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Σκοπός. Η παρούσα μελέτη σχεδιάστηκε με σκοπό τη διερεύνηση: α) της επίδρασης της νευροτενσίνης (ΝΤ), της εκλυτικής ορμόνης της κορτικοτροπίνης (CRH) και της ιντερλευκίνης-33 (IL-33) στον τρόπο με τον οποίο τα ανθρώπινα καλλιεργούμενα μαστοκυττάρα απελευθερώνουν ουσίες, όταν καλλιεργηθούν σε υλικό εμπλουτισμένο ή όχι με ιντερλευκίνη-4 (IL-4). Επίσης, β) την επίδραση των παραπάνω ουσιών και των συνδυασμών τους στη γονιδιακή έκφραση, ενδοκυττάρια σύνθεση και εξωκυττάρια απελευθέρωση της ιντερλευκίνης-31 (IL-31) από τα διεγερμένα μαστοκύτταρα όταν καλλιεργούνται σε υλικό εμπλουτισμένο ή όχι με ΙL-4 και ευαισθητοποιηθούν ή όχι με IgE/anti-IgE. Τελευταίος στόχος της παρούσας μελέτης αποτέλεσε γ) η μέτρηση των επιπέδων της NT, της CRH και της IL-33 στον ορό ασθενών με δερματική μαστοκυττάρωση (ΔΜ).Χαρακτηριστικά κυττάρων και ατόμων τα οποία μελετήθηκαν και μέθοδοι. Για την παρούσα μελέτη χρησιμοποιήθηκε κυτταρική σειρά ανθρώπινων λευχαιμικών μαστοκυττάρων (human LAD2 cells), η οποία χορηγήθηκε δωρεάν από το Δρ Kirshenbaum του Εθνικού Ινστιτούτου Υγείας (National Institute of Health,ΝΙΗ, Bethesda, MD, USA), από ασθενή ο οποίος έπασχε από λευχαιμία της σειράς των μαστοκυττάρων. Τα LAD2 καλλιεργήθηκαν σε ειδικές καλλιεργητικές φλάσκες, όπου τοποθετούνταν σε συγκέντρωση 105/ml θρεπτικού υλικού (StemPro-34 serum-free medium, Invitrogen, Carlsbad, CA, USA). Στην καλλιέργεια προστίθεντο 100 ng/ml ανασυνδυασμένου ανθρώπινου παράγοντα βλαστικών κυττάρων (recombinant human stem cell factor, rhSCF, Biovitrum, Sweden), 1% πενικιλλίνη, 1% στρεπτομυκίνη και 1% γλουταμίνη. Στις συνθήκες στις οποίες μελετήθηκε η συμπεριφορά των LAD2 όταν αυτά ευαισθητοποιήθηκαν με recombinant human IL-4, (rhIL-4, R&D systems, Mineapolis, MN) ή/και IgE, το καλλιεργούμενο θρεπτικό τους υλικού εμπλουτίστηκε με 100 ng/ml IL-4 για 2 εβδομάδες ή/και με 1 μg/ml IgE (human IgE, EMD Millipore, San Diego, CA) για 12 ώρες αντίστοιχα, προτού ξεπλυθούν με το αντίστοιχο ανά κατάσταση καλλιεργούμενο θρεπτικό υλικό και διεγερθούν για 24 ώρες με την ανάλογη ανά συνθήκη ουσία. Οι ουσίες, οι οποίες χρησιμοποιήθηκαν για την 24ωρη διέγερση των LAD2, ήταν η CRH 10 μΜ (Sigma-Aldrich, St Louis, MO), η ΝΤ 1 μM (Sigma-Aldrich, St Louis, MO), η IL-33 10 ng/ml, (R&D Sytsems, Minneapolis, MN), το anti-IgE 1 μg/ml, (Life Technologies, Carlsbad, CA) και η SP 2μΜ, (Sigma-Aldrich, St Louis, MO) καθώς και συνδυασμός αυτών. Οι τελικές συγκεντρώσεις στις οποίες χρησιμοποιήθηκαν οι τελευταίες ουσίες έγιναν στο αντίστοιχο για κάθε συνθήκη καλλιεργούμενο θρεπτικό υλικό, προτού χρησιμοποιηθούν, ενώ τα LAD2 αναπτύχθηκαν σε επωαστικό θάλαμο συγκέντρωσης 5% CO2 στους 37οC και χρησιμοποιήθηκαν κατά τη λογαριθμική φάση ανάπτυξής τους. Τα διεγερμένα ή μη με τις προαναφερόμενες ουσίες και ευαισθητοποίημένα ή όχι με IgE/anti-IgE μαστοκύτταρα μελετήθηκαν σε εμπλουτισμένο ή όχι με IL-4 καλλιεργητικό υλικό ως προς την ικανότητά τους να εκφράζουν γονιδιακά, να συνθέτουν ενδοκυττάρια και να απελευθερώνουν εξωκυττάρια τη νεοπεριγραφείσα ιντερλευκίνη-31 (IL-31), αφού πρώτα αποδείχθηκε αν οι μελετούμενες ουσίες αποκοκκιώνουν προσχηματισμένες ουσίες των μαστοκυττάρων ή απελευθερώνουν νεοσυντιθέμενες ουσίες από αυτά.Επίσης, για τη μέτρηση των επιπέδων της NT, της CRH και της IL-33 στον ορό ασθενών με ΔΜ, συμμετείχαν 20 παιδιά, Καυκάσιας φυλής, μέσης ηλικίας 5 + 4 έτη με κηλιδοβλατιδώδη (n=5), διάχυτη ΔΜ (n=4) ή μονήρες δερματικό μαστοκυττάρωμα (n=11), τα οποία παρουσιάστηκαν στο Παιδοδερματολογικό Ιατρείο της Α’ Πανεπιστημιακής Δερματολογικής Κλινικής του Πανεπιστημίου Αθηνών με έδρα το νοσοκομείο Ανδρέας Συγγρός καθώς και στη Β’ Μονάδα Αλλεργιολογίας και Κλινικής Ανοσολογίας του νοσοκομείου Παίδων Παναγιώτη και Αγλαίας Κυριακού του Πανεπιστημίου Αθηνών. Οι υγιείς μάρτυρες ήταν 10 παιδιά, Καυκάσιας φυλής, μέσης ηλικίας 4.5 + 3 ετών (n= 10), τα οποία προέρχονταν από το Παιδοδερματολογικό Ιατρείο της Α’ Πανεπιστημιακής Δερματολογικής Κλινικής του Πανεπιστημίου Αθηνών με έδρα το νοσοκομείο Ανδρέας Συγγρός και τα οποία προσήλθαν για άλλες δερματολογικές παθήσεις. Στους κηδεμόνες τόσο των υγιών όσο και των πασχόντων από ΔΜ παιδιών έγινε εκτενής αναφορά σχετικά με το σκοπό και τα σημεία της μελέτης και στο τέλος όλοι οι κηδεμόνες υπέγραψαν έντυπο συγκατάθεσης σύμφωνα με τη Διακήρυξη των Αρχών του Helsinki, όπως επίσης, ο κάθε κηδεμόνας συμπλήρωσε ένα ερωτηματολόγιο, το οποίο αφορούσε στο ατομικό ιστορικό των πασχόντων από ΔΜ παιδιών. Για τους παραπάνω σκοπούς, ακολουθήθηκαν οι παρακάτω μέθοδοι: α) απελευθέρωση της β-εξοζαμινιδάσης (β-hex), ώστε να μελετηθεί ο τρόπος με τον οποίο τα μαστοκύτταρα απελευθερώνουν τις ουσίες τους υπό την επίδραση των μελετούμενων ουσιών, έχοντας ως μάρτυρα τη SP, β) η λύση των LAD2 μαστοκυττάρων μετά από τις παραπάνω διαφορετικές συνθήκες, με φωτοευαίσθητο διάλυμα, το οποίο αποτελούνταν από ρυθμιστικό διάλυμα (Buffer 10x, Cell Signaling Technology), PBS και PMSF (Cell Signaling Technology). Με αυτήν την τεχνική συλλέχθηκε, σε συνθήκες φωτοπροστασίας, το ενδοκυττάριο υλικό των μαστοκυττάρων, προκειμένου να μετρηθεί η ενδοκυττάρια σύνθεση και αποθήκευση της IL-31, γ) η μέθοδος της αντίστροφης μεταγραφής ακολουθούμενης από την ποσοτική αλυσιδωτή αντιδραση της πολυμεράσης (quantitave PCR, qPCR), ώστε να μελετηθεί η δυνατότητα των καλλιεργούμενων μαστοκυττάρων αν εκφράζουν την IL-31 γονιδιακά, υπό την επίδραση των παραπάνω ουσιών και στις προαναφερόμενες συνθήκες και δ) η ενζυμοσύνδετη ανοσοπροσροφητική μέθοδος (Enzyme-Linked Immunosorbent Assay, ELISA), προκειμένου να μελετηθεί η ενδoκυττάρια σύνθεση και η εξωκυττάρια απελευθέρωση της IL-31 (human IL-31, R&D Systems, Minneapolis, MN) από τα μαστοκύτταρα κάτω από όλες τις προαναφερόμενες συνθήκες. Επίσης, η ELISA χρησιμοποιήθηκε και για τη μέτρηση των επιπέδων της NT (human Neurotensin, NT ELISA kit, CSB-E09144h, Cusabio), της CRH (human corticotropin releasing hormone, CRH ELISA Kit, CSB-E06872h, Cusabio) και της IL-33 (human IL-33 ELISA Kit, EA 100585, Origene) στον ορό τόσο πασχόντων από ΔΜ όσο και ανάλογων χαρακτηριστικών μαρτύρων.