Proinflammatory biomarkers' level and functional genetic polymorphisms in periprosthetic joint infection.

OBJECTIVE: The aims of this study were 1) to identify the level of inflammatory biomarkers interleukin (IL)-1α, IL-1ß, IL-6, IL-8, IL-17, C-reactive protein (CRP), granulocyte colony-stimulating factor (GCSF), ferritin, and tumor necrosis factor (TNF)-α in serum and synovial fluid samples of patients who underwent revision arthroplasty surgery; 2) to establish the relationship between serum and synovial fluid levels; 3) to determine if any of the 11 genetic polymorphisms of TNFα, IL-1, IL-6, IL-8, IL-17, and GCSF on the encoding genes was associated with periprosthetic joint infection (PJI). METHODS: Synovial fluid and serum was collected from 88 patients who underwent revision arthroplasty surgery. The Musculoskeletal Infection Society definition was used to classify these patients into 2 groups: 36 PJIs and 52 aseptic failures. Synovial fluid and serum samples were tested for 9 biomarkers using a micro enzyme-linked immunosorbent assay. Genetic polymorphisms were evaluated with polymerase chain reaction and restriction endonuclease analysis. RESULTS: Synovial fluid-derived IL-1α, IL-1ß, IL-8, IL-17, CRP, GCSF, TNFα, and serum-derived IL-6, IL-17, ferritin, CRP were found suitable to classify PJI and aseptic failure. In addition, IL-17 and CRP levels demonstrated a positive correlation between synovial fluid and serum. TNFα-238, IL6-174, GCSF3R, and IL1 RN-VNTR genetic polymorphisms occurred more frequently in individuals with septic failure. CONCLUSION: Significant differences between the two groups were observed in the functional polymorphisms of the genes encoding the cytokines investigated. These differences could be interpreted as indicating that there is an association between PJI and genetic polymorphisms. LEVEL OF EVIDENCE: Level III, diagnostic study.

Expression of granulocyte colony-stimulating factor receptor in rectal cancer.

AIM: To evaluate whether granulocyte colony-stimulating factor receptor (G-CSFR) expression before preoperative irradiation can predict the radiosensitivity of rectal cancer. METHODS: The expression of G-CSFR was examined, using immunohistochemistry, in biopsy specimens from 126 patients with locally advanced rectal adenocarcinoma before preoperative irradiation. Radiosensitivity was then evaluated according to the Rectal Cancer Regression Grading. Endoscopic inspection was used to detect the tumor area in each patient. General patient information, such as age, gender, lymph node status, tumor size and degree of differentiation was recorded. A statistical analysis was then performed to evaluate the correlation between clinical or pathological parameters and G-CSFR expression in tumors. RESULTS: According to endoscopic inspection, the tumor area ranged from 4 to 48 cm² (median, 15 cm²). Positive G-CSFR immunoreactions (G-CSFR⁺) were observed in 85 specimens, and negative (G-CSFR⁻) in 41. No significant differences were found in age, gender, tumor invasion, lymph node status and tumor size between G-CSFR⁺ and G-CSFR⁻ patients. G-CSFR expression was positively correlated with poor radiotherapy response (58.8% vs 75.6%, P = 0.014, r = 0.219). The proportion of well-differentiated tumors in G-CSFR⁺ and G-CSFR⁻ patients was 24.7% and 36.6%, respectively. Sphincter preservation was observed in 57.6% of G-CSFR⁺ patients and 78.5% of G-CSFR⁻ patients. Significant correlations were found between G-CSFR expression and tumor differentiation (24.7% vs 36.6%, P = 0.019, r = 0.210), as well as sphincter preservation (57.6% vs 78.5%, P = 0.044, r = 0.180). CONCLUSION: The expression of G-CSFR before preoperative irradiation may predict the radiosensitivity of rectal cancer.

Expression of functional markers in acute lymphoblastic leukemia.

