RESUMEN
The role of secretory IgM in protecting kidney tissue from immune complex glomerulonephritis induced by 4 mg horse spleen apoferritin and 0.05 mg lipopolysaccharide has been investigated in mutant mice in which B cells do not secrete IgM, but are capable of expressing surface IgM and IgD and secreting other Ig isotypes. Glomerular size, number of glomeruli per cross-section, glomerular cellularity and urine content of protein and creatinine was comparable in treated secreted IgM (sIgM)-deficient and wild-type mice. Assessment of urinary proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed a 30 kDa low molecular weight protein in treated sIgM-deficient animals only, reflecting dysfunction of proximal tubules. A shift of bound C3 from glomeruli to the tubulo-interstitial compartment in sIgM-deficient mice also suggests tubulo-interstitial damage. In contrast, local C3 synthesis within the kidney tissue did not differ between the two treated groups. Apoptosis physiologically present to maintain kidney cell homeostasis was increased slightly in treated wild-type mice. These results indicate that secretory IgM can protect the tubulo-interstitial compartment from immune complex-induced damage without having an effect on the glomerulus.
Asunto(s)
Complemento C3/metabolismo , Glomerulonefritis/inmunología , Enfermedades del Complejo Inmune/inmunología , Inmunoglobulina M/deficiencia , Glomérulos Renales/inmunología , Túbulos Renales Proximales/inmunología , Animales , Apoptosis , Complemento C3/genética , Femenino , Expresión Génica , Glomerulonefritis/patología , Enfermedades del Complejo Inmune/patología , Inmunohistoquímica , Glomérulos Renales/patología , Túbulos Renales Proximales/patología , Lipopolisacáridos , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Modelos Animales , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
OBJECTIVE: To investigate the presence of mesenchymal precursor cells (MPCs) in synovial surface projections of patients with osteoarthritis (OA), to characterize their phenotype and to show their localization. METHODS: Progenitor cells in synovial surface projections were identified by immunohistochemistry, morphometric analysis and confocal laser scanning microscopy using the following phenotypic markers: STRO-1, CD34, and alpha smooth muscle actin (alpha-SMA). RESULTS: In the synovial tissue of all 21 patients with OA MPCs were detected. Immunohistochemistry and subsequent morphometric analysis showed that approximately twice as many STRO-1+ cells/mm2 were observed in synovial tissue of patients with OA as compared to healthy organ donors and that number of STRO-1+ cells/mm2 correlated with total cell number/mm2. Interestingly, in the synovial tissue of patients with OA, twice as many STRO-1+ cells/mm2 were found in synovial surface projections as compared to the sublining area without villi. Using confocal laser scanning microscopy two populations of STRO-1+ MPCs could be detected in synovial surface projections. Single STRO-1+ cells that co-expressed alpha-SMA resemble a population of pericyte precursors required to stabilize the immature vasculature. The second STRO-1+ cell population that was found lacked alpha-SMA but co-expressed CD34 on their surface with low intensity. CONCLUSION: Here we can show that in the synovial tissue of patients with OA twice as many STRO-1+ MPCs can be found in synovial surface projections as compared to the sublining area. These cells are preferentially located at the basis and in the protruding end of the synovial surface projection.
