lactoferrin and Leukocyte-Disorders

Neutrophil-specific granule deficiency (SGD) is a rare autosomal recessive primary immunodeficiency characterized by bilobed neutrophil nuclei and lack of neutrophil-specific granule proteins such as lactoferrin. A deficiency of a myeloid-specific transcription factor, CCAAT/enhancer binding protein-epsilon (C/EBPε), has been identified as a cause of SGD. C/EBPε binds to DNA though its basic leucine zipper (bZIP) domain, and regulates terminal differentiation of neutrophils and expression of specific granule genes. Homozygous frameshift mutations resulting in loss of the bZIP domain have been reported in two patients with SGD. A recent observation showed that a homozygous 2-aa deletion in the bZIP domain with normal DNA-binding and dimerization abilities causes SGD by impairing protein-protein interactions with other transcription factors, indicating that multiple molecular mechanisms can lead to SGD. Studies of patient-derived mutations and analysis of C/EBPε knockout mice have shown the importance of the bZIP domain for the essential functions of C/EBPε.

Topics: Animals; CCAAT-Enhancer-Binding Proteins; Cytoplasmic Granules; Humans; Lactoferrin; Leucine Zippers; Leukocyte Disorders; Neutrophils; Protein Interaction Domains and Motifs

Studies devoted to evaluating the outcome of different doses of probiotics are very limited, especially for multistrain formulations. In this context, we performed an intervention study that aimed to compare the effect of the administration of two doses (7 billion and 70 billion bacterial cells) of a multistrain probiotic formulation on the persistence of the four probiotic strains that were present in the product in the fecal samples collected from healthy subjects. The overall persistence of the probiotic strains was significantly higher for the 70 billion formulation than for the 7 billion formulation. Furthermore, probiotic strains were detected earlier and for longer for the 70 billion formulation compared to those for the 7 billion formulation. All probiotic strains were recovered alive from the 70 billion preparation, whereas recovery was not possible in a few fecal samples upon administration of the 7 billion preparation. In addition, the overall number of viable probiotic cells recovered on day 14 (i.e., the last day of consumption) was significantly higher for the 70 billion formulation than that for the 7 billion formulation. Finally, we found that the viability of the probiotic cells was stable over the course of the trial independent of volunteers' handling, demonstrating good manufacturing of the product. In conclusion, this study demonstrated that strains belonging to different taxa may coexist in the human gastrointestinal tract upon ingestion of a multispecies probiotic formulation. Moreover, this study suggests that higher doses of bacterial cells in probiotic formulations may permit a higher, earlier, and longer recovery of the probiotics in the feces of healthy adults.

Topics: Bifidobacterium; Cell Survival; DNA, Bacterial; Feces; Gastrointestinal Tract; Humans; Lactobacillus; Lactoferrin; Leukocyte Disorders; Probiotics

Topics: CCAAT-Enhancer-Binding Proteins; Chromosomal Proteins, Non-Histone; Homozygote; Humans; Immunologic Deficiency Syndromes; Infant, Newborn; Lactoferrin; Leukocyte Disorders; Male; Neutrophils

SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2) has recently been shown to have a critical role in granulopoiesis in humans, mice, and zebrafish. Our patient presented with delayed cord separation, failure to thrive, and sepsis. Retrospective whole-exome sequencing confirmed a homozygous splice-site mutation in SMARCD2.. We sought to provide the second description of human SMARCD2 deficiency and the first functional analysis of human primary SMARCD2-deficient cells.. Heparinized venous blood and bone marrow were collected from the patient after obtaining informed consent. Patient leukocytes and CD34. Circulating neutrophils appeared phenotypically immature, lacking multilobed nuclei, and neutrophil granules lacked lactoferrin but showed normal levels of myeloperoxidase. Neutrophil oxidative burst was preserved in response to phorbol 12-myristate 13-acetate. Patient bone marrow-derived neutrophils and white blood cells showed a severely impaired chemotactic response. Furthermore, white blood cells showed defective in vitro killing of Staphylococcus aureus, consistent with a specific granule deficiency. Finally, patient bone marrow-derived CD34. This report highlights an important role for SMARCD2 in human myelopoiesis and the curative effect of hematopoietic stem cell transplantation for the hematopoietic features of SMARCD2 deficiency.

Topics: Biomarkers; Cell Differentiation; Chemotaxis, Leukocyte; Chromosomal Proteins, Non-Histone; Cytotoxicity, Immunologic; Female; Genetic Predisposition to Disease; Homozygote; Humans; Immunophenotyping; Infant, Newborn; Lactoferrin; Leukocyte Disorders; Mutation; NADPH Oxidases; Neutrophils; Pedigree; Phenotype; Respiratory Burst; RNA Splice Sites