We analyzed surface antigens, multidrug resistance (MDR) parameters (PGP, MRP, LRP), tissue infiltration parameters (CD18, CD44, VCAM, MMP2), receptors for colony stimulating factors (G-CSFr, GM-CSFr) and cell cycle parameters (Ki-67, topoisomerase IIalpha) in 86 patients with acute lymphoblastic leukemia (ALL). LRP, PGP and CD18 were associated with poor clinical outcome, and LRP expression was related with CD18, CD44 and G-CSFr. Of the cell cycle parameters, Ki-67 (+) fraction was increased in ALL with hepato-splenomegaly and extramedullary involvement. In conclusion, analysis of LRP, PGP, CD18 and Ki-67 could be helpful to predict the clinical behavior of ALL.

Varying expression levels of colony stimulating factor receptors in disease states and different leukocytes.

Administration of G-CSF may not always respond in rise of neutrophil counts in different patient population. In order to understand a possible inter-relationship between the G-CSF and GM-CSF induced leukocyte responses and expression levels of receptors for G-CSF (G-CSFr) and GM-CSF (GM-CSFr), the levels of each receptor and CSF were measured in patients with basophilia (8), eosinophilia (14) and bacterial infection showing neutrophilia (12) in comparison with normal healthy adults (12) and children (14). G-CSFr was expressed in neutrophils in the largest amount followed by monocytes, but GM-CSFr was expressed more in monocytes than neutrophils. Lymphocytes and basophils did not express G-CSFr or GM-CSFr. The amount of GM-CSFr in neutrophils was present less in patients with infection than normal control (P = 0.031). The neutrophils expressed more G-CSFr than GM-CSFr. The quantity of G-CSFr in eosinophil showed marked interval change, higher in acute stage. The plasma concentrations of G-CSF in patients with infection were much higher than normal adults or children (117.95 +/- 181.16 pg/ml, P < 0.05). Binding assay with excess amount of CSFs could discriminate the patient who did not show any response to G-CSF or GM-CSF administration. After incubation with excess CSFs, more receptors were blocked in children than in adults (G-CSF P = 0.024, GM-CSF P = 0.006). These results indicate that the amount of CSFr in leukocyte varies in different types of leukocyte, and changes according to the patients' condition even in the same type of leukocyte, and the CSFrs of children bind to CSFs more than those of adults.

Expression of functional markers in acute nonlymphoblastic leukemia.

Multidrug resistance parameters, tissue infiltration parameters, receptors for colony-stimulating factors (CSFr) and cell cycle parameters were analyzed using flow cytometry in 145, 109 initial and 36 relapsed or refractory, acute nonlymphoblastic leukemia (ANLL) patients to find out clinically more reliable functional parameters. Lung resistance-associated protein (LRP) was most frequently expressed in ANLL (44.1%) followed by P-glycoprotein (PGP) (35.9%) and multidrug resistance-associated protein (MRP) (8.3%). LRP and PGP were expressed more frequently in relapsed or refractory ANLL than initial ANLL cases. Complete remission rate after standard chemotherapy falls in PGP-positive cases (p = 0.001). CD44-positive ANLL cases relapsed more frequently. The organ tropism is different depending on the infiltration parameters, vascular cell adhesion molecule to splenomegaly, matrix metalloprotease-2 to hepatomegaly and to extramedullary infiltration other than spleen, liver or lymph node. The percentage of the granulocyte-macrophage-CSFr expression was high in M4 and M5, and granulocyte-CSFr-positive ANLL showed less extramedullary infiltration (p = 0.007) and more PGP expression. Ki-67 was expressed significantly less in refractory ANLL than initial ANLL and DNA topisomerase IIalpha was expressed significantly more in the surviving patients group. In conclusion, analysis of these new functional parameters could help to predict and overcome the clinical behavior of each ANLL at the time of diagnosis.

