Jill Moss

Mesenchymal precursor cells in the blood of normal individuals

Mesenchymal precursor cells found in the blood (BMPCs) of normal persons adhere to plastic and glass and proliferate logarithmically in DMEM-20% fetal calf serum (FCS) without growth factors. They form cells with fibroblast-like and stromal morphology, which is not affected by eliminating CD34, CD3, or CD14 cells. Osteogenic supplements (dexamethasone, ascorbic acid, and β-glycerophosphate) added to the culture inhibited fibroblast formation, and BMPCs assumed the cuboidal shape of osteoblasts. After 5 days in supplemented medium, the elutriated cells displayed alkaline phosphatase (AP), and the addition of bone morphogenetic protein (BMP)2 (1 ng) doubled AP production (P < 0.04). Two weeks later, 30% of the cells were very large and reacted with anti-osteocalcin antibody. The same cultures also contained sudanophlic adipocytes and multinucleated giant cells that stained for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptors. Cultured BMPCs immunostain with antibodies to vimentin, type I collagen, and BMP receptors, heterodimeric structures expressed on mesenchymal lineage cells. In addition, BMPCs stain with anti-CD105 (endoglin), a putative marker for bone-marrow mesenchymal stem cells (MSCs).

Complement Activation Contributes to Both Glomerular and Tubulointerstitial Damage in Adriamycin Nephropathy in Mice

Adriamycin nephropathy is a model of focal segmental glomerulosclerosis, characterized by proteinuria and progressive glomerulosclerosis and tubulointerstitial damage. In this study, we examined the role of complement in the etiology of adriamycin nephropathy in mice. We used mice deficient in C1q, factor D, C3, and CD59, and compared them with strain-matched controls. C3 deposition occurred in the glomeruli of wild-type mice as early as 48 h following a single i.v. injection of adriamycin. C3-deficient mice developed significantly less proteinuria and less podocyte injury at day 3 postadriamycin than controls, suggesting that complement is important in mediating the early podocyte injury. At later time points, C3-deficient mice were protected from glomerulosclerosis, tubulointerstitial injury, and renal dysfunction. Factor D-deficient mice were also protected from renal disease, confirming the importance of alternative pathway activation in this model. In contrast, C1q-deficient mice developed similar disease to controls, indicating that the complement cascade was not activated via the classical pathway. CD59-deficient mice, which lack adequate control of C5b-9 formation, developed significantly worse histological and functional markers of renal disease than controls. Interestingly, although more C9 deposited in glomeruli of CD59-deficient mice than controls, in neither group was tubulointerstitial C9 staining apparent. We have demonstrated for the first time that alternative pathway activation of complement plays an important role in mediating the initial glomerular damage in this in vivo model of focal segmental glomerulosclerosis. Lack of CD59, which regulates the membrane attack complex, led to greater glomerular and tubulointerstitial injury.

Muscle fibre characteristics and lactate responses to exercise in chronic fatigue syndrome

OBJECTIVES To examine the proportions of type 1 and type 2 muscle fibres and the degree of muscle fibre atrophy and hypertrophy in patients with chronic fatigue syndrome in relation to lactate responses to exercise, and to determine to what extent any abnormalities found might be due to inactivity. METHODS Quadriceps needle muscle biopsies were obtained from 105 patients with chronic fatigue syndrome and the proportions of type 1 and 2 fibres and fibre atrophy and hypertrophy factors were determined from histochemical preparations, using a semiautomated image analysis system. Forty one randomly selected biopsies were also examined by electron microscopy. Lactate responses to exercise were measured in the subanaerobic threshold exercise test (SATET). RESULTS Inactivity would be expected to result in a shift to type 2 fibre predominance and fibre atrophy, but type 1 predominance (23%) was more common than type 2 predominance (3%), and fibre atrophy was found in only 10.4% of cases. Patients with increased lactate responses to exercise did have significantly fewer type 1 muscle fibres (p<0.043 males, p<0.0003 females), but there was no evidence that this group was less active than the patients with normal lactate responses. No significant ultrastructural abnormalities were found. CONCLUSION Muscle histometry in patients with chronic fatigue syndrome generally did not show the changes expected as a result of inactivity. However, patients with abnormal lactate responses to exercise had a significantly lower proportion of mitochondria rich type 1 muscle fibres.

Transplantation of human fetal mesenchymal stem cells improves glomerulopathy in a collagen type I alpha 2-deficient mouse.