Αποτελέσματα. Τα μαστοκύτταρα εκφράζουν γονιδιακά, συνθέτουν ενδοκυττάρια και απελευθερώνουν τη νεοσυντιθέμενη IL-31 όταν σε αυτά επιδράσει η IL-33 μεμονωμένα, αλλά όχι η NT, η CRH και η SP. Ωστόσο, σε συνδιέγερση της NT ή της CRH ή της SP με την IL-33, και ιδιαίτερα όταν συνδιεγερθεί η SP με την IL-33, οι παραπάνω δραστηριότητες των μαστοκυττάρων σχετικά με την IL-31 παρατηρούνατι αυξημένες. Η γονιδιακή έκφραση, η ενδοκυττάρια σύνθεση και η εξωκυττάρια απελευθέρωση της IL-31 από τα μαστοκύτταρα ευνοείται εξαιρετικά όταν το καλλιεργητικό υλικό εμπλουτιστεί με την IL-4 ή όταν τα μαστοκύτταρα ευαισθητοποιηθούν με IgE και έπειτα διεγερθούν με anti-IgE. Αυτά τα αποτελέσματα υπεραυξάνονται, όταν τα ευαισθητοποιημένα με IgE και συνδιεγερμένα με anti-IgE μαστοκύτταρα καλλιεργηθούν, ταυτόχρονα, και σε καλλιεργητικό υλικό εμπλουτισμένο με IL-4, ξανά με ιδιαίτερα αυξημένα αποτελέσματα στη συνθήκη κατά την οποία η IL-33 αλληλεπιδράσει με την SP. Υποστηρίξαμε ότι τα μαστοκύτταρα δεν αποκοκκιώνουν την IL-31, λόγω του ότι στα πειράματα αποδείχθηκε ότι η NT, η CRH και η IL-33 δεν προκαλούν την αποκοκκίωση της γνωστής προσχηματισμένης ουσίας των μαστοκυττάρων, της β-εξοζαμινιδάσης, ενώ αντίθετα η SP προκαλεί την αποκοκκίωσή της τελευταίας. Στον ορό των πασχόντων από ΔΜ δεν παρατηρήθηκαν αυξημένα τα επίπεδα της NT, της CRH και της IL-33, συγκριτικά με τους μάρτυρες.Συμπεράσματα. Η επιδείνωση τόσο ως προς τη συμπτωματολογία φλεγμονωδών, αλλεργικών και αυτοανόσων νοσημάτων, με κυριότερο το σύμπτωμα του κνησμού, όσο και ως προς την πρόγνωσή τους, και κυρίως κάτω από καταστάσεις συναισθηματικού και σωματικού στρες, μπορεί να εξηγηθεί με την αύξηση των επιπέδων της IL-31 κάτω από την επίδραση την οποία δέχονται τα μαστοκύτταρα από το περιβάλλον τους, στο οποίο η IL-33, η CRH, η NT και η SP είναι αυξημένα στα παραπάνω νοσήματα, σύμφωνα με τη βιβλιογραφία. Τα μαστοκύτταρα διαδραματίζουν ουσιαστικό ρόλο στις προαναφερόμενες καταστάσεις, καθώς μπορούν να επάγουν και να ενισχύουν τόσο την ενδογενή όσο και την επίκτητη ανοσία, μέσω των πολλαπλών και ποικίλλων υποδοχέων που διαθέτουν και τα καθιστούν ικανά ως κύτταρα να συμμετέχουν, μεταξύ άλλων, και στη στρατολόγηση άλλων κυττάρων. Με τη στρατολόγηση, επιπλέον, κυττάρων απελευθερώνονται περισσότεροι μεσολαβητές και επομένως, συμβαίνουν περαιτέρω αλληλεπιδράσεις με τα μαστοκύτταρα, ανοσολογικά ή μη μεσολαβούμενες. Η μελέτη μας είναι μοναδική, γιατί είναι η πρώτη φορά που αποδεικνύεται ότι η IL-31, νεοπεριγραφείσα βασική κυτταροκίνη σε φλεγμονώδεις, αλλεργικές και αυτοάνοσες καταστάσεις, όπως προκύπτει από τη βιβλιογραφία, εκφράζεται γονιδιακά, συντίθεται ενδοκυττάρια και απελευθερώνεται εξωκυττάρια από τα μαστοκύτταρα όταν σε αυτά επιδράσουν ουσίες, όπως η IL-33, η CRH και η NT. Οι παραπάνω δράσεις των μαστοκυττάρων ως προς την IL-31 ενισχύονται όταν τα μαστοκύτταρα απαντήσουν με IgE-μεσολαβούμενους μηχανισμούς ή στην παρουσία της IL-4, αλλά μεγιστοποιούνται όταν τα μαστοκύτταρα απαντήσουν με IgE-μεσολαβούμενο μηχανισμό σε περιβάλλον στο οποίο, την ίδια στιγμή, υπάρχει η IL-4 και το άτομο φορτίζεται συναισθηματικά ή σωματικά. Η IL-4, η οποία μπορεί να απελευθερωθεί από τα μαστοκύτταρα, διεγείρει την επιπλέον παραγωγή των λευκοτριενιών από αυτά, με θετική ανατροφοδότηση και επομένως την περαιτέρω αντιδραστική υπερπλασία των μαστοκυττάρων. αλλά, επίσης, προωθεί τη μεγαλύτερη παραγωγή της IgE, πολλαπλασιάζοντας τα όποια αποτελέσματα των μαστοκυττάρων. H IL-4, όμως, απελευθερώνεται και από τα στρατολογημένα λευκά κύτταρα, μιας και τα Th2 κύτταρα θεωρούνται η βασική πηγή παραγωγής της IL-4 βιβλιογραφικά. Επίσης, στη μελέτη μας αποδείχθηκε, για πρώτη φορά στη βιβλιογραφία, ότι τα επίπεδα της NT, της CRH και της IL-33 στον ορό πασχόντων από ΔΜ δεν παρουσιάζονται αυξημένα συγκριτικά με αυτά των μαρτύρων. Ωστόσο, από τη βιβλιογραφία προκύπτει ότι και τα επίπεδα της IL-31 στον ορό ατόμων με ΔΜ δε μετρώνται αυξημένα, αποδεικνύοντας επιπρόσθετα τη στενή συσχέτιση μεταξύ των μελετούμενων ουσιών, NT, CRH, IL-33 με την IL-31, σε καταστάσεις στις οποίες τα μαστοκύτταρα κατέχουν κεντρικό ρόλο, όπως στη ΔΜ. Παρόλα αυτά τα επίπεδα των μελετούμενων ουσιών σε άτομα με συστηματική μαστοκυττάρωση θα μπορούσαν να είναι διαφορετικά, εφόσον, σύμφωνα με τη βιβλιογραφία, τα επίπεδα της IL-31 είναι αυξημένα στον ορό αυτών. Τα αποτελέσματά μας δείχνουν ότι τα μαστοκύτταρα είναι πολυδύναμα και ενεργοποιούνται μέγιστα από συνδυασμό ερεθισμάτων. Είναι, επομένως, απαραίτητο να βρεθεί αποτελεσματικός τρόπος για να ανασταλλούν η σύνθεση και έκκριση προφλεγμονωδών διαβιβαστών. Πρόσφατο παράδειγμα αποτελεί η πολλά υποσχόμενη θεραπεία αναστολέων της IL-4 (ντουπιλουμάμπη), σε νοσήματα, όπως η ατοπική δερματίτιδα, τα οποία αφενός χαρακτήριζονται από μεγάλο αριθμό μαστοκυττάρων και αφετέρου η IL-31 αποτελεί σε αυτά τόσο προγνωστικό δείκτη τους, όσο και δείκτη έντασης του κνησμού που τα χαρακτηρίζει.

