Generation and functional evaluation of novel monoclonal antibodies targeting glycosylated human stem cell factor.

Human stem cell factor (hSCF) is an early-acting growth factor that promotes proliferation, differentiation, migration, and survival in several tissues. It plays a crucial role in hematopoiesis, gametogenesis, melanogenesis, intestinal motility, and in development and recovery of nervous and cardiovascular systems. Potential therapeutic applications comprise anemia treatment, mobilization of hematopoietic stem/progenitor cells to peripheral blood, and increasing gene transduction efficiency for gene therapy. Developing new tools to characterize recombinant hSCF in most native-like form as possible is crucial to understand the complexity of its in vivo functions and for improving its biotechnological applications. The soluble domain of hSCF was expressed in HEK293 cells. Highly purified rhSCF showed great molecular mass variability due to the presence of N- and O-linked carbohydrates, and it presented a 2.5-fold increase on proliferative activity compared to bacteria-derived hSCF. Three hybridoma clones producing monoclonal antibodies (mAbs) with high specificity for the glycoprotein were obtained. 1C4 and 2D3 mAbs were able to detect bacteria-derived and glycosylated rhSCF and demonstrated to be excellent candidates to develop a sandwich ELISA assay for rhSCF quantification, with detection limits of 0.18 and 0.07 ng/ml, respectively. Interestingly, 1A10 mAb only recognized glycosylated rhSCF, suggesting that sugar moieties might be involved in epitope recognition. 1A10 mAb showed the highest binding affinity, and it constituted the best candidate for immunodetection of the entire set rhSCF glycoforms in western blot assays, and for intracellular cytokine staining. Our work shows that combining glycosylated rhSCF expression with hybridoma technology is a powerful strategy to obtain specific suitable immunochemical assays and thus improve glycoprotein-producing bioprocesses. KEY POINTS: • Soluble glycosylated human SCF exerted improved proliferative activity on UT-7 cells. • Three mAbs with high specificity targeting glycosylated human SCF were obtained. • mAbs applications comprise sandwich ELISA, western blot, and immunofluorescence assays.

Changes and correlations of anti-Müllerian hormone and stem-cell factors in different ovarian reserve patients during GnRH-antagonist protocol and the effects on controlled ovarian hyperstimulation outcomes.

PURPOSE: To explore the changes and correlations of anti-Müllerian hormone (AMH) and stem-cell factors (SCF) in different ovarian reserve patients during controlled ovarian hyperstimulation (COH) and the effects on COH outcomes. METHODS: Serum at six different timepoints during GnRH-antagonist protocol and follicular fluid (FF) on oocyte retrieval day of 52 patients with polycystic ovary syndrome (PCOS), 61 patients with normal ovarian reserve (NOR) and 42 patients with diminished ovarian reserve (DOR) were collected. AMH and SCF were assessed using enzyme-linked immunosorbent assay. RESULTS: During COH, AMH in the PCOS group was the highest, but SCF did the opposite, and serum AMH gradually decreased, while SCF inversely increased. In the PCOS group, SCF on the first and fourth days of gonadotropin (Gn) administration was negative with Gn dosage (r = - 0.362, P < 0.05; r = - 0.344, P < 0.05). In the NOR group, the basal AMH was also negative with Gn dosage (r = - 0.297, P < 0.05) and positive with COH outcomes (number of retrieved oocytes, MII oocytes, and 2PN fertilization) as well as serum SCF after Gn administration. In the DOR group, both AMH and SCF were significantly associated with COH outcomes. Serum AMH in the DOR group after Gn administration and FF AMH showed a negative correlation with SCF. CONCLUSIONS: Serum AMH decreased, while SCF increased during COH. AMH and SCF are effective for Gn time and dosage adjustment and predicting COH outcomes for NOR and DOR patients. In addition, serum AMH in DOR patients after Gn administration and FF AMH has a negative effect on SCF.

Combination of chick embryo and nutrient mixture prevent D-galactose-induced cognitive deficits, immune impairment and oxidative stress in aging rat model.