Varying expression levels of colony stimulating factor receptors in disease states and different leukocytes

Administration of G-CSF may not always respond in rise of neutrophil counts in different patient population. In order to understand a possible inter-relationship between the G-CSF and GM-CSF induced leukocyte responses and expression levels of receptors for G-CSF (G-CSFr) and GM-CSF (GM-CSFr), the levels of each receptor and CSF were measured in patients with basophilia (8), eosinophilia (14) and bacterial infection showing neutrophilia (12) in comparison with normal healthy adults (12) and children (14). G-CSFr was expressed in neutrophils in the largest amount followed by monocytes, but GM-CSFr was expressed more in monocytes than neutrophils. Lymphocytes and basophils did not express G-CSFr or GM-CSFr. The amount of GM-CSFr in neutrophils was present less in patients with infection than normal control (P = 0.031). The neutrophils expressed more G-CSFr than GM-CSFr. The quantity of G-CSFr in eosinophil showed marked interval change, higher in acute stage. The plasma concentrations of G-CSF in patients with infection were much higher than normal adults or children (117.95 +/- 181.16 pg/ml, P < 0.05). Binding assay with excess amount of CSFs could discriminate the patient who did not show any response to G-CSF or GM-CSF administration. After incubation with excess CSFs, more receptors were blocked in children than in adults (G-CSF P = 0.024, GM-CSF P = 0.006). These results indicate that the amount of CSFr in leukocyte varies in different types of leukocyte, and changes according to the patients' condition even in the same type of leukocyte, and the CSFrs of children bind to CSFs more than those of adults.

Localization and expression of CSF-1 receptor in rat dental follicle cells.

Colony-stimulating factor-1 (CSF-1) accelerates tooth eruption in rats and is localized in the dental follicle, a loose connective tissue sac that is necessary for eruption to occur. CSF-1 enhances the cellular events that occur in the follicle prior to eruption--namely, an influx of monocytes into the follicle early post-natally to form the osteoclasts needed to resorb bone for the eruption pathway. Because CSF-1 levels are at a peak at day 3 post-natally, and because CSF-1 has an autocrine effect on its own gene expression, the question remains as to what causes the subsequent decline in CSF-1 protein and mRNA after day 3 post-natally. To determine if the autocrine effect is inhibited through the CSF-1 receptor, analysis of the CSF-1 receptor mRNA levels in cultured dental follicle cells reveals that high concentrations of CSF-1 reduce the gene expression of the CSF-1 receptor. Interleukin 1 alpha, a molecule that enhances CSF-1 gene expression, has no effect on CSF-1 receptor mRNA levels. Immunostaining for the CSF-1 receptor protein shows that it is present in the dental follicle early post-natally and is either absent or greatly reduced by day 10 post-natally. Earlier studies showed that the mRNA levels of the CSF-1 receptor also parallel this time course. Thus, the above results suggest that the feedback inhibition of the autocrine effect of CSF-1 on its own expression is through the effect of CSF-1 inhibiting the translation and transcription of its receptor. In turn, these molecular interactions possibly regulate the cellular events that occur in the follicle prior to and during eruption.

Bone marrow stromal cells induce myeloid and lymphoid development of the sorted hematopoietic stem cells in vitro.

Regulation of development of hematopoietic stem cells was examined by culturing Lin- c-Kit+ Sca1+ stem cells sorted from bone marrow (BM) cells by fluorescence-activated cell sorting on a layer of TBR59, a BM stromal cell line established from simian virus 40 T-antigen gene transgenic mice. The sorted stem cells did not show self-renewal, but two waves (at 7 and 13 days) of a cobblestone formation were induced by the stromal cell layer. The cobblestones were formed by finite cell division (eight divisions on average) of sorted Lin- c-Kit+ Sca1+ stem cells, and divided cells were still immature. The c-Kithigh stem cell population was induced to form the first wave of cobblestone formation committed to myeloid lineage, whereas c-Kitlow population was induced to form the second wave of this formation committed to lymphoid lineage. Both cobblestone formations require c-Kit function, but very late activation antigen-4-vascular cell adhesion molecule-1 interaction plays different parts in the two lineages.