The pharmacological importance of recombinant human stem cell factor (rhSCF) has increased the demand to establish effective and large-scale production and purification processes. A good source of bioactive recombinant protein with capability of being scaled-up without losing activity has always been a challenge. The objectives of the study were the rapid and efficient pilot-scale expression and purification of rhSCF. The gene encoding stem cell factor (SCF) was cloned into pBV220 and transformed into Escherichia coli. The recombinant SCF was expressed and isolated using a procedure consisting of isolation of inclusion bodies (IBs), denaturation, and refolding followed by chromatographic steps toward purification. The yield of rhSCF reached 835.6 g/20 L, and the expression levels of rhSCF were about 33.9% of the total E. coli protein content. rhSCF was purified by isolation of IBs, denaturation, and refolding, followed by SP-Sepharose chromatography, Source 30 reversed-phase chromatography, and Q-Sepharose chromatography. This procedure was developed to isolate 5.5 g of rhSCF (99.5% purity) with specific activity at 0.96 × 106 IU/mg, endotoxin levels of pyrogen at 1.0 EU/mg, and bacterial DNA at 10 ng/mg. Pilot-scale fermentations and purifications were set up for the production of rhSCF that can be upscaled for industry.

Purpose Tunneling nanotubes (TNTs) are extremely thin (50–200 nm), actin-containing cell surface protrusions up to a few microns in length that can develop rapidly and connect various cell types. Mast cells (MCs) are unique immunomodulatory cells that are found perivascularly in all tissues. MCs communicate with many other cell types through the release of inflammatory, neurosensitizing, and vasoactive molecules, through which they are involved in the pathogenesis of many inflammatory diseases. We, therefore, investigated the possibility that MCs may form TNTs and communicate among themselves and with glioblastoma cells. Methods Laboratory Allergic Diseases (LAD)-2 human MCs were cultured in medium supplemented with 100 U/mL penicillin/streptomycin and 100 ng/mL recombinant human stem cell factor. They were incubated with 20 nmol/L deep red probe for 20 minutes and 50 nmol/L green probe for 30 minutes. Human glioblastoma cells were incubated with 20 nmol/L deep red probe only, moved to glass-bottom culture dishes, and observed using a substance P 2 confocal microscope. LAD2 MCs were stimulated with 2 µmol/L of the peptide substance P for 30 minutes at 37oC. Confocal digital images were processed. Findings MCs can rapidly (within 5 minutes) form TNTs, which appear to transport mitochondrial and secretory granule particles among themselves and with cocultured glioblastoma cells. Implications MCs are now accepted as having an important role in many diseases with an inflammatory component. TNTs provide a rapid and direct way for MCs to "alarm" other cell types with specificity not present when mediators are secreted into the tissue microenvironment. The identification of TNTs and their cargo could be important in the diagnosis and possible treatment of many inflammatory diseases.

Claudin-3 is a major protein of tight junctions (TJs) in the intestinal epithelium and is critical for maintaining cell-cell adhesion, barrier function, and epithelium polarity. Recent studies have shown high claudin-3 levels in several solid tumors, but the regulation mechanism of claudin-3 expression remains poorly understood. In the present study, colorectal cancer (CRC) tissues, HT-29 and DLD-1 CRC cell lines, CRC murine model (C57BL/6 mice) and c-kit loss-of-function mutant mice were used. We demonstrated that elevated claudin-3 levels were positively correlated with highly expressed c-kit in CRC tissues based upon analysis of protein expression. In vitro, claudin-3 expression was clearly increased in CRC cells by overexpressed c-kit or stimulated by exogenous recombinant human stem cell factor (rhSCF), while significantly decreased by the treatment with c-kit or c-Jun N-terminal kinase (JNK) inhibitors. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay showed that SCF/c-kit signaling significantly promoted activator protein-1 (AP-1) binding with CLDN-3 promoter and enhanced its transcription activity. Furthermore, decreased expression of claudin-3 was obtained in the colonic epithelium from the c-Kit loss-of-function mutant mice. In conclusion, SCF/c-kit-JNK/AP-1 signaling pathway significantly promoted claudin-3 expression in colonic epithelium and CRC, which could contribute to epithelial barrier function maintenance and to CRC development.

A method was developed to increase the recovery of recombinant human stem cell factor (rhSCF) from inclusion bodies using high performance hydrophobic interaction chromatography (HPHIC). The target protein was first solubilized in 8.0 mol/L urea solution, and was purified and refolded simultaneously by HPHIC with different chromatographic cakes. Experimental conditions, such as the ligand structures of stationary phase and the composition of mobile phase, were optimized. Under the optimal conditions, high mass recoveries and specific activities of rhSCF were acquired, the purities of rhSCF were above 95.5%, and the mass recoveries of rhSCF were above 49.6%. The final product was also verified as monomer by size exclusion chromatography and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). These results provided further evidence that HPHIC is an effective tool in the refolding and purification of recombinant proteins.

The mobilization and collection of hematopoietic stem and progenitor cells (HSPC) is central to many potentially curative treatments for cancer and some non-malignant conditions. Recombinant human cytokines have been the mainstay of therapeutic HSPC mobilization, particularly G-CSF. Even with currently used mobilization regimens using G-CSF with or without chemotherapy, up to 60% of patients can fail to mobilize enough HSPC for a transplant procedure. Recombinant human stem cell factor (ancestim, rhSCF, Stemgen) is another such cytokine, which has shown promising synergy when used in combination with G-CSF for HSPC mobilization. It provides a useful second-line option for prior failed-mobilizer patients and those who are anticipated to mobilize poorly due to recognised risk factors. It may also have utility in promoting bone marrow recovery in cases of refractory bone marrow failure such as aplastic anaemia and prolonged non-engraftment after allogeneic HSPC transplantation. We review the literature supporting the use of rhSCF in the context of HSPC mobilization and bone marrow failure. The emergence of other novel agents for HSPC mobilization such as plerixafor (AMD3100, Mozobil) will further demarcate the role of Ancestim as a second- or third-line mobilization agent for the mobilization-refractory patient.

We here present a new method for the expression and purification of recombinant human stem cell factor (rhSCF(164)) in endotoxin-free ClearColi(®) BL21(DE3) cells harboring codon-optimized Profinity eXact™-tagged hSCF cDNA. Previously, we demonstrated that co-expression with thioredoxin increased the solubility of rhSCF in Escherichia coli BL21(DE3), and addition of l-arginine enhanced chromatography performance by removing the endotoxin-masked surface of rhSCF. Initially, we tried to express rhSCF in an endotoxin-free strain using a thioredoxin co-expression system, which resulted in significantly lower expression, possibly due to the stress imposed by overexpressed thioredoxin or antibiotics susceptibility. Therefore, we developed a new expression system without thioredoxin. External redox coupling was tested using persulfides such as glutathione persulfide or cysteine persulfide for the in vivo-folding of hSCF in the cytoplasm. Persulfides improved the protein solubility by accelerating disulfide-exchange reactions for incorrectdisulfides during folding in E. coli. Furthermore, the persulfides enhanced the expression level, likely due to upregulation of the enzymatic activity of T7 RNA polymerase. The recombinant protein was purified via affinity chromatography followed by cleavage with sodium fluoride, resulting in complete proteolytic removal of the N-terminal tag. The endotoxin-free fusion protein from ClearColi(®) BL21(DE3) could bind to the resin in the standard protocol using sodium phosphate (pH 7.2). Furthermore, purified rhSCF enhanced the proliferation and maturation of the human mast cell line LAD2. Thus, we conclude that use of the protein expression system employing E. coli by disulfide shuffling with persulfide addition could be a very useful method for efficient protein production.

Ex vivo expanded peripheral blood progenitor cells (PBPC) have been shown to provide rapid neutrophil engraftment, and in some patients, to eliminate neutropenia after transplantation to support high-dose chemotherapy. However, the effect of expansion culture on stem cell content and potential loss of stem cells caused by induction of differentiation remains a concern. We have transplanted 21 patients with breast cancer with expanded autologous PBPC, with 11 patients receiving expanded PBPC as their sole hematopoietic cell source. In these studies, the CD34+ cells were selected and cultured for 10 days in defined media containing 100 ng/mL each of recombinant human stem cell factor (rhSCF), recombinant human granulocyte colony stimulating factor (rhG-CSF), and recombinant human megakaryocyte growth and developmental factor (rhMGDF) in 1-liter Teflon bags at 20,000 to 50,000 cells/mL. After culture the cells were washed and reinfused after high-dose chemotherapy followed by daily administration of rhG-CSF....

CD34(+) progenitor cells carrying human herpesvirus-8, Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV), have been described in the peripheral blood of AIDS patients suffering from Kaposi's sarcoma (KS). In this study, we investigated the influence of HHV-8 on the differentiation of CD34(+) progenitor cells. Native CD34(+) cells derived from cord blood could be infected by a laboratory strain of HHV-8, as shown by immunofluorescence staining and polymerase chain reaction, but no significant initial maturation/differentiation effects were observed. In addition, these infected cells were differentiated into immature and mature dendritic cells (DCs) using cytokine induction with recombinant human granulocyte-macrophage colony-stimulating factor (rhGm-CSF), recombinant human tumor necrosis factor (rhTNF-alpha) and recombinant human stem cell factor (rhSCF). Double immunofluorescence and flow cytometry studies demonstrated that virus infection did not impair the development of immature and mature DC populations. Subsequently, the immunostimulating capacity of DC populations was tested in a mixed lymphocyte reaction using allogeneic T-cells. The HHV-8-infected CD34(+) progenitor cell-derived mature DC population showed a significantly enhanced antigen-presenting capacity, compared to non-infected DCs, which was not observed with the immature DCs. This suggests stimulation of DC function by HHV-8 infection. Because there are only a small percentage of HHV-8-positive DCs in the preparations and because it is not clear whether infection is abortive or productive to some extent, this seems to be most likely due to an indirect viral effect.

Summary Background Mast cells (MCs) arise from haematopoietic stem cells. We have recently reported that CD34+ progenitors derived from human bone marrow (BM) develop into tryptase+, chymase+ MCs when cultured in the presence of recombinant human stem cell factor (rhSCF) and recombinant human IL-6 (rhIL-6). In an experiment for the expression of chymase during differentiation, chymase+ cells were detected in human BM, but tryptase+ cells were not found. Objective The purpose of this study was to show the appearance of chymase+ cells in CD34+ cells with an origin different from MC differentiation. Methods CD34+ cells from human BM were sorted with anti-CD117 monoclonal antibody (mAb), and cytospins of CD34+, CD34+CD117+, or CD34+CD117− were prepared. These cells were cultured with rhSCF+rhIL-6 for 12 weeks. Some of the cells were subjected to either histological stain with Wright–Giemsa or immunocytochemistry with anti-chymase mAb. Real-time RT-PCR was also performed to compare the transcriptional level of chymase from each cell preparation. Results Chymase was expressed in CD34+ cells as well as human MCs by immunocytochemistry. Substantial CD34+CD117− cells, but not CD34+CD117+ cells, were stained immunocytochemically with anti-chymase mAb. For 1 week culture with rhSCF+rhIL-6, no cells expressed chymase in any preparation. Real-time RT-PCR revealed positivity for chymase mRNA in CD34+ cells, but it reduced at 1 week of culture, and increased as cells developed into MCs. Chymase mRNA in CD34+CD117+ cells was negligible compared with that in CD34+CD117−. Tryptase mRNA was below the detectable level in CD34+ cells, and increased along with MC differentiation. After 12 weeks of culture, CD34+CD117+ developed predominantly into MCs, whereas CD34+CD117− developed into monocytes/macrophages. Conclusion Our findings suggested that chymase is present not only in MCs but also in CD34+CD117− BM progenitors, but that its origin is different from the MC lineage.