Fetal mesenchymal stem cell (fetal MSC) therapy has potential to treat genetic diseases with early onset, including those affecting the kidney and urinary tract. A collagen type Iα2‐deficient mouse has a deletion in the α2 chain of the procollagen type I gene, resulting in the synthesis of abnormal α1(I)3 homotrimers, which replace normal α1(I)2α2(I)1 heterotrimers and a glomerulopathy. We first confirmed that col1α2‐deficient homozygous mice show abnormal collagen deposition in the glomeruli, which increases in frequency and severity with postnatal age. Intrauterine transplantation of human MSCs from first trimester fetal blood led postnatally to a reduction of abnormal homotrimeric collagen type I deposition in the glomeruli of 4–12 week‐old col1α2‐deficient mice. Using bioluminescence imaging, in situ hybridization and immunohistochemistry in transplanted col1α2‐deficient mice, we showed that the damaged kidneys preferentially recruited donor cells in glomeruli, around mesangial cells. Real‐time RT–PCR demonstrated that this effect was seen at an engraftment level of 1% of total cells in the kidney, albeit higher in glomeruli. We conclude that intrauterine transplantation of human fetal MSCs improves renal glomerulopathy in a collagen type I‐deficient mouse model. These data support the feasibility of prenatal treatment for hereditary renal diseases. Copyright

The evolution of crescentic nephritis and alveolar haemorrhage following induction of autoimmunity to glomerular basement membrane in an experimental model of Goodpasture's disease

Goodpastures, or anti‐glomerular basement membrane (GBM), disease presents with rapidly progressive glomerulonephritis and lung haemorrhage, and is caused by autoimmunity to the NC1 domain of the α3 chain of type IV collagen (α3(IV)NC1). This study examines the development of crescentic nephritis and alveolar haemorrhage in a model of Goodpastures disease, experimental autoimmune glomerulonephritis (EAG), induced in WKY rats by immunization with rat GBM in adjuvant. An increase in circulating anti‐GBM antibodies and albuminuria was observed by week 2, which increased further by weeks 3 and 4, while a decrease in creatinine clearance was observed by week 2, which decreased further by weeks 3 and 4. The kidneys of animals with EAG showed linear deposits of IgG on the GBM and a transient glomerular infiltration by CD4+ T cells at week 2. By week 3 there were large deposits of fibrin in Bowmans space, and glomerular infiltration by CD8+ T cells and macrophages, accompanied by focal necrotizing glomerulonephritis with crescent formation. Ultrastructural studies showed glomerular endothelial cell swelling and epithelial cell foot process effacement at week 2. As the lesion progressed, capillary loops became occluded and the mesangium became expanded by mononuclear cells. By week 3 there was detachment of the endothelium from the GBM, and accumulation of fibrin beneath the disrupted endothelial cells and in Bowmans space. Occasional breaks were observed in the continuity of the basement membrane, and cytoplasmic projections from infiltrating mononuclear cells could be seen crossing the capillary wall between the lumen and the crescent. The lungs of animals with EAG showed patchy binding of IgG to the alveolar basement membrane (ABM) at week 2, and infiltration of the interstitium by CD8+ T cells and macrophages by weeks 3 and 4, accompanied by both interstitial and alveolar haemorrhage. Ultrastructural studies showed focal mononuclear cell infiltrates in alveolar walls at week 2. Occasional breaks were observed in the basement membrane and adjacent endothelium by weeks 3 and 4, together with accumulation of surfactant and erythrocytes within the alveolar spaces. This study defines for the first time the relationship between the immunological and pathological events during the evolution of EAG, and provides the basis for further work on the pathogenesis of Goodpastures disease. Copyright

Primary glial tumor of the retina with features of myxopapillary ependymoma.