Early CD34high cells can be separated into KIThigh cells in which transforming growth factor-beta (TGF-beta) downmodulates c-kit and KITlow cells in which anti-TGF-beta upmodulates c-kit.

We have previously shown that early human CD34high hematopoietic progenitors are maintained quiescent in part through autocrine transforming growth factor-beta 1 (TGF-beta 1). We also demonstrated that, in the presence of interleukin-3, interleukin-6, granulocyte colony-stimulating factor, and erythropoietin, TGF-beta 1 antisense oligonucleotides or anti-TGF-beta serum have an additive effect with KIT ligand (Steel factor [SF]), which suggests that they control different pathways of regulation in these conditions. This finding also suggests that autocrine TGF-beta 1 might suppress c-kit expression in primitive human hematopoietic progenitors. We have now distinguished two subpopulations of CD34high cells. One subpopulation expresses a c-kit mRNA that can be downmodulated by exogenous TGF-beta 1 within 6 hours. Another subpopulation of early CD34high cells expresses a low or undetectable level of c-kit mRNA, but its expression can be upmodulated within 6 hours by anti-TGF-beta. These effects disappear 48 hours after induction and cannot be maintained longer than 72 hours, even if TGF-beta 1 or anti-TGF-beta serum are added every day. Similar kinetics, although delayed, are observed with KIT protein expression. On the contrary, no specific effect of TGF-beta 1 was observed on c-fms, GAPDH, and transferrin receptor gene expression in these early progenitors. These results clarify the complex interaction between TGF-beta 1 and SF in normal early hematopoietic progenitors. SF does not switch off the TGF-beta 1 inhibitory pathway. Autocrine TGF-beta 1 appears to maintain these cells in a quiescent state, suppressing cell division by downmodulating the receptor of SF, a key cytokine costimulator of early progenitors.

Effects of stem cell factor on the growth and radiation survival of tumor cells.

Recombinant human stem cell factor (SCF) binds to the c-kit receptor on human bone marrow progenitor cells and enhances their survival following irradiation. Since the c-kit receptor has also been detected on malignant cells, experiments were performed to study the effect of SCF on the proliferation and radiation survival of a variety of both c-kit-positive and -negative human tumor cell lines using [3H]thymidine incorporation and colony formation assays. The addition of SCF to both c-kit-positive and -negative cell line cultures had no significant effect on the stimulation index (in [3H]thymidine assay). In contrast, colony formation by H69 (small cell lung cancer cell line), H128 (small cell lung cancer cell line), and HEL (erythroid leukemia cell line) cells was enhanced by SCF in a dose-dependent manner, but SCF did not promote the in vivo growth of H128 xenograft tumors in terms of graft rate, time from implantation to tumor detection, or tumor size. Furthermore, SCF did not significantly increase the surviving fraction of either c-kit-positive or -negative cell lines following radiation, and there were no statistically significant differences between D0 [defined by the slope of the terminal exponential region of the two-component (single-hit multitarget model) survival curve where slope = 1/D0], Dq (quasithreshold dose), n (extrapolation number), alpha, and beta values for any of the cell lines studied that were irradiated with and without SCF. Finally, nude mice with transplanted human LG425 cutaneous T-cell lymphoma (c-kit positive) were treated with 10 Gy with or without SCF (100 micrograms/kg i.p. 20 h before, 2 h before, and 4 h after irradiation). There were no significant differences in the median tumor quadrupling time between groups that received either no treatment or SCF alone, or between groups treated with 10 Gy and SCF or 10 Gy alone (P > 0.05). These results are encouraging and suggest that SCF does not stimulate tumor cell proliferation in vivo or enhance the survival of tumor cells following irradiation.

Detection of transcripts and binding sites for colony-stimulating factor-1 during bone development.