Asunto(s)
Biomarcadores/análisis , Células Madre Mesenquimatosas/patología , Osteoartritis/patología , Membrana Sinovial/patología , Actinas/análisis , Antígenos CD34/análisis , Estudios de Casos y Controles , Humanos , Inmunohistoquímica/métodos , Células Madre Mesenquimatosas/química , Microscopía Confocal , Osteoartritis de la Cadera/patología , Osteoartritis de la Rodilla/patologíaRESUMEN
The effect of granulocyte colony-stimulating factor (G-CSF) induced mobilization of peripheral blood progenitor cells (PBPC) on the endogenous serum levels of cytokines stem cell factor (SCF) and flt3-ligand (flt3-L) was studied in 18 healthy subjects undergoing allogeneic PBPC donation. Donors received a standardized mobilization regime consisting of a 4-day course of G-CSF, with leukapheresis on day 5. Endogenous serum flt3-L and SCF were determined prior to G-CSF administration, on the day of leukapheresis, and followed up until day +100 after cessation of G-CSF administration. The administration of G-CSF resulted in a transient elevation of endogenous flt3-L serum levels. At the day of leukapheresis serum flt3-L showed a median increase of 75% compared to serum flt3-L levels obtained before G-CSF treatment. The increase in serum flt3-L levels showed no correlation with the total number of progenitor cells mobilized. Cessation of G-CSF treatment led to normalization of serum flt3-L within 7 days post G-CSF administration. In contrast, serum CSF levels remained unchanged in response to G-CSF administration. Our results demonstrate a transient surge in serum flt3-L in relation to G-CSF-induced PBPC mobilization, although the assessment of endogenous flt3-L give no information regarding the ability for G-CSF-induced PBPC recruitment in healthy individuals.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Movilización de Célula Madre Hematopoyética/métodos , Donadores Vivos , Proteínas de la Membrana/sangre , Adolescente , Adulto , Femenino , Hematopoyesis , Humanos , Leucaféresis , Masculino , Persona de Mediana Edad , Proteínas Recombinantes , Factor de Células Madre/sangre , Factores de TiempoRESUMEN
Mice with constitutive transgenic (tg) expression of IL-4 develop autoimmune-type disorders resembling human lupus nephritis. The kidneys show progressive glomerulosclerosis with immunoglobulin (Ig) and complement deposition. This study investigated the roles of renal IL-4 expression and glomerular Ig deposition in the pathogenesis of glomerulosclerosis in IL-4 tg mice. Treatment of these mice with IL-4 neutralizing antibody prevented renal disease. IL-4 tg mice treated with methylprednisolone (MP) showed increased mesangial collagen deposition with only trace amounts of glomerular Ig. To analyze the relevance of Ig deposition in the development of the renal lesions, IL-4 tg mice were cross-bred with mu chain-deficient mice (muMT-/-), which are unable to produce Ig. IL-4 tg/muMT-/- mice developed progressive glomerulosclerosis with mesangial accumulation of collagen types I, IV and V despite complete absence of glomerular Ig deposits. Renal IL-4 expression was observed in both anti-IL-4- and MP-treated IL-4 tg mice as well as in IL-4 tg/muMT-/- mice. No statistical difference in the number of glomerular T cells and macrophages between any of the groups was evident. Our data demonstrate that in this model glomerulosclerosis can develop independently of and prior to Ig deposition, and suggest that the initial accumulation of glomerular extracellular matrix is due to renal IL-4 expression. Our results point to a novel mechanism for the development of glomerulosclerosis which may have implications for human disease.
Asunto(s)
Inmunoglobulinas/metabolismo , Interleucina-4/fisiología , Glomérulos Renales/patología , Animales , Colágeno/metabolismo , Femenino , Cadenas mu de Inmunoglobulina/fisiología , Metilprednisolona/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , EsclerosisRESUMEN
Several cellular and extracellular markers that distinguish the phases of the hemangioma life cycle have been described previously. However, details of the phenotypic changes of; the various cellular elements during hemangioma development have not been fully reported, and the extracellular matrix composition, especially in the vicinity of the proliferating endothelial cells, is poorly described. This study examined the expression of cellular and extracellular molecules and cytokines in the proliferative, involuting, and involuted phases of hemangioma. Paraffin-embedded hemangioma specimens, four from each phase, were examined histochemically and immunohistochemically. Throughout the three phases, vascular endothelial cells stained positive for CD31 and von Willebrand factor, although in the involuted phase, not all vessels in the tissue expressed these endothelial markers. Proliferating cell nuclear antigen was expressed by the majority of endothelial cells and pericytes in the proliferative and early involuting phases, but its expression was negligible in the involuted phase. In addition to finding that the total number of mast cells was highest in the involuting phase, the authors observed that the proportion of chymase-positive mast cells decreased with the progression of hemangioma and that virtually all mast cells expressed the biogenic amine phenotype throughout the hemangioma life cycle. The localization of vascular endothelial growth factor predominantly to the pericytes and endothelial cells during the proliferative phase and of basic fibroblast growth factor to the endothelial cells in both the proliferative and early involuting phases is consistent with previous reports, although the latter growth factorwas also observed in mast cells. Type IV collagen and the beta chain of laminin and perlecan were detected in the basement membranes in all phases. Interestingly, collagen types I, III, and V were present in basal membranes throughout the phases and with increasing density in the stromal areas with involution, although type I collagen was less prominent during the proliferative phase. Short-chain collagen type VIII was localized extracellularly throughout the development of hemangioma but, during the early proliferative phase, it was also detected within mast cells. The expression of specific cytokines and cellular and extracellular markers may help distinguish the different clinical phases of the hemangioima life cycle. These results provide further insight into the biology of hemangioma.
