Growth is one of the fundamental aspects in the development of an organism. Classical genetic studies have isolated four viable, spontaneous mouse mutants disrupted in growth, leading to dwarfism. Pygmy is unique among these mutants because its phenotype cannot be explained by aberrations in the growth hormone-insulin-like growth factor endocrine pathway. Here we show that the pygmy phenotype arises from the inactivation of Hmgi-c (ref. 6), a member of the Hmgi family which function as architectural factors in the nuclear scaffold and are critical in the assembly of stereospecific transcriptional complexes. Hmgi-c and another Hmgi family member, Hmgi(gamma) (ref. 10), were found to be expressed predominantly during embryogenesis. The HMGI proteins are known to be regulated by cell cycle-dependent phosphorylation which alters their DNA binding affinity. These results demonstrate the important role of HMGI proteins in mammalian growth and development.

Familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/ AML) is an autosomal dominant familial platelet disorder characterized by thrombocytopenia and a propensity to develop AML. Mutation analyses of RUNX1 in 3 families with FPD/AML showing linkage to chromosome 21q22.1 revealed 3 novel heterozygous point mutations (K83E, R135fsX177 (IVS4 ؉ 3delA), and Y260X). Functional investigations of the 7 FPD/ AML RUNX1 Runt domain point mutations described to date (2 frameshift, 2 nonsense, and 3 missense mutations) were performed. Consistent with the position of the mutations in the Runt domain at the RUNX1-DNA interface, DNA binding of all mutant RUNX1 proteins was absent or significantly decreased. In general, missense and nonsense RUNX1 proteins retained the ability to heterodimerize with PEBP2␤/CBF␤ and inhibited transactivation of a reporter gene by wild-type RUNX1. Colocalization of mutant RUNX1 and PEBP2␤/CBF␤ in the cytoplasm was observed. These results suggest that the sequestration of PEBP2␤/CBF␤ by mutant RUNX1 may cause the inhibitory effects. While haploinsufficiency of RUNX1 causes FPD/AML in some families (deletions and frameshifts), mutant RUNX1 proteins (missense and nonsense) may also inhibit wild-type RUNX1, possibly creating a higher propensity to develop leukemia. This is consistent with the hypothesis that a second mutation has to occur, either in RUNX1 or another gene, to cause leukemia among individuals harboring RUNX1 FPD/AML mutations and that the propensity to acquire these additional mutations is determined, at least partially, by the initial RUNX1 mutation. IntroductionThe most frequent mutations associated with leukemia are recurrent somatic chromosomal translocations or inversions, many of which involve the polyomavirus enhancer-binding protein or core-binding factor transcriptional regulation complex (PEBP2/CBF). Several translocations involve the ␣ subunit of this complex, the RUNX1 gene (also called AML1, CBF␣2, or PEBP2␣B) on chromosome 21q22.1 (t(8;21), t(3;21), and t(12;21)). Additionally, the ␤ subunit of the complex, PEBP2␤ also called CBF␤, is disrupted in inv(16)(p13;q22). 1 An abundance of evidence points to the existence of genes that predispose to hematologic malignancies. However, large multiple-generation families with hematologic malignancies alone are rare. 2 Only 2 loci for familial hematologic malignancies have been identified to date, 1 on chromosome 21q22.1 3 and the other on 16q22. 4,5 These loci contain RUNX1 and PEBP2␤/CBF␤, respectively.Studies of families that demonstrate single-gene inheritance for leukemia predisposition should help to identify the genes and mechanisms involved in the first steps of leukemia development. The autosomal dominant familial platelet disorder (FPD)/ AML (acute myelogenous leukemia; Online Mendelian Inheritance in Man no. 601399) is a good model to validate this hypothesis because, in addition to developing thrombocytopenia, patients show a propensity for progression to myelodysplasia and acute myeloid leuke...

Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic 1,2 . We therefore screened GFI1 as a candidate for association with neutropenia in affected individuals without mutations in ELA2 (encoding neutrophil elastase), the most common cause of severe congenital neutropenia (SCN; ref. 3). We found dominant negative zinc finger mutations that disable transcriptional repressor activity. The phenotype also includes immunodeficient lymphocytes and production of a circulating population of myeloid cells that appear immature. We show by chromatin immunoprecipitation, gel shift, reporter assays and elevated expression of ELA2 in vivo in neutropenic individuals that GFI1 represses ELA2, linking these two genes in a common pathway involved in myeloid differentiation.Low neutrophil numbers lead to opportunistic infections. There are two hereditary human neutropenia syndromes: cyclic hematopoiesis 4 , comprising three-week oscillations of blood cells, and SCN 3 , consisting of statically low neutrophil counts progressing to leukemia. Heterozygous mutations of ELA2 cause cyclic hematopoiesis and about two-thirds of SCN cases. Mutations in WAS (different from those that cause Wiskott-Aldrich thrombocytopenia) also cause SCN 5 . Owing to its severity, SCN usually arises from new mutations, and additional genes associated with neutropenia have not yet been identified.

Human cyclic haematopoiesis (cyclic neutropenia, MIM 162800) is an autosomal dominant disease in which blood-cell production from the bone marrow oscillates with 21-day periodicity. Circulating neutrophils vary between almost normal numbers and zero. During intervals of neutropenia, affected individuals are at risk for opportunistic infection. Monocytes, platelets, lymphocytes and reticulocytes also cycle with the same frequency. Here we use a genome-wide screen and positional cloning to map the locus to chromosome 19p13.3. We identified 7 different single-base substitutions in the gene (ELA2) encoding neutrophil elastase (EC 3. 4.21.37, also known as leukocyte elastase, elastase 2 and medullasin), a serine protease of neutrophil and monocyte granules, on unique haplotypes in 13 of 13 families as well as a new mutation in a sporadic case. Neutrophil elastase (a 240-aa mature protein predominantly found in neutrophil granules) is the target for protease inhibition by alpha1-antitrypsin, and its unopposed release destroys tissue at sites of inflammation. We hypothesize that a perturbed interaction between neutrophil elastase and serpins or other substrates may regulate mechanisms governing the clock-like timing of haematopoiesis.

Congenital neutropenia and cyclic neutropenia are disorders of neutrophil production predisposing patients to recurrent bacterial infections. Recently the locus for autosomal dominant cyclic neutropenia was mapped to chromosome 19p13.3, and this disease is now attributable to mutations of the gene encoding neutrophil elastase (the ELA2 gene). The authors hypothesized that congenital neutropenia is also due to mutations of neutrophil elastase. Patients with congenital neutropenia, cyclic neutropenia, or Shwachman-Diamond syndrome were referred to the Severe Chronic Neutropenia International Registry. Referring physicians provided hematologic and clinical data. Mutational analysis was performed by sequencing polymerase chain reaction (PCR)-amplified genomic DNA for each of the 5 exons of the neutrophil ELA2 gene and 20 bases of the flanking regions. RNA from bone marrow mononuclear cells was used to determine if the affected patients expressed both the normal and the abnormal transcript. Twenty-two of 25 patients with congenital neutropenia had 18 different heterozygous mutations. Four of 4 patients with cyclic neutropenia and 0 of 3 patients with Shwachman-Diamond syndrome had mutations. For 5 patients with congenital neutropenia having mutations predicted to alter RNA splicing or transcript structure, reverse transcriptase-PCR showed expression of both normal and abnormal transcripts. In cyclic neutropenia, the mutations appeared to cluster near the active site of the molecule, whereas the opposite face was predominantly affected by the mutations found in congenital neutropenia. This study indicates that mutations of the gene encoding neutrophil elastase are probably the most common cause for severe congenital neutropenia as well as the cause for sporadic and autosomal dominant cyclic neutropenia.

Cyclic hematopoiesis is a stem cell disease in which the number of neutrophils and other blood cells oscillates in weekly phases. Autosomal dominant mutations of ELA2, encoding the protease neutrophil elastase, found in lysosome-like granules, cause cyclic hematopoiesis and most cases of the pre-leukemic disorder severe congenital neutropenia (SCN; ref. 3) in humans. Over 20 different mutations of neutrophil elastase have been identified, but their consequences are elusive, because they confer no consistent effects on enzymatic activity. The similar autosomal recessive disease of dogs, canine cyclic hematopoiesis, is not caused by mutations in ELA2 (data not shown). Here we show that homozygous mutation of the gene encoding the dog adaptor protein complex 3 (AP3) beta-subunit, directing trans-Golgi export of transmembrane cargo proteins to lysosomes, causes canine cyclic hematopoiesis. C-terminal processing of neutrophil elastase exposes an AP3 interaction signal responsible for redirecting neutrophil elastase trafficking from membranes to granules. Disruption of either neutrophil elastase or AP3 perturbs the intracellular trafficking of neutrophil elastase. Most mutations in ELA2 that cause human cyclic hematopoiesis prevent membrane localization of neutrophil elastase, whereas most mutations in ELA2 that cause SCN lead to exclusive membrane localization.