Aging is spontaneous and inevitable processes that lead to changes in biological systems. The present paper was designed to investigate the anti-aging roles of chick embryo (CE) and nutrient mixture (NM) in aging rats. Aging was induced by administration of D-galactose (D-gal, 500 mg/kg/day for 90 days). CE and NM were administered to aging rats through different dose gavage once a day. Cognitive function assessment was performed using the Morris water maze test. At the end of experiment, serum and tissues were collected for immunity and antioxidation function. The organs and tissues were excised for histological study. The results demonstrated that CE plus NM was superior treatment to improve the histopathologic changes and reverse learning and memory impairment of the aging rats. CE plus NM also increased the spleen and thymus index as well as splenocyte proliferation, and reversed inflammatory cytokine levels. In addition, the biochemical index showed that CE plus NM could improve the antioxidant enzyme activity of the aging rats, decrease lipofuscin (LF) and glutamate content. CE plus NM also inhibited the activation of TLR4/NF-κB pathway stimulated by LPS in splenic B lymphocytes. Overall, these results seem to be implying that CE plus NM was used as potentially natural supplement or functional food for preventing aging.

Stem Cell Factor-Based Identification and Functional Properties of In Vitro-Selected Subpopulations of Malignant Mesothelioma Cells.

Malignant mesothelioma (MM) is an aggressive neoplasm characterized by a poor patient survival rate, because of rapid tumor recurrence following first-line therapy. Cancer stem cells (CSCs) are assumed to be responsible for initiating tumorigenesis and driving relapse after therapeutic interventions. CSC-enriched MM cell subpopulations were identified by an OCT4/SOX2 reporter approach and were characterized by (1) increased resistance to cisplatin, (2) increased sensitivity toward the FAK inhibitor VS-6063 in vitro, and (3) a higher tumor-initiating capacity in vivo in orthotopic xenograft and allograft mouse models. Overexpression of NF2 (neurofibromatosis 2, merlin), a tumor suppressor often mutated or lost in MM, did not affect proliferation and viability of CSC-enriched MM populations but robustly decreased the viability of reporter-negative cells. In contrast, downregulation of calretinin strongly decreased proliferation and viability of both populations. In summary, we have enriched and characterized a small MM cell subpopulation that bears the expected CSC characteristics.

Aberrant expressions of stem cell factor/c-KIT in rat testis with varicocele.

BACKGROUND/PURPOSE: Varicocele (VC) is considered by the World Health Organization as the main cause of male infertility. Studies have shown that VC can affect spermatogenesis and then result in male infertility. But the exact mechanism by which VC affects spermatogenesis is still unclear. Stem cell factor (SCF) and c-KIT receptor are crucial molecules during spermatogenesis in testis. This study aims to investigate whether SCF/c-KIT signaling is involved in the pathophysiology of VC on spermatogenesis. METHODS: Rat models of VC were built (n = 13), and sham-operated rats were used as controls (n = 8). The seminiferous tubules of the testis were observed with hematoxylin and eosin staining, expression of SCF was analyzed via enzyme-linked immunosorbent assay and Western blot, and expression of c-KIT was assessed with Western blot and immunofluorescence. RESULTS: Compared with controls, the seminiferous epithelium was disorganized and had significantly fewer cells in the testes of rats with VC. Expression of SCF increased in testes of VC rats, while expression of c-KIT was decreased. CONCLUSION: These results suggest that sperm counts in seminiferous epithelium are affected by VC, and the SCF/c-KIT system is aberrantly expressed in VC testis, which could be involved in male infertility caused by VC.

Low c-Kit Expression Level Induced by Stem Cell Factor Does Not Compromise Transplantation of Hematopoietic Stem Cells.

The c-Kit expression level is decreased in regenerating bone marrow, and such bone marrow performs poorly when co-transplanted with normal bone marrow. We asked whether diminished numbers of c-Kit receptors on hematopoietic stem and progenitor cells (HSPCs) after their internalization induced by the binding of the cytokine stem cell factor (SCF) would jeopardize transplantability of HSPCs. We used a battery of functional assays to evaluate the capacity of HSPCs with markedly different c-Kit expression levels to be transplanted. Surprisingly, our experiments testing the homing of transplanted HSPCs to bone marrow of recipient mice and their short-term and long-term engraftment did not reveal any defects in HSPCs with severely reduced numbers of c-Kit receptor molecules. This unexpected result can be ascribed to the fact that HSPCs exposed to SCF replace the consumed c-Kit receptors rapidly. This article demonstrates that exposure of HSPCs to SCF and diminished number of c-Kit receptors in their cell membranes do not compromise the capacity of HSPCs to reconstitute damaged hematopoietic tissue.