Umbilical cord blood (UCB) provides a rich source of stem cells for transplantation after myeloablative therapy. One major disadvantage of UCB transplantation is delayed platelet engraftment. We propose to hasten platelet engraftment by expanding the number of megakaryocyte (MK) precursors (CD34/CD41 cells) through cytokine stimulation within a closed, pre-clinical liquid culture system. Clinical engraftment data suggest a 5- to 10-fold increase in MK precursors in a UCB unit can accelerate platelet engraftment, so this was our goal. Thirteen UCB samples from full-term births were Ficoll-separated and frozen for subsequent use. On thawing, the mononuclear cell population was positively selected for CD34+ expression. The cells were cultured in gas-permeable Teflon-coated bags in serum-free medium containing the following cytokines: recombinant human interleukin-3, recombinant human Flt3 ligand, recombinant human stem cell factor, and recombinant human thrombopoietin. MK lineage cell expansion was assessed using mononuclear cell count and flow cytometry (CD34/41, CD41, CD34/61, and CD61 expression) on days 7, 11, and 14. Optimal expansion of CD34/41 and CD41 cells was observed at day 11, with a median 6-fold and 33-fold increase in the starting cell doses, respectively. CD34/61 and CD61 cell expansion at day 11 was 7-fold and 14-fold, respectively. MK precursors can be successfully expanded from CD34+ UCB cells in a closed liquid culture system using interleukin-3, recombinant human Flt3 ligand, recombinant human stem cell factor, and recombinant human thrombopoietin to a level that should have a clinical impact in the transplantation setting. Our ex vivo expansion technique needs to be further optimized before it can be used in a pilot UCB transplantation trial. © 2003 American Society for Blood and Marrow Transplantation Biology of Blood and Marrow Transplantation 9:151-156 (2003)

This study assessed the ability of recombinant human stem cell factor (rHuSCF) to mobilize stem cells in 44 patients who had failed a prior mobilization (CD34(+) yield 0.5-1.9 x 10(6)/kg BW) with filgrastim-alone or chemotherapy-plus-filgrastim. The same mobilization regimen was used with the addition of rHuSCF. In the filgrastim-alone group (n=13), rHuSCF 20 microg/kg was started 3 days before filgrastim and continued for the duration of filgrastim. In the chemotherapy-plus-filgrastim group (n=31), rHuSCF 20 microg/kg/day plus filgrastim 5-10 microg/kg/day were administered concurrently. Leukaphereses were continued to a maximum of four procedures or a target of >or=3 x 10(6) CD34(+) cells/kg. In both groups, CD34(+) yield (x 10(6)/kg BW) of the study mobilization was higher than that of the prior mobilization (median: 2.42 vs 0.84 P=0.002 and 1.64 vs 0.99 P=

Background The combination of recombinant human stem cell factor (rhSCF), rh interleukin (IL)-6 and rhIL-10 was reported to be optimal for mast cell development from cord blood progenitors and to induce chymase expression in all such mast cells earlier in their development than tryptase. Objective The effects of rhIL-6 and rhIL-10 in various combinations on the rhSCF-dependent development of human mast cells from fetal liver progenitors were examined in serum-free media. Methods Dispersed fetal liver cells were cultured in serum-free AIM-V medium with rhSCF alone, or with combinations of rhIL-6 and rhIL-10. Tryptase and chymase expression, surface Kit expression, metachromasia with toluidine blue and apoptosis were measured. Results Neither rhIL-6 nor rhIL-10 nor the two interleukins together, when included from day 0 of culture, affected the number or protease phenotype of mast cells at 1 or 3 weeks. Expression of tryptase paralleled the appearance of metachromasia and surface Kit, both of which preceded chymase expression, regardless whether a rabbit polyclonal or mouse monoclonal anti-chymase antibody preparation was used. On the other hand, rhIL-6 markedly attenuated baseline levels of apoptosis in the presence of rhSCF as well as apoptosis occurring after withdrawal of rhSCF, whereas rhIL-10 had no effect. Conclusion RhIL-6 protected fetal liver-derived mast cells from apoptosis, particularly after withdrawal of rhSCF, but neither rhIL-6 nor rhIL-10 nor the combination of these interleukins affected the numbers or protease phenotype of these mast cells.

Genetic modification of hematopoietic stem cells often results in the expression of foreign proteins in pluripotent progenitor cells and their progeny. However, the potential for products of foreign genes introduced into hematopoietic stem cells to induce host immune responses is not well understood. Gene marking and induction of immune responses to enhanced green fluorescent protein (eGFP) were examined in rhesus macaques that underwent nonmyeloablative irradiation followed by infusions of CD34+ bone marrow cells transduced with a retroviral vector expressing eGFP. CD34+ cells were obtained from untreated animals or from animals treated with recombinant human granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and recombinant human stem cell factor. Levels of eGFP-expressing cells detected by flow cytometry peaked at 0.1% to 0.5% of all leukocytes 1 to 4 weeks after transplantation. Proviral DNA was detected in 0% to 17% of bone marrow–derived colony-forming units at periods of 5 to 18 weeks after transplantation. However, 5 of 6 animals studied demonstrated a vigorous eGFP-specific cytotoxic T lymphocyte (CTL) response that was associated with a loss of genetically modified cells in peripheral blood, as demonstrated by both flow cytometry and polymerase chain reaction. The eGFP-specific CTL responses were MHC-restricted, mediated by CD8+lymphocytes, and directed against multiple epitopes. eGFP-specific CTLs were able to efficiently lyse autologous CD34+ cells expressing eGFP. Antibody responses to eGFP were detected in 3 of 6 animals. These data document the potential for foreign proteins expressed in CD34+ hematopoietic cells and their progeny to induce antibody and CTL responses in the setting of a clinically applicable transplantation protocol.

BACKGROUND Methods for assessing engraftment efficiency have been explored in a primate xenogeneic model of in utero hematopoietic stem cell transplantation. METHODS Human peripheral blood stem cells (PBSC) were obtained by leukapheresis from a human male donor after 4 days of administration of recombinant human granulocyte-colony stimulating factor (5 microg/kg/ day). PBSC were enriched for the CD34+ population with and without T-cell depletion. The resulting mononuclear cells consisted of two cell populations, one that was stem cell enriched (0.83% CD3+ cells, 95% CD34+; group 1) and one that was stem cell enriched and T-cell depleted (

Mast cells have been regarded as one of the most important effector cells in IgE-dependent allergic response. Recently the heterogeneity of mast cells in localization and species have been recognized. However, whether anti-allergic drugs possess inhibitory effects on histamine release from human mast cells still remains uncertain. Therefore, in the present study, effects of anti-allergic drugs on histamine release from human mast cells, which were derived by the culture of cord blood cells with 80 ng/ml recombinant human stem cell factor and 50 ng/ml interleukin 6. The human cultured mast cells presented functional IgE receptors on their cell surfaces and were effectively stimulated to release histamine in dose-dependent and time-dependent manners of anti-IgE antibody. Anti-allergic drugs, such as azelastine, ketotifen, and emedastin, were able to inhibit histamine release from the human mast cells in dose-dependent manners. Immunosuppressive agent, cyclosporin A, and flavonoid, quercetin, also showed inhibitory effects on the histamine release from the human cultured mast cells.

We performed an ultrastructural analysis of 10 skin biopsy specimens that had been obtained from three women who were undergoing daily subcutaneous dosing with recombinant methionyl-human stem cell factor (rhSCF) as part of a phase I clinical trial. The biopsy specimens were obtained at sites of subcutaneous administration of rhSCF, within approximately 1 to 2 hours of rhSCF injection, and, at the same time, at contralateral control sites that had not been directly injected with rhSCF. We previously reported that subcutaneous dosing with rhSCF in these subjects induced the local development of a wheal and flare response, which was associated with evidence of mast cell degranulation, as well as a systemic increase in numbers of cutaneous mast cells. The present electron microscopic analysis revealed that all biopsies of swollen, erythematous rhSCF-injected sites exhibited anaphylactic degranulation of both mature and immature mast cells, an acute inflammatory response characterized by the migration of neutrophils, basophils (some of which exhibited evidence of piecemeal degranulation), and eosinophils through blood vessel walls into the perivascular and extravascular spaces, and edema and fibrin deposition within the interstitium. By contrast, the control biopsies contained no evidence of mast cell degranulation or acute inflammation. However, both control and rhSCF-injected sites exhibited mast cells that were undergoing granule building and maturation. Thus at the doses tested in these subjects, subcutaneous injection of rhSCF induced anaphylactic-type degranulation of dermal mast cells at the injection site, with an acute inflammatory response that was associated with the recruitment of granulocytes. By contrast, mast cells at sites distant from those directly injected with rhSCF exhibited no evidence of enhanced secretion.