BackgroundThis study was performed to evaluate anti-inflammatory and immune modulating properties of the probiotic, spore-forming bacterial strain: Bacillus coagulans: GBI-30, (PTA-6086, GanedenBC30TM). In addition, cell wall and metabolite fractions were assayed separately to address whether biological effects were due to cell wall components only, or whether secreted compounds from live bacteria had additional biological properties. The spores were heat-activated, and bacterial cultures were grown. The culture supernatant was harvested as a source of metabolites (MTB), and the bacteria were used to isolate cell wall fragments (CW). Both of these fractions were compared in a series of in vitro assays.ResultsBoth MTB and CW inhibited spontaneous and oxidative stress-induced ROS formation in human PMN cells and increased the phagocytic activity of PMN cells in response to bacteria-like carboxylated fluorospheres. Both fractions supported random PMN and f-MLP-directed PMN cell migration, indicating a support of immune surveillance and antibacterial defense mechanisms. In contrast, low doses of both fractions inhibited PMN cell migration towards the inflammatory mediators IL-8 and LTB4. The anti-inflammatory activity was strongest for CW, where the PMN migration towards IL-8 was inhibited down to dilutions of 1010.Both MTB and CW induced the expression of the CD69 activation marker on human CD3- CD56+ NK cells, and enhanced the expression of CD107a when exposed to K562 tumor cells in vitro.The fractions directly modulated cytokine production, inducing production of the Th2 cytokines IL-4, IL-6, and IL-10, and inhibiting production of IL-2.Both fractions further modulated mitogen-induced cytokine production in the following manner: Both fractions enhanced the PHA-induced production of IL-6 and reduced the PHA-induced production of TNF-alpha. Both fractions enhanced the PWM-induced production of TNF-alpha and IFN-gamma. In addition, MTB also enhanced both the PHA- and the PWM-induced expression of IL-10.ConclusionThe data suggest that consumption of GanedenBC30TM may introduce both cell wall components and metabolites that modulate inflammatory processes in the gut. Both the cell wall and the supernatant possess strong immune modulating properties in vitro. The anti-inflammatory effects, combined with direct induction of IL-10, are of interest with respect to possible treatment of inflammatory bowel diseases as well as in support of a healthy immune system.

The impact of chronic inflammatory conditions on immune function is substantial, and the simultaneous application of anti-inflammatory and immune modulating modalities has potential for reducing inflammation-induced immune suppression. Sorghum-based foods, teas, beers, and extracts are used in traditional medicine, placing an importance on obtaining an increased understanding of the biological effects of sorghum. This study examined selected anti-inflammatory and immune-modulating properties in vitro of Jobelyn™, containing the polyphenol-rich leaf sheaths from a West African variant of Sorghum bicolor (SBLS). Freshly isolated primary human polymorphonuclear (PMN) and mononuclear cell subsets were used to test selected cellular functions in the absence versus presence of aqueous and ethanol extracts of SBLS. Both aqueous and nonaqueous compounds contributed to reduced reactive oxygen species formation by inflammatory PMN cells, and reduced the migration of these cells in response to the inflammatory chemoattractant leukotriene B4. Distinct effects were seen on lymphocyte and monocyte subsets in cultures of peripheral blood mononuclear cells. The aqueous extract of SBLS triggered robust upregulation of the CD69 activation marker on CD3- CD56+ natural killer (NK) cells, whereas the ethanol extract of SBLS triggered similar upregulation of CD69 on CD3+ CD56+ NKT cells, CD3+ T lymphocytes, and monocytes. This was accompanied by many-fold increases in the chemokines RANTES/CCL5, Mip-1α/CCL3, and MIP-1β/CCL4. Both aqueous and nonaqueous compounds contribute to anti-inflammatory effects, combined with multiple effects on immune cell activation status. These observations may help suggest mechanisms of action that contribute to the traditional use of sorghum-based products, beverages, and extracts for immune support.