Stem cell factor is a novel independent prognostic biomarker for hepatocellular carcinoma after curative resection.

Stem cell factor (SCF), a ligand of c-kit, is a hematopoietic growth factor. Uncontrolled activity of SCF/c-kit signaling pathway contributes to the formation of a variety of human malignancies. In this study, we determined whether SCF expression could risk-stratify patients with hepatocellular carcinoma (HCC) after curative resection. HCC tissues from 160 patients were collected during curative resection and stained with SCF and CD34, a marker for microvessel density (MVD), using immunohistochemistry. Two statistical analyses were performed: an independent continuous and a multivariate categorical analysis, with test/validation set-defined cut points, and Kaplan-Meier estimated outcome measures of overall survival (OS) and relapse-free survival (RFS). We found that higher levels of SCF confer worse OS (continuous P = 0.014; and categorical P = 0.009), and RFS (continuous P = 0.002; categorical P = 0.003) of patients with HCC. SCF varies independently from MVD-CD34, tumor node metastasis, histologic grade, age and gender, and retains prognostic significance when analysed as a categorical variable in a multivariate analysis . We confirmed that MVD-CD34 is also an independent prognostic marker for patients with HCC. The levels of SCF and CD34 showed a positive and significant correlation (P < 0.0001) and double low expression confers superior OS (median = 48 months) and RFS (median = 24 months), whereas double high expression confers shortest RFS (median = 10.5 months) compared with single measurements. The prognostic values of SCF and CD34 were independently determined in this study and we propose that both of them are independent prognostic markers for HCC.

Cyclic AMP and c-KIT signaling in familial testicular germ cell tumor predisposition.

BACKGROUND: Familial testicular germ cell tumors (FTGCTs) are hypothesized to result from the combined interaction of multiple low-penetrance genes. We reported inactivating germline mutations of the cAMP-binding phosphodiesterase 11A (PDE11A) as modifiers of FTGCT risk. Recent genome-wide association studies have identified single-nucleotide polymorphisms in the KITLG gene, the ligand for the cKIT tyrosine kinase receptor, as strong modifiers of susceptibility to both familial and sporadic testicular germ cell tumors. DESIGN: We studied 94 patients with FTGCTs and 50 at-risk male relatives from 63 unrelated kindreds, in whom the PDE11A gene had been sequenced by investigating the association between KITLG genome-wide association study single-nucleotide polymorphisms rs3782179 and rs4474514 and FTGCT risk in these patients and in 692 controls. We also examined cAMP and c-KIT signaling in testicular tissues and cell lines and extended the studies to 2 sporadic cases, one with a PDE11A defect and one without, as a comparison. RESULTS: We found a higher frequency of the KITLG risk alleles in FTGCT patients who also had a PDE11A sequence variant, compared with those with a wild-type PDE11A sequence. In NTERA-2 and Tcam-2 cells transfected with the mutated forms of PDE11A (R52T, F258Y, Y727C, R804H, V820M, R867G, and M878V), cAMP levels were significantly higher, and the relative phosphodiesterase activity was lower than in the wild-type cells. KITLG expression was consistently increased in the presence of PDE11A-inactivating defects, both at the RNA and protein levels, in familial testicular germ cell tumors. The 2 sporadic cases that were studied, one with a PDE11A defect and another without, agreed with the data in FTGTCT and in the cell lines. CONCLUSIONS: Patients with FTGCT and PDE11A defects also carry KITLG risk alleles more frequently. There may be an interaction between cAMP and c-KIT signaling in predisposition to testicular germ cell tumors.

Association of stem cell factor and high-sensitivity C reactive protein concentrations in crevicular fluid and serum in patients with chronic periodontitis with and without type 2 diabetes.