We have developed a long-term culture system using the murine bone marrow stromal cells MS-5 to support the growth of progenitor B cells with CD34-, CD10+, CD19+, and cytoplasmic mu chain (C mu)-negative surface phenotype from human CD34+ cells purified from umbilical cord blood (CB). When 10(3) CD34+ cells/well were seeded on MS-5 stromal cells at the beginning of culture in the absence of exogenously added cytokines, progenitor B cells first appeared after 14 days, and the maximal cell production was achieved during the 6th week of culture. Intriguingly, the addition of recombinant human stem cell factor (rhSCF) and granulocyte colony-stimulating factor (rhG-CSF), but not rhIL-7, strikingly enhanced the growth of progenitor B cells from CB CD34+ population cultured on MS-5 stromal cells. The culture of progenitor B cells could be maintained until the 6th week of culture when some cells were revealed to have a C mu phenotype, and a small number of cells had immunoglobulin mu chain on their cell surface in the presence of both rhSCF and rhG-CSF. When CD34+ cells were cultured physically separated from the stromal layer by membrane, supportive effects of MS-5 stromal cells for the growth of progenitor B cells were not observed. These results suggest that the present culture system could generate progenitor B cells to proliferate from CB CD34+ cells, that some of these progenitor B cells could differentiate into immature B cells in conjunction with rhSCF and rhG-CSF, and that a species-cross-reactive membrane-bound factor(s), which stimulates early human B lymphopoiesis, may exist in MS-5 stromal cells. Further studies are required to investigate the mechanism how rhG-CSF acts on progenitor B cells to allow their proliferation and differentiation.

Stem cell factor (SCF ) has a major role in hematopoiesis and in the regulation of mast cell development and function. For example, recombinant human SCF (rhSCF ) can induce the development of human mast cells from precursor cells in vitro, stimulate mediator release from human skin mast cells in vitro, and promote both the development and functional activation of human skin mast cells in vivo. In the present study, we used a new ultrastructural enzyme-affinity method, employing diamine oxidase (DAO)-conjugated gold particles (DAO-gold), to detect histamine in skin biopsies obtained from patients with breast carcinomas who were receiving daily subcutaneous (SC) injections of rhSCF in a phase I study of this cytokine. We examined control biopsies obtained at sites remote from rhSCF injection as well as biopsies of rhSCF-injected skin that were obtained within 2 hours and 30 minutes of the SC injection of rhSCF at that site. The rhSCF-injected sites (which clinically exhibited a wheal-and-flare response), but not the control sites, contained mast cells undergoing regulated secretion by granule extrusion. The DAO-gold-affinity method detected histamine in electron-dense granules of mast cells in control and injected skin biopsies; however, the altered matrix of membrane-free, extruded mast cell granules was largely unreactive with DAO-gold. Notably, DAO-gold bound strongly to fibrin deposits and collagen fibers that were adjacent to degranulated mast cells. These findings represent the first morphologic evidence of histamine secretion by classical granule exocytosis in human mast cells in vivo.

Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (Ka) of 2-4 x 10(8) M-1, using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum. Based on the dimerization Ka, greater than 90% of the circulating SCF would be in the monomeric form. When 125I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10-100 ng/ml, consistent with the Ka determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer Ka values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH2CONH2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys165-Cys165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.

Recombinant human stem cell factor (rhSCF) induced histamine release from human skin and lung mast cells but had no effect on human basophil leukocytes. More importantly, rhSCF enhanced the release of histamine in response to IgE-crosslinking of human mast cells. This potentiation was observed at rhSCF concentrations which induced histamine release, and also at lower concentrations of the ligand which by themselves produced no effect. Very limited potentiation was observed with human basophil leukocytes. The enhancing effect of SCF was unique to IgE-dependent stimulation and when SCF was incubated with the neurotransmitter substance P and the calcium ionophore A23187, no augmentation of histamine release was observed with any of the cell types tested. These findings suggest that endogenous SCF may contribute to the regulation of mast cell function and may hence play a role in diverse allergic and inflammatory reactions.

The dose intensity of chemotherapy is an important factor in the treatment of patients with ovarian cancer, and a role for hemopoietic growth factors in accelerating bone marrow recovery after intensive chemotherapy has been established. A clinical trial has been proposed introducing recombinant human stem cell factor (rhSCF) to improve peripheral blood progenitor cell mobilization following chemotherapy for ovarian malignancy. In view of this a study to examine the expression of c-kit, the receptor for SCF, in ovarian tumors, and also the effect of rhSCF on the growth of ovarian tumor cell lines has been carried out. Of the 46 ovarian tumors examined immunohistochemically only one, a malignant teratoma, demonstrated positive c-kit expression. None of the short term or established ovarian carcinoma cell lines treated with rhSCF showed significant growth acceleration of adenocarcinoma cells. Similarly, none possessed c-kit receptors detected immunohistochemically.

Stem cell factor (SCF), also known as mast cell growth factor, kit ligand, and steel factor, is the ligand for the tyrosine kinase receptor (SCFR) that is encoded by the c-kit proto-oncogene. We analyzed the effects of recombinant human SCF (r-hSCF, 5-50 micrograms/kg/day, injected subcutaneously) on mast cells and melanocytes in a phase I study of 10 patients with advanced breast carcinoma. A wheal and flare reaction developed at each r-hSCF injection site; by electron microscopy, most dermal mast cells at these sites exhibited extensive, anaphylactic-type degranulation. A 14-d course of r-hSCF significantly increased dermal mast cell density at sites distant to those injected with the cytokine and also increased both urinary levels of the major histamine metabolite, methyl-histamine, and serum levels of mast cell alpha-tryptase. Five subjects developed areas of persistent hyperpigmentation at r-hSCF injection sites; by light microscopy, these sites exhibited markedly increased epidermal melanization and increased numbers of melanocytes. The demonstration that r-hSCF can promote both the hyperplasia and the functional activation of human mast cells and melanocytes in vivo has implications for our understanding of the role of endogenous SCF in health and disease. These findings also indicate that the interaction between SCF and its receptor represents a potential therapeutic target for regulating the numbers and functional activity of both mast cells and cutaneous melanocytes.

Distinct from the noncovalently linked recombinant human stem cell factor (rhSCF) dimer, we report here the isolation and identification of an SDS-nondissociable dimer produced during folding/oxidation of rhSCF. Experimental evidence using various cleavage strategies and analyses shows that the isolated dimer is composed of two rhSCF monomers covalently linked by four disulfide bonds. The cysteines are paired as in the noncovalently associated dimer except that all pairings are intermolecular rather than intramolecular. Other structural models, involving intertwining of intramolecular disulfide loops, are ruled out. The molecule behaves similarly to the noncovalently associated dimer during ion-exchange or gel permeation chromatography. However, the disulfide-linked dimer exhibits increased hydrophobicity in reverse-phase columns and in the native state does not undergo spontaneous dimer dissociation-association as seen for the noncovalent dimer. Spectroscopic analyses indicate that the disulfide-linked and noncovalently associated rhSCF dimers have grossly similar secondary and tertiary structures. In vitro, the disulfide-linked dimer exhibits approximately 3-fold higher biological activity in supporting growth of a hematopoietic cell line and stimulating hematopoietic cell colony formation from enriched human CD34+ cells. The molecule binds to the rhSCF receptor, Kit, with an efficiency only half that of the noncovalently associated dimer. Formation of intermolecular disulfides in the disulfide-linked dimer with retention of biological activity has implications for the three-dimensional structure of noncovalently held dimer and disulfide-linked dimer.

Thrombopoietin (TPO) is a recently discovered hematopoietic growth factor which stimulates the production and maturation of megakaryocytes. In this study, we used a modified MTT assay to examine the in vitro growth-stimulatory effects of recombinant human (rh) TPO and recombinant human stem cell factor (rhSCF) on eight small cell lung cancer (SCLC) cell lines and one leukemic cell line, CMK, with megakaryocytic characteristics. rhTPO did not reveal any stimulatory effects on all eight SCLC cell lines, while rhSCF demonstrated a modest growth-stimulatory effect on one etoposide-resistant SCLC cell line (H69/VP). The transcripts of c-mpl, the receptor of TPO, was not detected in all SCLC cell lines by RT-PCR analysis, while those of c-kit, the receptor of SCF, were detected in five of eight SCLC cell lines. Our data suggest that rhTPO does not promote the growth of SCLC cell lines and may be clinically applicable for patients with this disease. Moreover, rhSCF may cause adverse effects in part of the SCLC patients.

We have previously reported on the stimulation of clonal growth of a glioblastoma cell line by rhSCF (Berdel et al., Cancer Res 1992, 52, 3498–3502). Within an extensive screening programme of haematopoietic growth factor activity on malignant cells, the effects of rhSCF were further tested on the growth of 29 different human cell lines derived from a wide range of solid tumours, among them six lung cancers and five melanomas. RhSCF (0,1,10, 100 ng/ml) was tested in a human tumour cloning assay (HTCA) which reliably detects growth modulation of tumour cells by cytokines. Additionally, a tritiated thymidine uptake test was used. Growth of 27 of the 29 cell lines tested was not affected by rhSCF. However, growth of the small cell lung cancer (SCLC) cell line HTB 120 was slightly stimulated (1.5 fold that of controls), and that of the melanoma cell line Me Wowas stimulated up to 1.3-fold. This activity was eliminated dose-dependently by the tyrosine kinase inhibitor, genistein. We further analysed the cell lines for expression of the proto-oncogene C-KIT and its ligand SCF. All melanoma and lung cancer cell lines expressed SCF as assessed at the mRNA level. Northern blotting also revealed clear C-KIT mRNA expression in three melanoma (HAS, MeWo, SK-MEL-28), one NSCLC (HTB 53), and four SCLC cell lines (HTB 119, HTB 120, HTB 171, HTB 175). Furthermore, C-KIT protein expression was detected by flow cytometric analysis on the cell surface of MeWo, HTB 119 and HTB 120 cells. Our data indicate that SCF can be operative in growth modulation of non-haematopoietic malignant cells, especially SCLC and melanoma. However, our extensive screening of SCF/tumour cell interaction shows that this interaction is rare and makes potential hazards, such as tumour stimulation upon clinical use of rhSCF in conjunction with chemotherapy in cancer patients, unlikely for the majority of other tumour histologies.