Asunto(s)
Biomarcadores de Tumor/análisis , Hemangioma/metabolismo , Hemangioma/patología , Neoplasias de los Tejidos Blandos/metabolismo , Neoplasias de los Tejidos Blandos/patología , Antígenos Nucleares , Recuento de Células , Niño , Preescolar , Colágeno/análisis , Citocinas/análisis , Endotelio Vascular/patología , Femenino , Histocitoquímica , Humanos , Inmunohistoquímica , Lactante , Recién Nacido , Laminina/análisis , Mastocitos/patología , Proteínas Nucleares/análisisRESUMEN
Hemangioma is the most common tumor of infancy. This vascular tumor is characterized by an initial rapid proliferation followed by an inevitable regression. The life cycle of hemangioma is divided into proliferative, involuting, and involuted phases. The cellular and molecular mechanisms responsible for controlling the biological behavior of hemangioma are largely unknown. Differential display analysis using mRNA isolated from biopsy specimens representative of the 3 different phases showed increased expression of clusterin/apoJ (clust/apoJ) in the involuting samples. Clust/apoJ is a multifunctional glycoprotein that has been associated with apoptosis. Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry showed that both the transcription and protein expression of clust/apoJ were increased in hemangioma as the tumor progressed from the proliferative to the involuting and involuted phases. This suggests that clust/apoJ is involved in regulating apoptosis during the spontaneous regression of hemangioma. It has been suggested that mast cells (MC) play a role in the regression of hemangioma. The increase in the number and proportion of clust/ apoJ-positive MC with progression of hemangioma, along with the localization of clust/apoJ to MC granules, supports this hypothesis. We suggest that MC may be synthesizing/releasing this apoptotic modulator, leading to the regression of the tumor. Better understanding of the pathogenesis of hemangioma by identification of the relevant factors involved in its regression such as clust/apoJ will result in the development of novel therapies for this condition and tumors that do not undergo spontaneous regression.
Asunto(s)
Proteínas Inactivadoras de Complemento/genética , Expresión Génica , Glicoproteínas/genética , Hemangioma/genética , Chaperonas Moleculares , Biopsia , División Celular , Niño , Preescolar , Clusterina , Endotelio Vascular/patología , Femenino , Hemangioma/patología , Humanos , Inmunohistoquímica , Lactante , Recién Nacido , Masculino , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The short chain collagen variant, type VIII, is considered to be comprised of two distinct gene products, the alpha1 and alpha2 polypeptide chains. However, recent in vitro translation studies suggest that these chains can form homotrimers. We report here data from biochemical, immunohistochemical and molecular biological experiments, which together provide evidence that alpha1 and alpha2 polypeptides of type VIII collagen exist as homotrimers in cells and tissues. High-performance liquid chromatographic separation of type VIII collagen isolated from Descemet's membrane consistently demonstrated equimolar quantities of the two chains (alpha1:alpha2 1. 03+/-0.02 (S.E.M.); n=41). The availability of highly specific antibodies for the two polypeptides has assisted the in vivo characterisation of type VIII collagen. Immunoprecipitation of trimeric type VIII collagen from Descemet's membrane with purified anti-alpha1(VIII) and anti-alpha2(VIII) yielded fractions that contained only the alpha1(VIII) and alpha2(VIII) chains, respectively. Cultured human mesangial cells synthesised both polypeptides, but the alpha1(VIII) chain was found exclusively in the cell pellet, while the media contained only the alpha2(VIII) chain. The RNA from human mesangial cells and cornea showed message for both chains. However, in peritoneal fibroblast and mesothelial cell RNA, only alpha1(VIII) mRNA was detectable, demonstrating that the transcription of these two genes was not always co-ordinated. Immunohistochemistry showed that both polypeptides were present in cornea, optic nerve, aorta and umbilical cord but did not always co-localise. These results indicate the alpha1(VIII) and alpha2(VIII) chains preferentially form pepsin-resistant, homotrimeric molecules and so can exist as two distinct proteins.