The aim of the present study was to clarify whether there is any correlation between the levels of high-sensitivity C reactive protein (hs-CRP) and stem cell factor (SCF) in serum and gingival crevicular fluid (GCF) of patients with chronic periodontitis (CP) with and without type 2 diabetes mellitus (DM). A total of 40 subjects were divided into 3 groups: 10 periodontally healthy subjects (Group 1), 15 CP patients (Group 2), and 15 type 2 DM patients with CP (Group 3). Levels of hs-CRP and SCF in GCF and serum were quantified using different techniques. The clinical outcomes evaluated were gingival index (GI), probing depth (PD) and clinical attachment level (CAL), and the correlations of the two inflammatory mediators with clinical parameters were evaluated. The levels of these inflammatory mediators increased continuously from group 1 to group 2, and to group 3. The serum levels of both hs-CRP and SCF were correlated with PD in patients with CP (P < 0.05). SCF levels were correlated with PD in Group 3 (P < 0.05). The fact that the levels of hs-CRP and SCF were highest in DM patients with CP suggests that the presence of a systemic condition has a profound effect on the levels of inflammatory mediators, both locally at sites of periodontal disease, and elsewhere.

Airway inflammatory responses to diesel exhaust in allergic rhinitics.

CONTEXT: Proximity to traffic, particularly to diesel-powered vehicles, has been associated with inducing and enhancing allergies. To investigate the basis for this association, we performed controlled exposures of allergic rhinitics to diesel exhaust (DE) at a dose known to be pro-inflammatory in healthy individuals. OBJECTIVE: We hypothesized that diesel-exhaust exposure would augment lower airway inflammation in allergic rhinitics. MATERIALS AND METHODS: Fourteen allergic rhinitics were exposed in a double-blinded, randomized trial to DE (100 µg/m³ PM10) and filtered air for 2 h on separate occasions. Bronchoscopy with endobronchial mucosal biopsies and airway lavage was performed 18 h post-exposure, and inflammatory markers were assessed. RESULTS: No evidence of neutrophilic airway inflammation was observed post-diesel, however, a small increase in myeloperoxidase was found in bronchoalveolar lavage (p = 0.032). We found no increases in allergic inflammatory cells. Reduced mast cell immunoreactivity for tryptase was observed in the epithelium (p = 0.013) parallel to a small decrease in bronchial wash stem cell factor (p = 0.033). DISCUSSION AND CONCLUSION: DE, at a dose previously shown to cause neutrophilic inflammation in healthy individuals, induced no neutrophilic inflammation in the lower airways of allergic rhinitics, consistent with previous reports in asthmatics. Although there was no increase in allergic inflammatory cell numbers, the reduction in tryptase in the epithelium may indicate mast cell degranulation. However, this occurred in the absence of allergic symptoms. These data do not provide a simplistic explanation of the sensitivity in rhinitics to traffic-related air pollution. The role of mast cells requires further investigation.

Mechanism and efficacy of mobilization of granulocyte colony-stimulating factor in the treatment of chronic hepatic failure.

BACKGROUND/AIMS: To explore the efficacy of G-CSF mobilization in the treatment of chronic liver failure (CLF) and the mechanism of its action. METHODOLOGY: The proportions of cluster-of-differentiation (CD)-34+ cells and their receptor-CXCR4 were detected by flow cytometry in patients with different types of chronic HBV infection. The levels of chemokines and cytokines were measured by enzyme-linked immunosorbent assay. RESULTS: The proportion of CD34+ cells in patients with cirrhosis was significantly increased compared with the healthy controls (p<0.05) and was increased obviously after treatment by G-CSF mobilization (p<0.01). The expression levels of SDF-1, SCF and MMP-9 were significantly elevated in patients with chronic hepatitis B and liver cirrhosis (p<0.01). The expression levels of SCF and MMP-9 were significantly elevated after treatment with G-CSF (p<0.05). No significant differences were found in the levels of total bilirubin, albumin and prothrombin time between the treated and control groups; furthermore, no significant differences were observed in the cure and improvement rates between the two groups. CONCLUSIONS: The basal levels of stem cell mobilization in patients with liver cirrhosis might be associated with the repair of liver injury. G-CSF could promote hematopoietic stem cell mobilization through regulation of the expression levels of stem-cell-mobilization-related factors in patients with liver cirrhosis. No apparent effects of G-CSF therapy on both liver function and short-term prognosis in patients with liver cirrhosis were confirmed.

