Walter R. Paukovits

Isolation and synthesis of a hemoregulatory peptide.

Abstract A peptide was isolated in pure form from human leukocytes which strongly inhibits the proliferation of immature meyloid cells in vitro (committed stem cells). Structural investigations yielded pGlu-Asp or Glu-Asp or Glu-Cys-Lys-OH as the probable sequence of this peptide. The Glu2,Asp3-analog, prepared synthetically, displayed similar activities and when applied in vivo showed effects on the hemopoietic system ranging from an inhibition of pluripotent and committed stem cells to variations in the bone marrow proliferation and alterations in peripheral blood counts.

Molecular weight and some chemical properties of the Granulocytic Chalone

ZusammenfassungEin Peptid, welches sich wie ein Granulozytenchalon verhält, wurde aus durch normale menschliche Leukozyten konditioniertem Medium gewonnen. Die Reinigung erfolgte durch Säulenchromatographie an Sephadex G-25, G-10, Rechromatographie an G-10, Ionenaustauschchromatographie an Dowex 50, sowie durch präparative Papier- und Dünnschichtchromatographie. Die Hemmwirkung und Spezifität wurden mittels der Koloniebildung in Agar sowie durch3H-Thymidineinbau in Knochenmarks- und Thymuszellen nachgewiesen. Das gelchromatographische Verhalten von GCH wird im Hinblick auf das Molgewicht diskutiert. Demnach scheint es sich um ein saures Peptid vom Molgewicht 500–600 zu handeln, welches neben anderen Aminosäuren Asparaginsäure und Glutaminsäure enthält. Die N-terminale Aminogruppe scheint blockiert zu sein.SummaryGranulocytic Chalone (GCh) has been highly purified from culture medium conditioned by human peripheral leukocytes. Purification was performed by column chromatography on Sephadex G-25 and G-10, rechromatography on G-10, preparative paper chromatography and thin-layer-chromatography. The inhibitory activity and specificity of GCh was monitored by agar colony formation and3H-thymidine incorporation into bone marrow and thymic cells. The gelchromatographic behavior of GCh is discussed in detail with regard to its molecular weight.The results obtained indicate that GCh may be a small (MW 500–600), acidic and highly polar peptide containing aspartic and glutamic acid among others. The N-terminal aminogroup seems to be blocked.

Haemoprotection against cytostatic drugs by stem cell inhibition.

Cytostatic drug-induced haematopoietic damage is a major problem in tumour chemotherapy, due to the intensive proliferation of many bone marrow constituents and to the drug-induced recruitment of immature pluripotent haematopoietic cells (spleen colony-forming units, CFU-S). Marie-Hélène Moser and Walter Paukovits discuss how it should be possible to minimize such proliferation-associated damage by inhibiting CFU-S during the most dangerous treatment phases, with factors such as transforming growth factor beta, tumour necrosis factor alpha, macrophage inflammatory protein 1 alpha, and the CFU-S-inhibitory peptides N-acetyl-Ser-Asp-Lys-Pro and pyroGlu-Glu-Asp-Cys-Lys (pEEDCK). Clinically relevant data are available for pEEDCK, showing that application of this peptide leads to a delayed, shorter, and less severe neutropenia, combination of pEEDCK with a stimulator avoids neutropenia, and stem cell preservation with pEEDCK improves long-term reconstitution of the haematopoietic system. Stem cell inhibition by synthetic peptides like pEEDCK may provide a useful strategy for bone marrow protection.

A synthetic hemoregulatory peptide (HP5B) inhibits human myelopoietic colony formation (CFU-GM) but not leukocyte phagocytosis in vitro.

A synthetic analog of a hemoregulatory peptide associated with mature human granulocyte (HP5B) has been investigated for inhibitory effects on human myelopoietic stem cells in vitro. In addition, it has been tested for effects on phagocytosis by human granulocytes and monocytes by use of an automatic flow cytometric method. A dose‐dependent inhibition of colony formation was found after preincubation of bone marrow cells for 1 h at 37°C in the range 10−7‐10−11 mol/l. Above or below these concentrations, no inhibitory effects were seen. The degree of inhibition varied from experiment to experiment, indicating variable responsiveness of the donor cells. Maximal effect was of magnitude 90% inhibition, and the optimal dose was 10−7 mol/l. The peptide had no effect on the kinetics of phagocytosis by measurements of the uptake of fluorescent Zymosan particles or Staphylococcus aureus. This may indicate a selective effect on the precursor cells, with no effect on the functional state of their progeny, the granulocytes and monocytes.