Colony-stimulating factor-1 (CSF-1), originally characterized as the growth factor for the cells of the mononuclear phagocytic system, has been shown to be essential for osteoclast formation. The aim of the present study was twofold: (i) to investigate the expression of transcripts encoding CSF-1; and (ii) to detect binding sites for CSF-1 during bone development. As a model, metatarsal rudiments from embryonic mice of different ages were used, an in vivo system allowing one to follow osteoclast formation. In 16-day-old embryos, proliferating osteoclast precursors are located on the outer surface of the rudiments. They differentiate subsequently to post-mitotic precursors. At 18 days, the precursors fuse and the mature osteoclasts invade the mineralized cartilage of the rudiments to excavate the future bone marrow cavity. Within this study, in situ hybridization on sections of whole paws from 17-day-old embryos revealed CSF-1 transcripts to be present in cells lining the outside of the midregion of the metatarsals. One day later, cells containing CSF-1 mRNA were found within the mineralized cartilage. The levels of transcripts encoding CSF-1 were further increased in the bone rudiments of newborn animals. Binding sites for CSF-1 on cells in close proximity of the metatarsals were detected at embryonic age 17 days, but not before. At this stage, cells binding CSF-1 were located on the periosteum of the midregion of the metatarsal rudiment. At 18 days, cells expressing high levels of CSF-1 binding sites had invaded the mineralized cartilage.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of hematopoietic stem and progenitor cell populations in cytomegalovirus-infected mice.

We have studied the effects of murine cytomegalovirus (MCMV) infection on bone marrow stem and progenitor cell populations to find an explanation for the defects in hematopoiesis that accompany CMV infections in patients. Sublethal MCMV infection of BALB/c mice resulted in a 5- to 10-fold decrease in the numbers of myeloid (colony-forming unit-granulocyte-macrophage [CFU-GM]) and erythroid (burst-forming unit-erythroid [BFU-E]) progenitor cells in the marrow, but not in primitive myeloerythroid progenitor cell (colony-forming unit-spleen [CFU-S]) numbers. In contrast, we observed a 10- to 20-fold reduction in CFU-S as well as CFU-GM and BFU-E in lethally infected mice. Depletion of marrow CFU-GM was less severe in C57BL/10 and C3H/HeJ mice, which are more resistant to the effects of MCMV infection. Treatment of bone marrow cells with MCMV preparations in vitro did not reduce the numbers of CFU-GM, although up to 10% of the cells were productively infected. This finding suggests that CFU-GM were not susceptible to lytic MCMV infection in vitro and are probably not eliminated by lytic infection in vivo. Increases in the frequencies of Sca-1+Lin- marrow cells, a population that includes cells with the characteristics of pluripotential stem cells, were observed in MCMV-infected BALB/c, C57BL/10, and DBA/2J mice. Increases in the frequencies of c-kit+Lin- marrow cells were only seen in DBA/2J mice. MCMV infection did not impair the function of pluripotential stem cells because transplantation of marrow from MCMV-infected donors into irradiated recipient mice resulted in successful reconstitution of the T, B, and myeloid cell lineages.

Interleukin-3 treatment of rhesus monkeys leads to increased production of histamine-releasing cells that express interleukin-3 receptors at high levels.