Post-treatment levels of stem cell factor and hs-CRP in serum and crevicular fluid of chronic periodontitis subjects with type 2 diabetes.

OBJECTIVE: The purpose of this study is to evaluate the stem cell factor (SCF) and high sensitive C reactive protein (hs-CRP) concentration in gingival crevicular fluid (GCF) and serum of chronic periodontitis subjects with type 2 diabetes, and to evaluate the effect of nonsurgical periodontal therapy on their GCF and serum concentrations. MATERIALS AND METHODS: A total of (age and gender matched) 22 subjects were evaluated. Pre- and post-treatment levels of SCF and hs-CRP in GCF and serum were measured and compared using enzyme linked immunosorbant assay. Clinical parameters including probing depth and clinical attachment level were also measured. Paired t-test was used to compare the before- and after-treatment levels of the two molecules. RESULTS: A highly significant difference (P < 0.001) was found in the GCF and serum concentrations of SCF and hs-CRP before and after treatment. CONCLUSION: Our observations indicated that short-term nonsurgical therapy resulted in a significant improvement in periodontal indices and in a marked decrease of SCF and hs-CRP serum and GCF levels.

Evaluation of the KIT/stem cell factor axis in renal tumours.

AIM: To investigate the expression of the KIT/stem cell factor (SCF) axis in different renal cell carcinoma subtypes with regard to targeted therapies. MATERIALS AND METHODS: The expression of KIT and SCF were immunhistochemically assessed in 40 clear cell (ccRCC), 25 papillary (pRCC) and 19 chromophobe carcinomas (chRCC); 27 oncocytomas and 32 benign kidney parenchyma specimens differentiated into distal tubules (DT) and proximal tubules (PT). RESULTS: The expression of KIT was significantly higher in chRCC and oncocytoma compared to ccRCC and pRCC. All tumours exhibited a significant increase of membranous to cytoplasmic KIT expression, with the highest in ccRCC and pRCCs. SCF was expressed in all tumour subgroups, with the highest in oncocytomas and pRCC. SCF correlated positively with the cytoplasmic expression of KIT. A higher tumour stage correlated to lower KIT expression in ccRCC. CONCLUSION: Simultaneous expression of SCF and KIT in renal tumours, which seems to undergo a shift from the cytoplasm to the cell membrane, suggests paracrine and autocrine mechanisms in KIT activation, with different, as yet unknown, regulatory mechanisms in the different tumour entities.

Optimization of SCF feeding regimen for ex vivo expansion of cord blood hematopoietic stem cells.

Stem cell factor (SCF) plays important roles in ex vivo expansion of hematopoietic stem cells (HSCs). In this study, the effects of dose and feeding time of SCF on ex vivo expansion of CD34(+) cells were investigated in serum-free medium supplemented with a cytokine cocktail composed of SCF, thrombopoietin (TPO) and flt3-ligand (FL). Among the four tested doses (0, 5, 50 and 500ng/mL), a SCF dose of 50ng/mL was demonstrated to be most favorable for ex vivo expansion of CD34(+) cells, which resulted in 34.22±10.80 and 8.89±1.25 folds of expansion regarding total cells and CD34(+) cells, respectively. Meanwhile, the specific growth rate of cells, the consumption rate of SCF and the percentage of CD34(+)c-kit(+) cells during the 21-day culture process were analyzed. The results indicated that initial 4-day period was a critical stage for SCF functioning on CD34(+) cells during ex vivo expansion. Based on this, a modified SCF feeding regimen was proposed, in which SCF (50ng/mL) was only supplemented on day 0 in the cytokine cocktail and cells were then fed with TPO and FL till the end of culture. It was found that this SCF feeding regimen could expand CD34(+) cells efficiently, thus providing a cost-effect expansion protocol for HSCs.

High density of tryptase-positive mast cells in patients with renal cell carcinoma on hemodialysis: correlation with expression of stem cell factor and protease activated receptor-2.

