce the formation of ectopic host-derived neural tissues but these tissues rarely expressed anterior neural genes and did not show any organization of an ectopic axis. Therefore, although the anterior endoderm and epiblast together may display some inductive activity, they do not act like a classical organizer. Induction of the anterior neural genes in the ectopic axis was achieved only when a combination of the posterior epiblast fragment, anterior visceral endoderm and the anterior epiblast was transplanted to the host embryo. The formation of anterior neural structures therefore requires the synergistic interaction of the early gastrula organizer and anterior germ layer tissues.

Misexpression of the pancreatic homeodomain protein IDX-1 by the Hoxa-4 promoter associated with agenesis of the cecum

Background & Aims: The endoderm-specific homeodomain transcription factor IDX-1 is critical for pancreas development and for the regulation of islet cell–specific genes. During development, IDX-1 is expressed in the epithelial cells of the endoderm in the pancreatic anlage of the foregut. The aim of this study was to determine whether IDX-1 may have potential properties of a master homeotic determinant of pancreas and/or gut development. Methods: Transgenic mice were generated in which the expression of IDX-1 was misdirected by a promoter of the mesoderm-specific homeodomain protein Hoxa-4 known to express in the stomach and hindgut during development. The expectation was the formation of ectopic pancreatic tissue or alterations of gut patterning or morphology. Results: Although no ectopic induction of pancreatic markers was found in these transgenic mice, they manifested an altered midgut-hindgut union and agenesis of the cecum. Further, IDX-1 binds to the gut-specific homeodomain protein Cdx-2 and inhibits transactivation of the sucrase-isomaltase promoter by Cdx-2. Conclusions: These findings further support the emerging understanding that interactions among different classes of homeodomain proteins, expressed in a spatially and temporally restricted manner during development, determine the pattern of organogenesis. A possible mechanism for the dysmorphogenesis of the proximal colon may be an inhibition of Cdx-2 actions by IDX-1. GASTROENTEROLOGY 1998;115:381-387

Expression of Collagen Genes during Ectopic Hard Tissue Formation Induced by BMP and Type Ⅰ Atelocollagen in Mouce Subcutaneous Tissue

Expression of Collagen Genes during Ectopic Hard Tissue Formation Induced by BMP and Type Ⅰ Atelocollagen in Mouce Subcutaneous Tissue

Modulation of commitment, proliferation, and differentiation of chondrogenic cells in defined culture medium.

The factors regulating the growth and development of mesenchymal precursor cells toward chondrogenesis are not well identified. We have developed a defined serum-free culture system that allows the proliferation of chick embryo chondrogenic cells and their maturation toward hypertrophic chondrocytes. Proliferation is obtained in adhesion in medium supplemented with insulin (Ins), Dexamethasone (Dex), and either basic fibroblast growth factor (FGF-2), platelet-derived growth factor bb, epithelial growth factor, or GH; the highest mitogenic response is induced by FGF-2 in synergy with Ins. Ins can be substituted by Ins-like growth factor I. When these cells are transferred into suspension culture in Ins/Dex and T3 without growth factor supplement, they undergo the complete chondrogenic development characterized by type X collagen synthesis and cellular hypertrophy. During differentiation, Ins cannot be substituted by Ins-like growth factor I. Chondrogenesis is also evidenced by the formation of hypertrophic cartilage when the medium is supplemented with ascorbic acid. If T3 is introduced in the proliferation phase, the cells fail to differentiate to hypertrophy in suspension unless bone morphogenetic protein-2 is added. Assays of ectopic tissue formation in nude mice, with cells implanted sc after adsorption on collagen sponge or porous hydroxyapatite ceramics, indicate that cells grown in Ins/FGF-2 reform mainly cartilage in vivo, whereas expansion in Ins/T3/Dex/FGF-2 leads to the formation of cartilage, bone, and adipose tissue.

XASH genes promote neurogenesis in Xenopus embryos.

Neural development in Drosophila is promoted by a family of basic helix-loop-helix (bHLH) transcription factors encoded within the Achaete Scute-Complex (AS-C). XASH-3, a Xenopus homolog of the Drosophila AS-C genes, is expressed during neural induction within a portion of the dorsal ectoderm that gives rise to the neural plate and tube. Here, we show that XASH-3, when expressed with the promiscuous binding partner XE12, specifically activates the expression of neural genes in naive ectoderm, suggesting that XASH-3 promotes neural development. Moreover, XASH-3/XE12 RNA injections into embryos lead to hypertrophy of the neural tube. Interestingly, XASH-3 misexpression does not lead to the formation of ectopic neural tissue in ventral regions, suggesting that the domain of XASH proneural function is restricted in the embryo. In contrast to the neural inducer noggin, which permanently activates the NCAM gene, the activation of neural genes by XASH-3/XE12 is not stable in naive ectoderm, yet XASH-3/XE12 powerfully and stably activates NCAM, Neurofilament and type III beta-tubulin gene expression in noggin-treated ectoderm. These results show that the XASH-3 promotes neural development, and suggest that its activity depends on additional factors which are induced in ectoderm by factors such as noggin.

Porous Acrylic Cement: Evaluation of a Novel Implant Material

The preparation and some properties of in situ curing porous acrylic cement, a modification of the traditional bone cement, are described. Through numerous animal experiments, the behavior of the porous cement in a biological environment has been studied. The histological evaluation reveals that hard and soft tissues readily grow into the pores of the cement, thus anchoring the implant to the host tissues. Some inflammatory foci, decreasing in number with residence time, together with varying numbers of multinucleated giant cells were found occasionally in the tissues in and around implants. When implanted in soft tissues, the porous cement behaved favorably with respect to solid acrylic implants because of the immobility of the implants and the absence of ectopic hard tissue formation. The biocompatibility of porous acrylic cement is judged to justify clinical trials in plastic and reconstructive surgery.