An attempt was made to establish an SCID-hu murine model of long-term human hematopoiesis by coimplantation of a bone fragment and bone marrow (BM) cells from an adult human. The SCID-hu mice were treated with a cytokine mixture (recombinant human stem cell factor, interleukin-3, granulocyte/macrophage colony-stimulating factor, and granulocyte colony-stimulating factor) for 4 months and were then maintained for further 8 months under cytokine-free conditions. In the peripheral blood, spleen, and implanted bone fragments in the SCID-hu mice that had received both a bone fragment and BM cells, human CD59+ cells were detected 1 year after transplantation; however, they were not detectable in SCID-hu mice that had received either a bone fragment or BM cells only. Thus, implantation of both a bone fragment and BM cells appears to provide a model of long-term adult human hematopoiesis in SCID mice.

Bone marrow (BM) aspirate and biopsy specimens from seven female patients with advanced or metastatic breast cancer and preserved marrow function treated on a phase I trial of recombinant methionyl human stem cell factor (r-metHuSCF; SCF) were evaluated by immunohistochemical staining before and after treatment with SCF. Doses of SCF included 10 g/kg/day in 2 patients, 25 micrograms/kg/day in 2 patients, and 50 micrograms/kg/day in 3 patients administered as subcutaneous bolus injections for 14 days. Following treatment, bone marrow cellularity increased up to 1.6-fold (P = NS), with an increased frequency of promyelocytes (P < .002), but an unchanged relative frequency of other marrow hematopoietic cells. The mean relative frequency of BM CD34+ progenitor cells increased from 0.9% to 1.8% (P < .001). The mean proportion of BM cells stained by Ki-67/MIB 1 and PCNA/PC10, monoclonal antibodies (MoAb) recognizing proliferation-associated nuclear proteins, increased from 18.6% to 35.4% (P < .003) and from 32.4% to 49.4% (P < .01), respectively. Most of the Ki-67 and PCNA positive cells were represented by promyelocytes, proerythroblasts, and myeloblasts. SCF therapy was not associated with marrow fibrosis or increases in the number of macrophages. Peripheral white blood cell counts increased 1.3- to 3.6-fold following SCF. The mean absolute neutrophil counts increased from 3.9 x 10(9)/L (range 2.6-5.3) to 7.2 x 10(9)/L (range 4.7-12.3), and reticulocyte counts increased by a mean of 1.5 fold (range 1.2-fold to 2.0-fold). No consistent difference in platelet counts was seen. These results suggest that SCF given in vivo is effective in increasing the frequency of CD34+ BM progenitor cells, and has the capacity to increase the proliferation and differentiation rate of hematopoietic precursor cells. These effects indicate that SCF may represent a cytokine capable of affecting multiple hematopoietic lineages.

We have previously shown that administration of low-dose recombinant human stem cell factor (rhSCF) plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) to baboons mobilizes greater numbers of progenitor cells in the blood than does administration of rhG-CSF alone. The purpose of the present study was to determine whether marrow repopulating cells are present in the blood of nonhuman primates administered low-dose rhSCF plus rhG-CSF, and if present, whether these cells engraft lethally irradiated recipients as rapidly as blood cells mobilized by treatment with rhG-CSF alone. One group of baboons was administered low-dose rhSCF (25 micrograms/kg/d) plus rhG- CSF (100 micrograms/kg/d) while a second group received rhG-CSF alone (100 micrograms/kg/d). Each animal underwent a single 2-hour leukapheresis occurring the day when the number of progenitor cells per volume of blood was maximal. For baboons administered low-dose rhSCF plus rhG-CSF, the leukapheresis products contained 1.8-fold more mononuclear cells and 14.0-fold more progenitor cells compared to the leukapheresis products from animals treated with rhG-CSF alone. All animals successfully engrafted after transplantation of cryopreserved autologous blood cells. In animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells, we observed a time to a platelet count of > 20,000 was 8 days +/- 0, to a white blood cell count (WBC) of > 1,000 was 11 +/- 1 days, and to an absolute neutrophil count (ANC) of > 500 was 12 +/- 1 days. These results compared with 42 +/- 12, 16 +/- 1, and 24 +/- 4 days to achieve platelets > 20,000, WBC > 1,000, and ANC > 500, respectively, for baboons transplanted with rhG-CSF mobilized blood cells. Animals transplanted with low-dose rhSCF plus rhG-CSF mobilized blood cells had blood counts equivalent to pretransplant values within 3 weeks after transplant. The results suggest that the combination of low-dose rhSCF plus rhG-CSF mobilizes greater numbers of progenitor cells that can be collected by leukapheresis than does rhG-CSF alone, that blood cells mobilized by low-dose rhSCF plus rhG-CSF contain marrow repopulating cells, and finally that using a single 2-hour leukapheresis to collect cells, the blood cells mobilized by low-dose rhSCF plus rhG-CSF engraft lethally irradiated recipients more rapidly than do blood cells mobilized by rhG- CSF alone.

The SR-1 monoclonal antibody (MoAb) recognizes an epitope of the c-kit receptor (KR), present on normal hemopoietic CD34+ stem cells as well as on blasts from patients with acute leukemia. Cytometric analysis by indirect immunofluorescence with the SR-1 MoAb was performed in 98 patients with acute myeloblastic leukemia (AML) and in 37 patients with acute lymphoblastic leukemia (ALL) in order to detect the presence of the KR and to examine its prognostic significance. Sixty-nine of 98 (70%) AML patients were SR-1 positive independently of the FAB subtype, although a higher incidence of SR-1 positive cases was observed in M4 and M5 AML and in those cases that also coexpressed lymphoid antigens. Fourteen AML samples were studied by Northern blot analysis and the KR mRNA was detected in the majority of SR-1 positive cases and also in 2 of 3 SR-1 negative samples. Furthermore, "in vitro" cultures from 15 cases showed that recombinant human Stem cell factor (rhSCF) induced an increased proliferative activity in most tested cases (11/15); this was further enhanced when rhSCF was combined with rhIL-3 + rhGM-CSF (p = 0.007) and with the GM-CSF/IL-3 fusion protein PIXY321 (p = 0.003). Thirty-seven ALL cases were also studied and all but one were SR-1 negative. Interestingly, the only SR-1 positive case also coexpressed myeloid antigens and showed an "in vitro" response when stimulated with rhSCF. Finally, the complete remission (CR) rate, survival and event-free survival were evaluated in 75 AML patients who received standard and identical chemotherapy; unlike previous studies which utilized a different anti-KR MoAb (YB5.B8) and which showed a poor prognosis for KR positive patients, we were unable to document any significant difference in CR rate, survival and event-free survival.

Mast cells may be cultured from human peripheral blood in the presence of recombinant human stem cell factor (rhSCF). The characteristics of the cells in peripheral blood that give rise to mast cells are unknown. Because mast cell precursors in human marrow are CD34+, human peripheral blood mononuclear cells from patients with mastocytosis and normal controls were sorted on the basis of CD34 expression and the positive and negative cell populations were cultured in rhSCF, recombinant human interleukin-3 (rhIL-3), or both for 6 weeks. Cell cultures were examined every 2 weeks for total and mast cell number and cell differential using Wright Giemsa and acid toluidine blue stains and antibodies to mast cell tryptase and chymase, cell-associated histamine, and expression of CD34, c-kit, Fc epsilon RI, and Fc gamma RII using flow cytometric analysis. The ultrastructural anatomy of mast cells was examined by electron microscopy. Peripheral blood CD34+ cells cultured in rhSCF with or without rhIL-3 gave rise to cell cultures consisting of greater than 80% mast cells by 6 weeks. CD34+ cells cultured in rhIL-3 alone did not give rise to mast cells, whereas rhIL- 3 plus rhSCF increased the final mast cell number eightfold when compared with cells cultured in rhSCF alone. Mast cells increased concomitantly with a decrease in large undifferentiated mononuclear cells. CD34- cells did not give rise to mast cells. Histamine content per cell at 6 weeks was approximately 5 pg. Electron microscopy of 4- week cultures showed immature mast cells containing predominantly tryptase-positive granules that were either homogeneous or contained lattice structures, partial scroll patterns, or central dense cores and mixtures of vesicles, fine granular material, and particles. The CD34+ population at day 0 expressed Kit (65%) and Fc gamma RII (95%), but not Fc epsilon RI, by fluorescence-activated cell sorter analysis. At 6 weeks, CD34+-derived mast cells exhibited Fc epsilon RI in addition to Kit and Fc gamma RII, and were negative for CD34 antigen. Patients with mastocytosis showed a higher number of mast cells per CD34+ cell cultured compared with normal controls. Thus, the mast cell precursor in human peripheral blood is CD34+/Fc epsilon RI- and gives rise to mast cells in the presence of rhSCF with or without rhIL-3, and the number of mast cells arising per CD34+ cell in culture is greater when the CD34+ cells are obtained from patients with mastocytosis compared with normal subjects.