Patients on hemodialysis are at higher risk of renal cell carcinoma probably because of inflammatory and immune system disorders. The aim of this study was to clarify the pathologic roles of 2 phenotypes of mast cells, mast cell tryptase and mast cell chymase, and their correlation with stem cell factor and protease-activated receptor 2 in patients with renal cell carcinoma on hemodialysis. The densities of mast cell tryptase and mast cell chymase and expressions of stem cell factor and protease-activated receptor 2 were examined in 35 patients with hemodialysis-renal cell carcinoma and 39 with non-hemodialysis-renal cell carcinoma who were diagnosed and treated in our hospital. Protein expression was examined by immunohistochemistry. The proliferation index represented the number of Ki-67-positive cells. There were no significant differences in clinicopathologic features between the 2 groups. Mast cell tryptase densities in intratumoral (8.3 per high-power field) and peritumoral areas (8.7 per high-power field) were higher in hemodialysis-renal cell carcinoma than non-hemodialysis-renal cell carcinoma (2.7 and 5.3 per high-power field). No such significant correlations were detected in mast cell chymase. In hemodialysis-renal cell carcinoma, intratumoral mast cell tryptase density correlated with the proliferation index (P = .039 and P = .008, respectively) and also with stem cell factor and protease-activated receptor 2 expression. Our results emphasize the important roles of mast cell tryptase in cancer cell proliferation and recurrence in hemodialysis-renal cell carcinoma. Stem cell factor and protease-activated receptor 2 seem to up-regulate mast cell tryptase functions in these patients. The results suggest collaborative effects of stem cell factor, mast cell tryptase, and protease-activated receptor 2 on the malignant potential of hemodialysis-renal cell carcinoma.

Rapid and sensitive detection of cytokines using functionalized gold nanoparticle-based immuno-PCR, comparison with immuno-PCR and ELISA.

Reliable and simple methods are required for detection of low concentrations of cytokines and some other proteins in complex biological fluids. This is especially important when monitoring the immune responses under various physiological and pathophysiological conditions in vivo or following production of these compounds in in vitro systems. Cytokines and other immunologically active molecules are being predominantly detected by enzyme-linked immunosorbent assays (ELISA) and newly also by immuno-polymerase chain reactions (iPCR). New simplified variants of iPCR have recently been described where antibodies are connected with multiple DNA templates through gold nanoparticles (Au-NPs) to form a new class of detection reagents. In this study we compared functionalized Au-NP-based iPCR (Nano-iPCR) with standard ELISA and iPCR for the detection of interleukin (IL)-3 and stem cell factor (SCF). The same immunoreagents (IL-3- and SCF-specific polyclonal antibodies and their biotinylated forms) were used throughout the assays. The obtained data indicate that both Nano-iPCR and iPCR are superior in sensitivity and detection range than ELISA. Furthermore, Nano-iPCR is easier to perform than the other two methods. Nano-iPCR was used for monitoring changes in concentration of free SCF during growth of mast cells in SCF-conditioned media. The results show that growing cultures gradually reduce the amount of SCF in supernatant to 25% after 5 days. The combined data indicate that Nano-iPCR assays may be preferable for rapid detection of low concentrations of cytokines in complex biological fluids.

Maternal and fetal circulating sKL and ET-1 levels as function of normal labor at term.

OBJECTIVES: To determine whether labor is associated with alterations of the levels of soluble c-kit ligand (sKL) and endothelin-1 (ET-1) in maternal plasma and umbilical cord blood. METHODS: The sKL and ET-1 levels were investigated in umbilical cord and maternal plasma on the day of delivery in 18 pregnant women with vaginal delivery during labor, 18 non-pregnant women and 9 pregnant women before cesarean delivery, using an ELISA assay. RESULTS: Umbilical cord plasma sKL levels were significantly higher than the maternal plasma in both types of delivery (p = 0.0001, p < 0.0001, respectively). However, maternal plasma ET-1 levels in the presence of labor were significantly higher than the cesarean delivery group (p < 0.0001). No difference was noted for sKL and ET-1 in umbilical cord vessels of both groups. Furthermore, a highly significant inverse correlation was documented between the individual levels of cord plasma ET-1 and the levels of cord plasma sKL (r = -0.6269, p = 0.0054). CONCLUSIONS: The sKL levels found in umbilical cord plasma are consistent with the pleiotropic effects of sKL in facilitating the transition of the fetus to the neonatal stage. The reduced ET-1 maternal plasma levels, compared to non-pregnant women, probably are indicative of a putative mechanism for embryo protection from vasoconstriction sequelae. This assumption is strengthened by the corresponding ET-1 levels in umbilical cord plasma.