The Granulocytic Chalone – A Specific Inhibitor of Granulopoiesis: Molecular Weight and Chemical Nature

Chalones specifically inhibit cell rpoliferation not only in their respective normal cell system, but also in the malignant descendants of these cells. Consequently, a number of hypotheses have been put forward as to how chalones could be used in cancer treatment or related areas. As chalones are not cytotoxic themselves, but merely inhibit proliferation, the regression of tumors which have been observed in some cases must have been caused by other mechanisms, not directly related to the increased chalone level. Some of the potential advantages and drawbacks of this direct approach will be discussed. Some models in which chalones serve as adjuncts to other forms of therapy will be discussed. The experimental testing of these procedures and hypothesis is hampered severly by the unavailability of satisfactory chalone preparations. A possible approach to overcome this by using synthetic granulocytic (and possibly other) chalone(s) is discussed in detail.

Peripheral blood leukocyte alterations in mice induced by a hemoregulatory pentapeptide (HP 5b)

The effects of different doses of a synthetic hemoregulatory pentapeptide, analoguous to an inhibitory factor associated with human granulocytes, on the numbers of peripheral blood leukocytes have been investigated in female C3H mice. Following single injections as well as continuous infusion for 6 days, the numbers of granulocytes in peripheral blood were reduced to about one half of the normal. Maximal effect with single injection was seen with 120 micrograms (10(-5) M), while 1.2 mg (10(-4) M) as one dose only had a slight, temporary effect on the granulocyte numbers in peripheral blood. Upon continuous infusion of 14 micrograms/h (less than 10(-5) M) for 19 days, a stimulation with doubling of the granulocyte numbers was seen instead. The substance also decreased the monocyte numbers in peripheral blood. With prolonged exposure, a relative monocytosis was seen instead. After both single injection and continuous infusion, the pentapeptide increased the lymphocyte numbers in peripheral blood. The thrombocyte numbers were not altered. The decreased granulocyte numbers seemed to be due to a dual mechanism, one reducing proliferation in the maturing compartment which gives a rapid cell reduction, and one mediated through inhibition of the committed stem cells for myelopoiesis, giving a long-lasting reduction in the granulocyte numbers. It is postulated that the pentapeptide has a complex regulatory effect in vivo.

The use of haemoregulatory peptides (pEEDCK monomer and dimer) for reduction of cytostatic drug induced haemopoietic damage

We have previously shown that the stem cell inhibitory peptide pGlu-Glu-Asp-Cys-Lys (pEEDCK monomer) leads to a good tolerance of otherwise lethal multiple ara-C doses and an increased survival of ara-C + peptide treated mice. This effect was due to the prevention of drug-induced CFU-S proliferation, thus keeping stem cells in a quiescent state insensitive to ara-C. Here we show that the pEEDCK monomer also inhibits stem cell proliferation after clinically relevant (non-lethal) ara-C doses. This leads to a sustained (100%) stem cell number in the femoral bone marrow, which was greatly reduced without protective peptide treatment (27%). We have measured the kinetics of influx of CFU-S into the empty S-phase (after two consecutive ara-C injections). This influx reached peak levels of 60-70%; pEEDCK treatment reduced it to 25-30%. Due to its cysteine content the pEEDCK monomer is easily oxidized and forms a symmetric disulfide-bonded dimer (pEEDCK)2. This dimer is a potent stimulator of haemopoiesis. Various modes of protective peptide treatment (monomer and dimer) were investigated in conjunction with a standardized protocol of 2 x 300 mg/kg ara-C given 12 h apart. (a) ara-monomer-ara: The administration of pEEDCK-monomer 2 h before the second ara-C injection retarded the onset of neutropenia, shortened its duration and improved recovery after depression. The degree of short-term neutropenia was not changed. (b) ara-ara-HN2-dimer: Post chemotherapy infusion of the stimulatory (pEEDCK)2 dimer led to considerable increases of progenitor levels (6.8 CFU-GM/1000 bone marrow cells vs. 1.2 CFU-GM/1000 in normal mice) 2 days after cytostatic treatment when CFU-GM were not detectable in unprotected mice. This increase was followed by greatly elevated granulocyte counts (8000 PMN/mm3 vs. 750 PMN/mm3 in normal mice). In the dimer-treated mice, up to 75% of the peripheral leukocytes were mature PMN (normal, 10%). (c) ara-monomer-ara-dimer: ara-C and monomer treatment as above (a) followed by dimer infusion led to complete protection of haemopoiesis. Mice treated with the protective pEEDCK monomer plus stimulatory dimer did not develop the leukocyte depression seen in unprotected animals. Our results show that the haemoregulatory peptide monomer and dimer can be used to improve the haematological status of mice treated with clinically relevant doses of cytostatic drugs (anti-metabolite and alkylating, alone and in combination). The pEEDCK monomer and dimer are equally active also on human haemopoietic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibitors of hematopoiesis. Report on the 2nd International Conference on Negative Regulators of Hematopoiesis, Providence, R.I., 22-25 August 1990.