We report a primary retinal tumor with features of myxopapillary ependymoma. The lesion occurred in a 33-year-old man with a long history of phthisis bulbi and a more recent history of pain to the right eye. Enucleated ocular globe revealed a lesion occupying most of the retinal surface. Histologically, the retina was replaced by a tumor composed of spindle cells with fibrillary cytoplasm and round to ovoid nuclei forming fascicles, perivascular pseudorosettes, microcysts, and deposition of extracellular mucins. Calcifications, metaplastic bone, and lymphoplasmacytic inflammatory infiltrate were also seen. Tumor cells expressed GFAP and S-100 and to lesser extent carbonic anhydrase II. The immunoreaction for EMA showed diffuse granular positivity, decorated a few extracellular lumina, and highlighted intracytoplasmic lumina in a few cells. Ultrastructurally, there was accumulation of extracellular material between cells and around capillaries, long interdigitating cytoplasmic processes, extracellular lumina packed with microvilli, a few junctions evident around lumina, and some ciliary basal bodies and ciliary basal rootlets. As control cases, we also investigated expression of EMA and carbonic anhydrase II in an ocular globe with retinal gliosis and three cases of myxopapillary ependymoma of the cauda equina. The lesion described here represents the first example of retinal tumor with features of myxopapillary ependymoma. Pathologic features and particularly expression of carbonic anhydrase II suggest a derivation from intrinsic glial cells of retina otherwise known as Muller cells.

Phenotypic variability in siblings with type III spinal muscular atrophy

Autosomal recessive spinal muscular atrophy (SMA) shows substantial phenotypic variability, presenting at a variety of ages from infancy to adult life. Diagnostic difficulties may arise because SMA sometimes produces a dystrophic or myopathic phenotype rather than classical neurogenic abnormalities. Two brothers are described who illustrate this principle and highlight the increasing importance of molecular genetics in investigating patients with neuromuscular diseases. The findings are discussed in the light of recent observations in a mouse model of SMA.

Increased incidence of anti-GBM disease in Fcgamma receptor 2b deficient mice, but not mice with conditional deletion of Fcgr2b on either B cells or myeloid cells alone.

Fcgamma receptor 2b (Fcgr2b) is the only inhibitory Fcgamma receptor in both humans and mice, and is implicated in both antibody production and effector responses to antibody complexes. Reduced function of Fcgr2b has previously been associated with anti-glomerular basement membrane antibody (anti-GBM) disease in mice. However, the mice used had 129 genetic elements flanking the deleted Fcgr2b gene, which are known to increase susceptibility to autoimmunity. In order to confirm a role for Fcgr2b in protection from anti-GBM disease, wild type (WT) mice, mice lacking Fcgr2b on a pure C57BL/6 background, or mice lacking Fcgr2b on a C57BL/6 background with 129 flanking sequences, were immunized with the recombinant NC1 domain of alpha 3 Type IV collagen. Twenty two weeks after immunization, there was a higher incidence of crescentic glomerulonephritis, macrophage infiltration and renal dysfunction in both groups of Fcgr2b-/- mice, indicating an important role of Fcgr2b in regulating the development of anti-GBM disease, on both genetic backgrounds. In order to determine the cellular origin of the Fcgr2b-associated effect, disease was induced in mice with deficiency of Fcgr2b on either B cells alone (CD19Cre), or a subset of myeloid cells (LysozymeMCre). Neither B cell nor myeloid specific knockout mice developed crescentic glomeruonephritis with higher incidence than WT mice indicating that Fcgr2b deficiency on either B cells or a subset of myeloid cells alone is not sufficient to increase susceptibility to anti-GBM disease, but that a combination of cell types, or deficiency of Fcgr2b in a different cell type, is also required.

An ultrastructural study of the colocalization of biglycan and decorin with AA amyloid fibrils in human renal glomeruli.

An investigation was undertaken on paraformaldehyde-fixed, Lowicryl resin-embedded renal biopsies from patients with AA amyloidosis to study the association of two small chondroitin sulphate/dermatan sulphate proteoglycans, decorin and biglycan, with amyloid fibrils using an ultrastructural immunogold technique. Biglycan was present in glomerular endothelial cells in both normal kidney and in amyloidosis, but little biglycan or decorin was present in the normal mesangial matrix. By contrast, conspicuous amounts of both biglycan and decorin were seen to be associated with amyloid fibrils in the glomerular matrix in cases of renal AA amyloidosis. The results further emphasise the close association between amyloid and extracellular matrix components which are now considered to be an integral part of the amyloid fibrils.

Myopathy in acquired partial lipodystrophy

We describe two women with acquired partial lipodystrophy, one with significant myopathic symptoms and signs. Muscle biopsy of deltoid and quadriceps was performed in each case. The light microscopy findings were of type 1 and type 2 fibre hypertrophy, with an increase in intracytoplasmic fat in both cases. Electron microscopy showed normal fibres, with accumulations of electron-lucent fat droplets between the myofibrils. The cause of the lipodystrophies is uncertain, but myopathy may be a feature, and muscle biopsy studies may help in further defining the syndrome.