Localization of c-kit and stem cell factor (SCF) in ovarian follicular epithelium of a lizard, Podarcis s. sicula.

We performed an immunohistochemical study to determine the immunolocalization of c-kit and stem cell factor (SCF) in ovarian follicles during the reproductive cycle of the lizard, Podarcis s. sicula. Follicles were serially cut and used for histological and histochemical characterization and also for immunohistochemical detection of both c-kit and SCF. C-kit and SCF were localized in the follicles with a differing pattern with regard to the stage of sexual cycle or the cell type forming the follicular epithelium (granulosa). In pre-reproductive follicles, where the granulosa consists of three main different cytotypes, the c-kit receptor was prevalently localized on the plasmalemma of small cells, although some pyriform and intermediate cells also appeared positive. C-kit was also localized in the theca. In pre-reproductive follicles, SCF was markedly observed in the cytoplasm of some pyriform cells. Small cells and theca also stained moderately positive, whereas the intermediate cells were mostly negative. In reproductive follicles, where granulosa cells are morphologically rearranged, c-kit was observed in small cells and in some thecal elements, while SCF was weakly immunostained. At the site of follicular layer invaginations evident c-kit/SCF immunostaining was observed in the granulosa epithelium and in the theca. These observations suggest that the expression of c-kit and SCF changes as a function of follicular development and may reflect the involvement of this system in the maturation of the oocyte.

MMP9 limits apoptosis and stimulates branching morphogenesis during kidney development.

Early events in kidney organogenesis involve reciprocal interactions between the ureteric bud and the metanephric mesenchyme, which lead to remodeling of the extracellular matrix. This remodeling involves matrix metalloproteases (MMPs), but the specific roles of individual MMPs in kidney development are not completely understood. Here, we analyzed MMP9-deficient mice at the first step of kidney development and found that MMP9 deficiency delayed embryonic kidney maturation and increased apoptosis ex vivo by 2.5-fold. These early defects resulted in a 30% decrease in nephron number, a 20% decrease in adult kidney weight, and altered kidney function and morphology at 12 mo. The membrane form of stem cell factor (SCF) increased, whereas the activated form of the SCF receptor, c-kit, decreased in MMP9-deficient embryonic kidneys. In organotypic culture, MMP9-deficient kidneys failed to secrete SCF, and addition of recombinant SCF partially rescued both apoptosis and the branching defect. In conclusion, these data show that MMP9 protects mesenchymal cells from apoptosis during kidney development and stimulates ureteric bud branching morphogenesis, most likely by releasing the soluble form of SCF, suggesting that normal renal development requires MMP9.

A kindred with familial progressive hyperpigmentation-like disorder: implication of fibroblast-derived growth factors in pigmentation.

Two patients with a generalized, progressive dyschromatosis disorder are described and investigated as a model to study the role of fibroblast-derived mediators on skin pigmentation. The patients (father and daughter) had had a widespread hyperpigmentation since early life which then progressively worsened with the appearance of hyperpigmented macules, café-au-lait macules and freckles, also involving the lips, palms and soles, intermixed with small hypopigmented spots. These features resembled those of familial progressive hyperpigmentation (FPH). Histology revealed a normal epidermis with pronounced keratinocyte hyperpigmentation and the presence of dermal melanophages. Ultrastructural analysis showed basal and suprabasal keratinocytes enriched in melanosome complexes. Immunohistochemical staining displayed an increased expression of hepatocyte growth factor (HGF), stem cell factor (SCF) and keratinocyte growth factor (KGF) in fibroblast-like cells of the upper dermis in hyperpigmented lesions of both patients, compared to control healthy skin. Our data suggest that a persistent activation of fibroblasts abnormally stimulating melanocyte functions is involved in hyperpigmentation disorders.