Asunto(s)
Colágeno/química , Animales , Bovinos , Células Cultivadas , Colágeno/genética , Colágeno/inmunología , Colágeno/aislamiento & purificación , Córnea/metabolismo , Lámina Limitante Posterior/química , Humanos , Pruebas de Precipitina , ConejosRESUMEN
Hemangioma, the most common tumor of infancy, is characterized by a proliferation of capillary endothelial cells with multilamination of the basement membrane and accumulation of cellular elements, including mast cells. The initial rapid growth is followed by an inevitable but slow involution. The currently available therapies are empirical and unsatisfactory because what is known of the cellular and molecular basis of hemangioma development is rudimentary. Advances in the understanding of its programmed biologic behavior has been hampered by the lack of a valid human model. We report here a novel in vitro culture system that is a useful human model of hemangioma. A small fragment of hemangioma biopsy is embedded in fibrin gel in a well of culture plates and incubated in a serum-free, buffered-salt, minimal medium. A complex network of microvessels grows out from the tissue fragments. Biopsies taken from all three phases of hemangioma development were cultured successfully; proliferative phase samples developed microvessels in 1 to 4 days, involuting phase in 5 to 7 days, and involuted phase in 7 to 12 days. The relative growth rates of the microvessels in the culture of biopsies taken from different stages of hemangioma development reflect the growth patterns seen clinically. This model has been validated using histochemistry, immunohistochemistry, and reverse transcriptase-polymerase chain reaction. Comparison of the number, localization, and phenotype of endothelial and mast cells and the distribution of basement membrane constituents (type IV collagen, perlecan, and laminins) and growth factors (basic fibroblast growth factor, vascular endothelial growth factor, transforming growth factor-betas) in the biopsy and the tissue after culture shows that many of the characteristics of the original tissues were retained in culture. This in vitro human model of hemangioma overcomes some of the deficiencies associated with earlier models. It offers an opportunity for studying the precise cellular, biochemical, and molecular basis of hemangioma It may also help to elucidate the mechanisms of action of existing therapies and may lead to the identification of novel treatments for hemangioma.
Asunto(s)
Hemangioma/patología , Biomarcadores de Tumor/análisis , Recuento de Células , Niño , Preescolar , Cartilla de ADN/análisis , Endotelio Vascular/química , Endotelio Vascular/patología , Femenino , Hemangioma/química , Hemangioma/genética , Humanos , Técnicas para Inmunoenzimas , Lactante , Recién Nacido , Masculino , Mastocitos/química , Mastocitos/citología , Modelos Biológicos , Neovascularización Patológica/patología , ARN Neoplásico/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
It has been suggested that progeria, a congenital disorder associated with clinical features that resemble premature aging, may be the result of a connective tissue abnormality. Although to date the clinical and pathologic features for 14 autopsied cases of progeria have been reported, details as to the renal changes in progeria are scanty. We investigated the histological features from a male and female with progeria who died aged 11 years and 20 years respectively. In our young male subject there was no glomerulosclerosis, while the kidney from the older subject showed focal renal scarring with focal glomerulosclerosis and associated tubular atrophy. Two small papillary adenomas were present within the renal cortex of the latter subject. In both cases non-sclerotic glomeruli were moderately enlarged with expansion of mesangial matrix. Immunohistochemical detection of collagens showed absence of collagen I and III within the mesangium of non-sclerotic glomeruli, while there was moderate to marked expression of collagen IV, V and VI. Collagen V is thought to be involved in matrix assembly while collagen VI probably has a regulatory role in extracellular matrix development and these are either not seen or are very weakly expressed in normal renal mesangium. The distribution of collagen within the mesangium of progeria kidney is evidence in support of the concept that progeria is a primary connective tissue disorder.