To understand the hematopoietic and nonhematopoietic responses to interleukin-3 (IL-3), expression of cell-surface IL-3 receptors (IL-3R) was examined on bone marrow (BM) cells and peripheral blood (PB) cells of rhesus monkeys during the course of in vivo IL-3 treatment. Whereas IL-3R expression is low in untreated monkeys, IL-3 administration led to a gradual increase in both low- and high-affinity binding sites for IL-3. This increase reflected the total number of cells expressing IL-3Rs, as detected by flow cytometry using biotinylated IL-3. Most of these IL-3R+ cells in both BM and PB could be characterized as basophilic granulocytes that contained high levels of histamine. In contrast to the effect on these differentiated cells, IL-3 administration did not significantly alter the low level IL-3R expression on immature, CD34+ cells. Further flow cytometric analysis using biotinylated growth factors showed that the IL-3R+ basophils also expressed receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), but not for IL-6 or Kit ligand. These findings indicated that the IL-3R+ cells included neither monocytes, which express GM-CSFRs and IL-6Rs abundantly, nor mast cells, which express c-kit. By combining flow cytometric and Scatchard data, it was calculated that the basophils contain as many as 1 to 2 x 10(3) high-affinity IL-3Rs and 15 to 30 x 10(3) low-affinity sites. The finding that in vivo IL-3 treatment leads to the production of large numbers of cells that express high levels of IL-3R and are capable of producing histamine provides an explanation for the often severe allergic reactions that occur during prolonged IL-3 administration. It also indicates that IL-3, in addition to its direct effects on hematopoietic cells, may also stimulate hematopoiesis through the release of secondary mediators such as histamine by IL-3-responsive mature cells.

An improved panning technique for the selection of CD34+ human bone marrow hematopoietic cells with high recovery of early progenitors.

The human hematopoietic pluripotent repopulating "stem cell" is thought to be present within a minor subpopulation of CD34+ cells. This has not been definitively shown, although the more primitive CD34+ cell subset contains precursors for all lymphoid and nonlymphoid cell lineages. When purifying CD34+ cells, it is important to recover these early progenitors, which are more strongly immunoadsorbent to the separation devices. Using a commercialized panning system (AIS CELLector flasks), we observed that a high degree of purity requires a thorough washing procedure so that cells not binding or weakly binding to CD34 antibodies are removed. High recoveries can be obtained if the adherent cells are then efficiently detached by a 2-hour incubation in culture medium without added cytokines. In this way, we can routinely obtain 93.5 +/- 3.4% purity of CD34+ cells with a 74% yield of the multipotent colony-forming units (CFU-GEMM). Complete recovery of the putative "stem cell," or at least the early progenitor cell compartment (CD34+ CD38low/- CD34+ Thy-1+ cells), is also obtained. More than 30% of these cells can generate day-14 colonies in vitro. Comparable results were obtained when the separation was scaled up for clinical application using appropriate large-scale devices. The various incubation times of the procedure can be easily adjusted to the work schedule. This renders the procedure easy to handle, efficient, safe, and, because the cells can be observed under light microscopy, easy to control.

Characterization of enriched CD34+ cells from healthy volunteers and those from patients treated with chemotherapy plus granulocyte colony-stimulating factor (G-CSF).

CD34+ cells were enriched, using a panning method, from peripheral blood (PB) and bone marrow (BM) of healthy volunteers and of patients treated with chemotherapy plus granulocyte colony-stimulating factor (G-CSF). In healthy volunteers, PB CD34+ cells expressed CD33 and CD13 at a higher frequency than BM CD34+ cells, and PB CD34+ cells contained a greater number of burst-forming units-erythroid (BFU-E) than colony-forming units granulocyte-macrophage (CFU-GM). Administration of G-CSF to healthy volunteers induced a marked increase in the number of PB CD34+ cells, although the proportions of those expressing CD33, CD13, and c-kit among these cells as well as colony-forming ability were not changed before and after G-CSF administration. There were no significant differences in surface antigens on PB CD34+ cells between healthy volunteers and patients after chemotherapy plus G-CSF, except for low expression of c-kit in the PB of patients. However, PB CD34+ cells from patients contained almost the same number of CFU-GM as BFU-E. These results indicate that there were clear differences in the features of CD34+ cells from BM and from PB, and between healthy volunteers and patients after chemotherapy plus G-CSF. Enriched CD34+ cells are useful for analyzing the characteristics of hematopoietic progenitor cells, and such analysis may predict the usefulness of autologous or allogeneic peripheral blood stem cell transplantation.

Interleukin-3 and interleukin-7 are alternative growth factors for the same B-cell precursors in the mouse.