Recombinant human stem cell factor (rhSCF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) are synergistic in vitro in stimulating the proliferation of hematopoietic progenitor cells and their precursors. We examined the in vivo synergy of rhSCF with rhG-CSF for stimulating hematopoiesis in vivo in baboons. Administration of low-dose (LD) rhSCF (25 micrograms/kg) alone did not stimulate changes in circulating WBCs. In comparison, administration of LD rhSCF in combination with rhG-CSF at 10 micrograms/kg or 100 micrograms/kg stimulated increases in circulating WBCs of multiple types up to twofold higher than was stimulated by administration of the same dose of rhG-CSF alone. When the dose of rhG-CSF is increased to 250 micrograms/kg, the administration of LD rhSCF does not further increase the circulating WBC counts. Administration of LD rhSCF in combination with rhG-CSF also stimulated increased circulation of hematopoietic progenitors. LD rhSCF alone stimulated less of an increase in circulating progenitors, per milliliter of blood, than did administration of rhG-CSF alone at 100 micrograms/kg. Baboons administered LD rhSCF together with rhG-CSF at 10, 100, or 250 micrograms/kg had 3.5- to 16-fold higher numbers per milliliter of blood of progenitors cells of multiple types, including colony-forming units granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming and burst-forming units-megakaryocyte (CFU- MK and BFU-MK) compared with animals given the same dose of rhG-CSF without rhSCF, regardless of the rhG-CSF dose. The increased circulation of progenitor cells stimulated by the combination of rhSCF plus rhG-CSF was not necessarily directly related to the increase in WBCs, as this effect on peripheral blood progenitors was observed even at an rhG-CSF dose of 250 micrograms/kg, where coadministration of LD rhSCF did not further increase WBC counts. Administration of very-low- dose rhSCF (2.5 micrograms/kg) with rhG-CSF, 10 micrograms/kg, did not stimulate increases in circulating WBCs, but did increase the number of megakaryocyte progenitor cells in blood compared with rhG-CSF alone. LD rhSCF administered alone for 7 days before rhG-CSF did not result in increased levels of circulating WBCs or progenitors compared with rhG- CSF alone. Thus, the synergistic effects of rhSCF with rhG-CSF were both dose- and time-dependent. The doses of rhSCF used in these studies have been tolerated in vivo in humans.(ABSTRACT TRUNCATED AT 400 WORDS).

The use of growth factors to stimulate haemopoietic proliferation in various clinical conditions such as renal failure-associated anaemia, post-chemotherapy cytopenia and enhancement of bone marrow engraftment has been well described. Of the 3 haemopoietic lineages, the use of growth factors has been most beneficial in the erythroid and myeloid compartments. Currently, there are no cytokines available specifically for the purpose of stimulating thrombopoiesis. However, expanding knowledge of haemopoiesis in general and megakaryocytopoiesis in particular has led to an appreciation of the fact that multiple growth factors with overlapping or specific activities are involved in various stages of megakaryocyte proliferation and differentiation. Although application of early acting, multiline-age cytokines such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) has resulted in significant augmentation of thrombopoiesis in vitro, little such activity has been seen in vivo. The IL-3/GM-CSF fusion protein PIXY-321 (pixykine), also active in vitro, similarly has limited thrombopoietic effect in vivo. On the other hand, interleukin-6 (IL-6) and, in early studies, interleukin-11 (IL-11), which both augment IL-3-dependent progenitor proliferation and have significant megakaryocytopoietic activity in vitro, produce a more pronounced thrombopoietic effect. Further analyses and trials will be required to establish proper cytokine dosages, regimens and combinations in order to achieve maximal platelet enhancement.

A growing number of in vitro studies suggest that recombinant human stem cell factor (SCF) is capable of augmenting the proliferative capacity of human hematopoietic progenitor cells (HPC) and stem cells (HSC). We further evaluated this biologic effect by analyzing the response of bone marrow (BM) HPCs and HSCs to the administration of SCF in eight patients with locally advanced or metastatic breast cancer who were enrolled in an ongoing phase I study. SCF was administered for 14 days by daily subcutaneous injection at dosages of 10, 25, or 50 micrograms/kg/d. BM CD34+ HLA-DR+ and CD34+ HLA-DR- CD15- cells, previously shown by our laboratory to be enriched for various classes of differentiated and primitive HPCs, respectively, were quantitated in BM samples on day 0 (pretreatment) and day 15 (posttreatment). These CD34+ HLA-DR+ and CD34+ HLA-DR- CD15- cells were then isolated by cell- sorting and assayed for several classes of HPCs, including the high-- proliferative potential colony-forming cell (HPP-CFC), the burst- forming unit--megakaryocyte (BFU-MK), and the long-term BM culture-- initiating cell (LTBMC-IC). SCF administration resulted in a 3.3-fold (range, 1.4- to 18.8-fold; P = .018) increase in the absolute numbers of CD34+ cells, a 3.7-fold (range, 1.2- to 8.2-fold; P = .028) increase in the absolute numbers of CD34+ HLA-DR+ cells, and a 2.4-fold (range, 1.1- to 29.3-fold; P = .010) increase in the absolute numbers of CD34+ HLA-DR- CD15- cells. Following the infusion of SCF, a statistically significant increase in the absolute numbers of HPP-CFC (P = .018), BFU- MK (P = .046), CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM: P = .043), BFU-erythrocyte (BFU-E; P = .043), CFU- granulocyte, macrophage (CFU-GM; P = .045), and CFU-megakaryocyte (CFU- MK; P = .028) per milliliter of marrow was observed. Stromal cell-free LTBMCs supplemented with SCF and interleukin-3 (IL-3), initiated with CD34+ HLA-DR- CD15- cells obtained on day 0, produced viable cells for 9.6 weeks, compared with 11.5 weeks for LTBMCs initiated with CD34+ HLA- DR- CD15- cells obtained on day 15. Cumulative cellular production by LTBMCs initiated with day 15 CD34+ HLA-DR- CD15- cells was statistically greater than that by day 0 LTBMCs (P = .031). These same cultures produced CFU-GM for 6.3 weeks (day 0) versus 9 weeks (day 15).(ABSTRACT TRUNCATED AT 400 WORDS)

We tested the ability of CD34+lin- precursor cells isolated from marrow after treatment with 4-hydroperoxycyclophosphamide (4HC) to generate colony-forming cells (CFC). In liquid cultures, recombinant human stem cell factor (SCF), in combination with interleukin-1 (IL-1), IL-3, IL- 6, granulocyte-macrophage colony-stimulating factor, or granulocyte colony-stimulating factor caused untreated, but not 4HC-treated, CD34+lin- cells to form CFC. However, generation of CFC from CD34+lin- cells treated with 60 micrograms/mL of 4HC was possible in the presence of an irradiated allogeneic stromal cell layer. This generation was increased when combinations of hematopoietic growth factors including SCF and IL-3 were added. Maximal generation of CFC was seen after 11 to 21 days of culture. At that time, generation of CFC from CD34+lin- 4HC- treated cells equalled that from untreated cells. The phenotype of these 4HC-resistant CD34+lin- precursors was also further defined as CD38-. These studies show that the generation of CFC from the 4HC- resistant, highly immature population of CD34+lin- cells requires an as yet undefined interaction with marrow stroma in addition to known hematopoietic growth factors.

Peripheral blood hematopoietic progenitors (PBHP) are capable of colony growth in vitro. The effect of stem cell factor (SCF), interleukin-6 (IL-6), and basic fibroblast growth factor (bFGF) on myeloid colony proliferation of PBHP was determined. PBHP purified by positive selection with CD34-specific antibody were plated in semisolid agarose with reported plateau doses of interleukin-3 (IL-3), granulocyte- macrophage colony-stimulating factor (GM-CSF), and granulocyte colony- stimulating factor (G-CSF) to enhance myeloid colony growth. Experiments then were done to examine colony growth in response to SCF or with SCF and bFGF and/or IL6. SCF alone in the absence of any other growth factors did not support colony growth. SCF at a determined optimum concentration of 100 ng/mL added to the combination of IL-3, GM- CSF, and G-CSF enhanced colony growth and size relative to proliferation in response to the latter three factors alone (from 78 to 188 total colonies/10(4) PBHP plated and from 10 to 93 large [> 200 cells] colonies/10(4) PBHP plated). Furthermore, addition of bFGF and/or IL-6 to the combination of optimum concentrations of SCF, IL-3, GM-CSF, and G-CSF further enhanced colony number and size in a dose- dependent fashion. Using the optimum combination of all growth factors, we determined that the number of myeloid colony-forming PBHP in whole blood was similar between individuals at about three colonies per milliliter whole blood. We conclude that progenitors capable of responding to the early-acting growth factor, SCF, are represented in PBHP and that the number of circulating myeloid colony-forming PBHP is likely a regulated parameter that may have an important biologic function.