It has become increasingly clear that the regulation of cell proliferation involves stimulatory as well as inhibitory influences on the molecular machinery of growth-related processes. While recent years have seen an extraordinary increase of our knowledge about the properties and actions of stimulatory growth factors, the corresponding rise in the field of inhibitory regulators has come more slowly, although the idea of negative growth control has been around for many years. In the field of hemopoiesis the well-known stimulators, like the various colonystimulating factors (CSF) and interleukins, have now received well-characterized functional counterparts in various inhibitory factors, cytokines, and small peptides, acting by inhibiting the proliferation of hemopoietic cells at various levels during their multistep differentiation and maturation journey through the compartments of the hemopoietic system. This increase in our knowledge about the inhibitors of hemopoiesis is reflected by an increasing interest of the scientific community in this field. Proliferation inhibitors are now well-respected beasts in the factor zoo and in the special field of hemopoiesis the second international meeting solely devoted to this topic was recently held in Providence, R.I. The Proceedings of this conference will be published in the Annals of the New York Academy of Sciences. The Proceedings of the previous meeting, held in Paris in 1987, were published by Najman A. et al. (eds) The Inhibitors ofhematopoiesis, J. Libbey Eurotext, London. In his introductory lecture H. Broxmeyer (Indianapolis, Ind.) presented data on a variety of molecules known to be inhibitory for hemopoiesis. Lactoferrin decreased

Human lung tissue as a source of colony stimulating activity

ZusammenfassungMedium, welches durch menschliches Lungengewebe konditioniert wird, enthält kolonienstimulierende Aktivität (= Colony Stimulating Activity, CSA). Diese Präparation wurde an Menschen- und Mäuseknochenmark getestet. Kolonienbildende Einheiten von beiden Species wurden in Agarkulturen zu klonalem Wachstum aktiviert. Es zeigte sich eine sigmoide Dosisabhängigkeit des Wachtums der CFUc von der Konzentration an lungenkonditioniertem Medium. Die Molekulargewichtsbestimmung der kolonienstimulierenden Faktoren in Lungenmedium ergab vier Molekulargewichtsfraktionen, nämlich mit annähernd 79000,40000,23000und 2000 Daltons. Die 23 000 Dalton-Fraktion aktivierte ausschließlich menschliche Zellen, die übrigen Fraktionen aktivierten Menschen- und Mäuseknochenmarkzellen.SummaryMedium conditioned by human lung tissue was found to contain colony stimulating activity (CSA). This material was tested against mouse and human bone marrow as target system. Colony forming units (CFUc)from both species responded and gave rise to clonal growth in agar cultures. This colony formation was dose dependent and the relationship was a sigmoid one. Experiments to determine the molecular weight of human lung derived colony stimulating Factors brought evidence for four active molecular weight fractions with approximately 79000, 40000, 23000 and 2000 daltons. The 23000 dalton fraction activated human cells only, whereas the other fractions were active on both human and mouse bone marrow cells.

Endotoxin-induced myeloid reactions in dogs

The response to artificial endotoxinemia was studied in adult dogs. Granulocyte-macrophage colony stimulating activity (CSA) in lung tissue and blood was measured along with the number of circulating granulocytes and myeloid committed stem cells (colony forming units, CFUc). Acute endotoxinemia induced a measurable CSA increase in lung tissue before being detectable in blood. Granulocytes, rapidly removed from the circulation, showed no release of CSA during sequestration. These experiments demonstrate that the process of endotoxin recognition and subsequent transition into a myelopoietic stimulus is medicated by cells belonging to tissue; mature granulocytes, involved in the defence against bacterial infection, do not release activity that promotes growth of immature myeloid cells.