Clones and lines of precursor (pre) B cells can be established by limiting dilutions of unseparated cell suspensions of fetal liver or bone marrow on stromal cells in the presence of interleukin (IL)-7. When IL-3 is used instead of IL-7, cultures are regularly overgrown by different precursor cells of the myeloid lineage, as well as by adherent cells that inhibit pre-B-cell expansion. However, in the presence of either IL-7 or IL-3, clones of pre-B cells can be established on stroma cells at frequencies near one in one when the cultures are initiated with cell sorter purified CD45RO (B220)+/c-kit+ fetal liver or bone marrow derived pre-B cells. Clones grown on stromal cells in the presence of IL-7 can be regrown in IL-3, and vice versa. Pre-B cells that proliferate on stromal cells in the presence of IL-7 or IL-3 have the same phenotype, ie, are B220+ c-kit+, CD43+, and surrogate light chain+. Removal of the growth factors (IL-7, respectively IL-3) from the cultures results in differentiation to surface immunoglobulin (slg) positive, c-kit-, CD43-, surrogate light chain- B cells, a fraction of which is lipopolysaccharide (LPS) responsive as shown by IgM secretion. These results show that IL-7 and IL-3 stimulate largely overlapping populations of precursor B cells from bone marrow to proliferate for long periods of time in the presence of stromal cells. Thus, IL-7 and IL-3 are alternative growth factors for the same pre-BI cell.

The Wsh and Ph mutations affect the c-kit expression profile: c-kit misexpression in embryogenesis impairs melanogenesis in Wsh and Ph mutant mice.

The receptor tyrosine kinases (RTKs) c-kit and platelet-derived growth factor receptor alpha chain (PDG-FRa) are encoded at the white spotting (W) and patch (Ph) loci on mouse chromosome 5. While W mutations affect melanogenesis, gametogenesis, and hematopoiesis, the Ph mutation affects melanogenesis and causes early lethality in homozygotes. W-sash (Wsh) is an expression mutation and blocks c-kit expression in certain cell types and enhances c-kit expression in others, including at sites important for early melanogenesis. We have determined the effect of Ph on c-kit expression during embryogenesis in Ph heterozygotes. Immunohistochemical analysis revealed enhanced c-kit expression in several cell types, including sites important for early melanogenesis. We propose that in both Wsh and Ph mutant mice c-kit misexpression affects early melanogenesis and is responsible for the pigment deficiency. Moreover, we have defined the organization of the RTKs in the W/Ph region on chromosome 5 and characterized the Wsh mutation by using pulsed-field gel electrophoresis. Whereas the order of the RTK genes was determined as Pdgfra-c-kit-flk1, analysis of the Wsh mutation revealed that the c-kit and Pdgfra genes are unlinked in Wsh, presumably because of an inversion of a small segment of chromosome 5. The Ph mutation consists of a deletion including Pdgfra and the 3' deletion endpoint of Ph lies between Pdgfra and c-kit. Therefore, positive 5' upstream elements controlling c-kit expression in mast cells and some other cell types are affected by the Wsh mutation and negative elements are affected by both the Wsh and the Ph mutation.

Transformation of thyroid epithelium is associated with loss of c-kit receptor.

The receptor for the stem cell factor encoded by the c-kit proto-oncogene is expressed by a number of epithelial cells including thyrocytes. Since malignant transformation may be associated with loss of this receptor (melanoma and breast cancer), we have analyzed its expression in benign (38 cases) and malignant (31 cases) thyroid lesions. While low levels of c-kit are expressed in normal thyroids and in 60% of benign lesions, the receptor is undetectable in 60 and 90% of the follicular and papillary carcinomas, respectively. Northern blot analysis from surgical specimens of carcinomas and from carcinoma cell lines has demonstrated a lack of specific c-kit transcripts. These findings indicate that the c-kit receptor may be involved in the growth control of thyroid epithelium and that this function may be lost following malignant transformation.