Recombinant human stem cell factor (SCF), the ligand for c-kit, has been shown to stimulate increased numbers of hematopoietic progenitor cells of multiple types to circulate in the blood of baboons, but it was not known if the cells stimulated to circulate by SCF contained cells capable of engrafting and rescuing lethally irradiated baboons. Peripheral blood mononuclear cells (PBMNC) were collected by leukapheresis from four untreated control baboons and from three baboons on the 10th or 11th day of treatment with SCF (200 micrograms/kg/d). All animals were transplanted with 1.00 to 1.04 x 10(8)/kg of cryopreserved autologous PBMNC after treatment with a single dose of 1,020 cGy total body irradiation (TBI). Three animals were transplanted with PBMNC that had been collected during SCF treatment, 24 to 38 days after the last dose of SCF. Rapid trilineage engraftment was documented by bone marrow biopsy in all three. The mean time to a total white blood cell count (WBC) > or = 500/microL, WBC > or = 1,000/microL, and an absolute neutrophil count (ANC) > or = 500/microL was 15 +/- 3 (mean +/- SD), 19 +/- 1, and 19 +/- 2 days, respectively. Two animals remain alive with stable engraftment more than 180 and 245 days posttransplant. The third died of sepsis 32 days posttransplant with a hypercellular marrow showing trilineage engraftment. The surviving animals were transfusion independent by 10 and 59 days posttransplant. Four control animals were transplanted with PBMNC collected in the absence of SCF stimulation. One was treated for 11 days with SCF (200 micrograms/kg/d) after PBMNC were collected. This animal was transplanted 25 days after the last dose of SCF. None of the four control animals engrafted and they died 13, 16, 28, and 38 days posttransplant with marrow aplasia. Treatment with SCF stimulates the circulation of cells that engraft and rescue lethally irradiated baboons. The characteristics of the transplantable cells present in the circulation are now amenable to direct study.

This report describes the structure of soluble human stem cell factor isolated from the conditioned medium of Chinese hamster ovary (CHO) cells transfected with stem cell factor (SCF) cDNA, which encodes a leader sequence plus 248 additional amino acids. The 248 amino acids include a hydrophobic transmembrane region at positions 190-212. The isolated material is glycosylated and three bands (apparent M(r) 28,000, M(r) 35,000, and M(r) 40,000) are evident by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After complete deglycosylation, the molecular weight by SDS-polyacrylamide gel electrophoresis is 18,000-19,000. Structural analyses of the intact SCF, the deglycosylated SCF, and a deglycosylated C-terminal peptide were performed by laser desorption, fast atom bombardment, or electrospray mass spectrometry. Pulse-labeling of cells with 35S-labeled Met and Cys resulted in cell-associated glycosylated SCF of M(r) 33,000-45,000 which was converted to M(r) 33,000 by in vitro treatment with glycosidases. During a chase with unlabeled Met and Cys, labeled SCF of M(r) 28,000, M(r) 35,000, and M(r) 40,000 appeared in the medium; it was converted to M(r) 18,000-19,000 by glycosidase treatment. SCF at the surface of the transfected CHO cells could be demonstrated by immunofluorescence. The data obtained indicate that the recombinant human stem cell factor, as isolated, represents proteolytically processed forms containing amino acids 1-165, derived from the initially synthesized membrane-bound form of 248 amino acids. Further characterization indicated that the M(r) 28,000 form is glycosylated at Asn120, the M(r) 35,000 form at Asn120 and Asn65, and the M(r) 40,000 form at Asn120, Asn93, and Asn65. Each form also contains O-linked carbohydrate. The N-linked glycosylation, particularly that at Asn93 and at Asn65, adversely affects in vitro biological activity and receptor binding.

The ligand for the human c-kit, recombinant human stem cell factor (SCF), was administered to baboons at doses of 200, 100, 50, 25, and 10 micrograms/kg/d. SCF induced a dose-dependent expansion of hematopoietic colony-forming cells (CFC) of multiple types in both blood and marrow, including colony-forming unit (CFU) granulocyte- monocyte, burst-forming unit-erythroid, CFU-MIX, and high proliferative potential-CFC. These changes were associated with a dose-dependent leukocytosis, involving all leukocyte lineages, a reticulocytosis, and increases in marrow cellularity. At 200 micrograms/kg/d of SCF, CFC in blood were increased 10-fold to greater than 100-fold. This correlated with an increased frequency of CD34+ cells in blood. The frequency of CFC in blood approached that of marrow in some animals. These changes were reversed within 7 to 14 days of stopping SCF. The results of these studies suggest a role for the c-kit ligand in stimulating the expansion of multiple CFC types in blood and marrow for potential therapeutic purposes.

Human cord blood was used as a source of progenitor and stem cells to evaluate the effect of recombinant human stem-cell factor (SCF) on colony formation and the generation of colony-forming cells (CFC) under highly defined, serum-deprived conditions. SCF interacted with a number of hematopoietic growth factors to stimulate colony growth and was particularly effective in stimulating the formation of mixed-cell colonies from CD34+ soybean agglutinin negative (SBA-) cells. In suspension culture of CD34+, SBA- cells, SCF alone was unable to maintain cell numbers or CFC but, in combination with interleukin-3 (IL- 3), increased input numbers of cells by 10-fold and increased CFC of all kinds by nearly 20-fold. This included erythroid burst-forming cells (BFU-E), granulocyte/macrophage (GM) CFC, and mixed-cell CFC. In contrast, CD34- SBA- cells neither gave rise to CFC nor were maintained by combinations of growth factors including SCF. SCF interacted with erythropoietin (Epo) and granulocyte colony-stimulating factor (G-CSF) to maintain large numbers of cells as well as to generate a twofold to threefold increase in CFC in the case of Epo, and a 10-fold increase in CFC in the case of G-CSF. With Epo, the predominant CFC generated were BFU-E and erythroid CFC and many of the cells in suspension were erythroblasts. In contrast, SCF plus G-CSF resulted in large numbers of granulocytes at various stages of maturation and the CFC generated were almost exclusively granulocytic-CFC. IL-1 and IL-6, alone or in combination with SCF, showed little or no ability to increase cell numbers or generate CFC. In summary, SCF interacts with a variety of hematopoietic growth factors to promote colony formation, particularly mixed-cell colony formation, and also, in suspension culture, SCF interacts with IL-3, G-CSF, and Epo to generate large numbers of differentiated cells as well as a variety of CFC for up to 1 month.

The molecular weight of recombinant human stem cell factor (SCF) was determined using a low-angle laser light scattering combined with a differential refractometer and a uv detector. The protein samples were applied to these detectors through a gel filtration column by a high-performance liquid chromatographic pump. The Chinese hamster ovary (CHO) cell-derived SCF gave a molecular weight of 53,000 for the entire molecule and 35,000 for the protein moiety only at pH 7.0, indicating that the CHO cell-derived protein is glycosylated by 34%. Since the molecular weight of the polypeptide is 18,600, the results demonstrate that the CHO cell-derived SCF forms a dimer. The molecular weight of Escherichia coli-derived SCF was determined to be 39,000, similar to the above value (35,000). Essentially identical molecular weights were obtained at pH 3.0, indicating no dissociation of the dimer.

The c-kit proto-oncogene encodes a receptor tyrosine kinase that is thought to play an important role in hematopoiesis. In a series of human acute myeloblastic leukemia (AML), the expression of the c-kit proto-oncogene and its product was studied by means of Northern blot and immunoblot analyses. The c-kit mRNA was expressed in 20 of 25 cases of AML, and in those cases the product of the c-kit proto-oncogene was detected by immunoblotting with anti-c-kit antibody. The expression of c-kit transcripts and protein was barely detectable in normal bone marrow cells as a control. The expression of c-kit transcript did not correlate with the French-American-British classification nor clinical manifestations. In 6 of 11 cases that expressed c-kit product, AML cells were found to proliferate in response to recombinant human stem cell factor (rhSCF), the ligand for c-kit, and the synergistic stimulation of AML cells was observed by rhSCF and granulocyte- macrophage colony-stimulating factor. Immunoblotting with anti- phosphotyrosine antibody showed that the c-kit receptor protein was detectably phosphorylated in 7 of 12 cases tested before the stimulation with rhSCF, while the rhSCF treatment resulted in an increased tyrosine phosphorylation of c-kit in AML cells. These results indicate that c-kit proto-oncogene is expressed in most cases of AML and is functional in terms of supporting proliferation.

To understand the factors that regulate the early growth and development of immature erythroid progenitor cells, the burst-forming units-erythroid (BFU-E), it is necessary to have both highly purified target cells and a medium free of serum. When highly purified human blood BFU-E were cultured in a serum-free medium adequate for the growth of later erythroid progenitors, BFU-E would not grow even with the addition of recombinant human interleukin-3 (rIL-3), known to be essential for these cells. However, the addition of recombinant human stem cell factor (rSCF), which supports germ cell and pluripotential stem cell growth, stimulated BFU-E to grow equally well in serum-free as in serum-containing medium. Limiting dilution studies showed that rSCF acts directly on the BFU-E that do not require accessory cells for growth. Furthermore, rSCF was necessary for BFU-E development during the initial 7 days of culture, until these cells reached the stage of the late progenitors, the colony-forming units-erythroid (CFU-E). These studies indicate that early erythropoiesis is dependent on the direct action of SCF that not only affects early stem cells but is continually necessary for the further development of committed erythroid progenitor cells until the CFU-E stage of maturation.

Recombinant human stem cell factor (SCF) is homologous with recombinant rat SCF (rrSCF) and is a ligand for c-kit. We determined the influence of SCF on hematopoiesis in vitro and in vivo in baboons. In vitro, SCF alone stimulated little growth of hematopoietic colony-forming cells from baboon marrow, but did increase the number of colonies formed in response to erythropoietin (Epo), interleukin-3 (IL-3), and granulocyte- macrophage colony-stimulating factor (GM-CSF). In vivo, SCF caused an increase in the peripheral blood of the number of erythrocytes, neutrophils, lymphocytes, monocytes, eosinophils, and basophils. In marrow, it caused an increase in marrow cellularity and in the absolute number of colony-forming unit-granulocyte-monocyte (CFU-GM) and burst- forming unit-erythroid (BFU-E) in marrow following infusion of SCF. The in vivo stimulation of multiple lymphohematopoietic lineages corroborates previous in vitro studies and suggests a potentially important clinical role for SCF.