CCAAT/enhancer binding protein epsilon (C/EBPε), a myeloid-specific transcription factor, plays an important role in granulopoiesis. A loss-of-function mutation in this protein can result in an abnormal development of neutrophils and eosinophils, known as neutrophil-specific granule deficiency (SGD). The transcriptional activity of C/EBPε is regulated by interactions with other transcription factors and/or post-translational modification, including acetylation. Previously, we reported a novel SGD patient who had a homozygous mutation for two amino acids, arginine (R247) and serine (S248), which were deleted in the basic leucine zipper domain of C/EBPε (ΔRS) and exhibited loss of transcriptional activity with aberrant protein-protein interactions. In the present study, we found that a single amino acid deletion of either R247 (ΔR) or S248 (ΔS) was sufficient for the loss of C/EBPε transcriptional activity, while an amino acid substitution at S248 to alanine in C/EBPε (SA) had comparable transcriptional activity with the wild-type C/EBPε (WT). Although acetylation at lysine residues (K121 and K198) is indispensable for C/EBPε transcriptional activity, an acetylation mimic form of ΔRS (ΔRS-K121/198Q) did not exhibit the transcriptional activity. Interestingly, we discovered that ΔRS, ΔR, ΔS, and ΔRS-K121/198Q interacted with histone deacetylase 1 (HDAC1), whereas WT and SA did not. Furthermore, the proteoglycan 2/eosinophil major basic protein induction activity of ΔRS, ΔR, and ΔS could be restored by the HDAC inhibitor, trichostatin A (TSA), and protein-protein interactions between ΔRS and Gata1 could also be recovered by TSA treatment. Taken together, our results show that TSA has the potential to restore the transcriptional activity of ΔRS, indicating that the inhibition of HDAC1 could be a molecularly targeted treatment for SGD with ΔRS.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; CCAAT-Enhancer-Binding Proteins; GATA1 Transcription Factor; HEK293 Cells; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lactoferrin; Leukocyte Disorders; Mice; NIH 3T3 Cells; Protein Interaction Maps; Sequence Deletion

Neutrophil-specific granule deficiency (SGD) is a rare autosomal recessive primary immunodeficiency characterized by neutrophil dysfunction, bilobed neutrophil nuclei and lack of neutrophil-specific granules. Defects in a myeloid-specific transcription factor, CCAAT/enhancer binding protein-ε (C/EBPε), have been identified in two cases in which homozygous frameshift mutations led to loss of the leucine zipper domain. In this study, we report a 55-y-old woman affected with SGD caused by a novel homozygous 2-aa deletion (ΔRS) in the leucine zipper domain of the C/EBPε gene. The patient showed characteristic neutrophil abnormalities and recurrent skin infections; however, there was no history of deep organ infections. Biochemical analysis revealed that, in contrast to the two frameshift mutations, the ΔRS mutant maintained normal cellular localization, DNA-binding activity, and dimerization, and all three mutants exhibited marked reduction in transcriptional activity. The ΔRS mutant was defective in its association with Gata1 and PU.1, as well as aberrant cooperative transcriptional activation of eosinophil major basic protein. Thus, the ΔRS likely impairs protein-protein interaction with other transcription factors, resulting in a loss of transcriptional activation. These results further support the importance of the leucine zipper domain of C/EBPε for its essential function, and indicate that multiple molecular mechanisms lead to SGD.

Topics: Adult; Base Sequence; CCAAT-Enhancer-Binding Proteins; Cytoplasmic Granules; Eosinophil Major Basic Protein; Female; GATA1 Transcription Factor; Gene Expression Regulation; Homozygote; Humans; Lactoferrin; Leukocyte Disorders; Male; Middle Aged; Molecular Sequence Data; Neutrophils; Protein Binding; Protein Structure, Tertiary; Proteoglycans; Proto-Oncogene Proteins; Sequence Deletion; Signal Transduction; Trans-Activators; Transcription, Genetic

Avian pathogenic Escherichia coli (APEC) causes respiratory and systemic disease in poultry. Sequencing of a multilocus sequence type 95 (ST95) serogroup O1 strain previously indicated that APEC resembles E. coli causing extraintestinal human diseases. We sequenced the genomes of two strains of another dominant APEC lineage (ST23 serogroup O78 strains χ7122 and IMT2125) and compared them to each other and to the reannotated APEC O1 sequence. For comparison, we also sequenced a human enterotoxigenic E. coli (ETEC) strain of the same ST23 serogroup O78 lineage. Phylogenetic analysis indicated that the APEC O78 strains were more closely related to human ST23 ETEC than to APEC O1, indicating that separation of pathotypes on the basis of their extraintestinal or diarrheagenic nature is not supported by their phylogeny. The accessory genome of APEC ST23 strains exhibited limited conservation of APEC O1 genomic islands and a distinct repertoire of virulence-associated loci. In light of this diversity, we surveyed the phenotype of 2,185 signature-tagged transposon mutants of χ7122 following intra-air sac inoculation of turkeys. This procedure identified novel APEC ST23 genes that play strain- and tissue-specific roles during infection. For example, genes mediating group 4 capsule synthesis were required for the virulence of χ7122 and were conserved in IMT2125 but absent from APEC O1. Our data reveal the genetic diversity of E. coli strains adapted to cause the same avian disease and indicate that the core genome of the ST23 lineage serves as a chassis for the evolution of E. coli strains adapted to cause avian or human disease via acquisition of distinct virulence genes.

Topics: Animals; Biological Evolution; DNA, Bacterial; Escherichia coli; Fimbriae Proteins; Gene Expression Regulation, Bacterial; Genome, Bacterial; Lactoferrin; Leukocyte Disorders; Molecular Sequence Annotation; Molecular Sequence Data; Mutation; Phylogeny; Poultry Diseases; Turkeys; Virulence

Topics: Adult; Humans; Lactoferrin; Leukocyte Disorders; Male; Neutrophils