Asunto(s)
Colágeno/metabolismo , Mesangio Glomerular/metabolismo , Riñón/metabolismo , Progeria/metabolismo , Adulto , Niño , Femenino , Mesangio Glomerular/patología , Humanos , Inmunohistoquímica , Riñón/patología , Masculino , Progeria/patologíaRESUMEN
Recent reports have suggested the involvement of interleukin-4 (IL-4) in glomerular pathophysiology. Using immunohistochemistry and reverse transcriptase polymerase chain reaction we investigated the renal lesions in transgenic (tg) mice with widely distributed IL-4 expression including the kidney, and measured the serum levels of the cytokines transforming growth factor-beta (TGF-beta) and IL-4 by ELISA. Transgenic animals exhibited glomerular hypertrophy with progressive mesangial sclerosis leading to renal failure. Renal IL-4 transcript expression, mesangial accumulation of collagen types I, III, IV and V, and immune deposition accompanied by increased expression of TGF-beta1 protein and mRNA were observed. Seven day-old transgenic animals showed early renal fibrotic changes in the absence of immune deposits or TGF-beta1 upregulation. The sera of transgenic mice not only showed elevated levels of circulating IL-4 (tg: 76.6 pg/ml +/- 7.1 vs wildtype (wt): < 3 pg/ml), but significantly decreased TGF-beta1 levels (tg: 18.9 ng/ml +/- 4.1 vs wt: 38.7 ng/ml +/- 2.9; P < 0.005). The disease severity correlated with the serum IL-4/TGF-beta1 ratio rather than with the IL-4 concentration. These data suggest that renal IL-4 production results in matrix accumulation prior to any immunological insult, that increased circulating IL-4/TGF-beta1 ratios are associated with renal immunopathological manifestations and that upregulation of renal TGF-beta1 expression following glomerular Ig deposition accelerates the sclerosis and exacerbates disease development.
Asunto(s)
Glomerulonefritis/metabolismo , Interleucina-4/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Progresión de la Enfermedad , Glomerulonefritis/patología , Glomerulonefritis/fisiopatología , Técnicas para Inmunoenzimas , Fallo Renal Crónico/inmunología , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Renal injury in diabetes mellitus is associated with progressive interstitial fibrosis and extracellular matrix accumulation. However, the phenotypes of cells forming the interstitial infiltrate in diabetic nephropathy have not been precisely defined. There is increasing evidence for the association of mast cells with angiogenesis, chronic inflammatory conditions and fibrosis. We have recently shown that human mast cells can produce the non-fibrillar short chain type VIII collagen in vivo. Using immunohistochemistry, in situ hybridisation and reverse transcriptase-polymerase chain reaction, we examined the contribution of mast cells and type VIII collagen to the fibrotic changes occurring in biopsy-proven diabetic nephropathy. We observed that the number of interstitial mast cells was significantly increased in diabetic nephropathy compared with normal kidney tissue. In specimens from diabetic subjects, intense immunohistochemical staining for type VIII collagen was detected in mast cells, on periglomerular fibres and in perivascular and interstitial sites. The expression of type VIII collagen in periglomerular and interstitial sites coincided with that of alpha smooth muscle actin, a marker for myofibroblastic differentiation mRNA for type VIII collagen was detected by reverse transcriptase-polymerase chain reaction in diabetic nephropathy and in a human mast cell line. By in situ hybridisation the transcripts for type VIII collagen were localised to renal mast cells. The increased number of mast cells and the elevated type VIII collagen deposition in human diabetic nephropathy provides a potential link between the extracellular matrix accumulation and the fibrosis observed in this condition.
Asunto(s)
Colágeno/biosíntesis , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Nefropatías Diabéticas/metabolismo , Riñón/metabolismo , Mastocitos/metabolismo , Adulto , Anciano , Biopsia , Colágeno/análisis , Cartilla de ADN , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/patología , Nefropatías Diabéticas/patología , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Riñón/patología , Masculino , Mastocitos/patología , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Transcripción GenéticaRESUMEN
The role of tumor necrosis factor alpha (TNF-alpha) was examined in biopsy-proven glomerulonephritis by immunohistochemistry, in situ hybridization, immunogold electron microscopy, immunoassay in serum and urine, and urinary immunoblot. Striking glomerular capillary wall and visceral glomerular epithelial cell TNF-alpha protein staining was observed in all cases of membranous nephropathy and membranous lupus nephropathy. Staining was less frequently observed in crescentic glomerulonephritis and in isolated cases of other histological subtypes of glomerulonephritis, usually in association with glomerular macrophages. By immunogold electron microscopy TNF-alpha was localized in membranous nephropathy within the visceral glomerular epithelial cells, and also in the glomerular basement membrane, especially in relation to immune deposits. In situ hybridization localized TNF-alpha mRNA exclusively to glomerular epithelial cells in all biopsies with membranous morphology but not in other histological subtypes. Concentrations of TNF-alpha were significantly increased compared with normal controls in the urine of patients with membranous nephropathy and with crescentic glomerulonephritis. The expression of TNF-alpha by glomerular epithelial cells exclusively and universally in biopsies showing a membranous morphology strongly suggests this cytokine has a role in the pathogenesis of membranous nephropathy.