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Lactoferrin (Lf), a multiple functional natural immune protein, is widely distributed in mammalian milk and glandular secretions (bile, saliva, tears and nasal mucosal secretions, etc.). In the previous study, we found that Lf plays an anti-inflammatory and anti-tumorigenesis role in AOM/DSS (azoxymethane/dextran sulfate sodium) induced mouse colitis-associated colon cancer model. Although we found that Lf has anti-inflammatory effects in chronic inflammation, its specific role and mechanisms in acute inflammation have not been clarified. Here, we reported that the expression levels of Lf were significantly increased when the organism was infected by Gram-negative bacteria. We then explored the role and potential mechanism of Lf in lipopolysaccharide (LPS)-induced acute inflammation. In the LPS-induced acute abdominal inflammation model, Lf deficiency aggravated inflammatory response and promoted macrophage chemotaxis to the inflammation site. Lf inhibited macrophage chemotaxis by suppressing the expression of macrophage-associated chemokines Ccl2 and Ccl5. Highly activated NF-κB signaling in Lf -/- mice was responsible for the high expression of Ccl2 and Ccl5. Our results suggested that the anti-inflammatory effect of Lf offers a new potential treatment for acute inflammatory diseases., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Lactoferrin ( Ltf ), a naturally active glycoprotein, possesses anti-inflammatory, anti-microbial, anti-tumor, and immunomodulatory activities. Many published studies have indicated that Ltf modulates the proliferation of stem cells. However, the role of Ltf in the proliferation of satellite cells, an important cell type in muscle regeneration, has not yet been reported. Here, by using Ltf systemic knockout mice, we illustrate the role of Ltf in skeletal muscle. Results shows that Ltf deficiency impaired proliferation of satellite cells (SCs) and the regenerative capability of skeletal muscle. Mechanistic studies showed that ERK1/2 phosphorylation was significantly downregulated after Ltf deletion in SCs. Simultaneously, the cell cycle-related proteins cyclin D and CDK4 were significantly downregulated. Intervention with exogenous recombinant lactoferrin ( R-Ltf ) at a concentration of 1000 μg/mL promoted proliferation of SCs. In addition, intraperitoneal injection of Ltf effectively ameliorated the skeletal muscle of mice injured by 1.2% BaCl 2 solution. Our results suggest a protective effect of Ltf in the repair of skeletal muscle damage. Ltf holds promise as a novel therapeutic agent for skeletal muscle injuries.

Background: Specific granule deficiency (SGD) is a rare immunodeficiency associated with CCAT/enhancer-binding protein epsilon (CEBPE) gene variants. It can cause severe recurrent infections and is lethal without successful stem cell transplantation. Few cases with SGD of both type 1 and type 2 have been described in the literature. In this study, we present the first report of a case with a novel homozygous c.511 C > T (p.Gln171Ter) mutation in the SMARCD2 gene of SGD type 2, which was successfully treated with bone marrow transplantation. Case: A male infant presented to our neonatal intensive care unit on the second day of life with an icteric appearance and mild hypotonia. He was evaluated for immunodeficiency as the cause of delayed cord separation and refractory neutropenia. At 6 weeks of age, SGD type 2 with a new variant was diagnosed and successfully treated by bone marrow transplantation. Conclusion: SGD is an immunodeficiency disease that is quite rare. However, we believe that SGD diagnosis and associated new variants can be detected more frequently with the widespread use of all whole-exome sequencing techniques.

Background: 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., Objective: We sought to provide the second description of human SMARCD2 deficiency and the first functional analysis of human primary SMARCD2-deficient cells., Methods: Heparinized venous blood and bone marrow were collected from the patient after obtaining informed consent. Patient leukocytes and CD34 + cells were then isolated, phenotyped, and assessed functionally., Results: 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 + cells showed markedly impaired in vitro expansion and differentiation toward the neutrophil lineage. Before her molecular diagnosis, our patient underwent hematopoietic stem cell transplantation and is well 8 years later., Conclusions: 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., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Lactoferrin, an innate immunity molecule, is involved in anti-inflammatory, anti-microbial, and anti-tumor activities. We previously reported that lactoferrin is downregulated in specimens of nasopharyngeal carcinoma and negatively associated with tumor progression and metastasis of patients with nasopharyngeal carcinoma. However, the relationship between lactoferrin and the pro-metastatic microenvironment has not been reported yet. Here, by using the lactoferrin knockout mouse, we found that lactoferrin deficiency facilitated melanoma cells metastasizing to lungs, through recruiting myeloid-derived suppressor cells (MDSCs) in the lungs. Mechanistic studies showed that in the lung microenvironment of the lactoferrin knockout mice, the TLR9 signaling was the most repressed signaling. Lactoferrin can induce MDSCs differentiation and apoptosis, as well as upregulate TLR9 expression. TLR9 agonist or lactoferrin treatment can rescue this phenotype in the tumor metastasis mouse model. Our results suggest a protective role of lactoferrin in cancer metastasis, along with a deficiency in certain components of the innate immune system, may lead to a pro-metastatic tumor microenvironment.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

Specific granule deficiency (SGD) is a rare disorder characterized by abnormal neutrophils evidenced by reduced granules, absence of granule proteins, and atypical bilobed nuclei. Mutations in CCAAT/enhancer-binding protein-ε ( CEBPE ) are one molecular etiology of the disease. Although C/EBPε has been studied extensively, the impact of CEBPE mutations on neutrophil biology remains elusive. Here, we identified two SGD patients bearing a previously described heterozygous mutation (p.Val218Ala) in CEBPE . We took this rare opportunity to characterize SGD neutrophils in terms of granule distribution and protein content. Granules of patient neutrophils were clustered and polarized, suggesting that not only absence of specific granules but also defects affecting other granules contribute to the phenotype. Our analysis showed that remaining granules displayed mixed protein content and lacked several glycoepitopes. To further elucidate the impact of mutant CEBPE , we performed detailed proteomic analysis of SGD neutrophils. Beside an absence of several granule proteins in patient cells, we observed increased expression of members of the linker of nucleoskeleton and cytoskeleton complex (nesprin-2, vimentin, and lamin-B2), which control nuclear shape. This suggests that absence of these proteins in healthy individuals might be responsible for segmented shapes of neutrophilic nuclei. We further show that the heterozygous mutation p.Val218Ala in CEBPE causes SGD through prevention of nuclear localization of the protein product. In conclusion, we uncover that absence of nuclear C/EBPε impacts on spatiotemporal expression and subsequent distribution of several granule proteins and further on expression of proteins controlling nuclear shape.

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.

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., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Aggregatibacter actinomycetemcomitans, a periodontopathogen, has been associated with several systemic diseases. Herein, we report the protective effect of human lactoferrin (hLF) during A. actinomycetemcomitans bacteremia in lactoferrin knockout (LFKO(-/-)) mice. The prophylactic, concurrent, and therapeutic intravenous (i.v.) administrations of hLF significantly cleared the bacteria from blood and organs. Nevertheless, all modes of hLF administration significantly decreased the concentrations of serum proinflammatory cytokines, such as interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, and IL-12p70. Additionally, hLF administration significantly decreased hepatic and splenic proinflammatory cytokine expression levels compared to those in the non-hLF-treated group. Furthermore, administration of hLF decreased the serum C-reactive protein level, inducible nitric oxide synthase (iNOS) and myeloperoxidase (MPO) gene expression levels in liver and spleen. hLF treatment has also resulted in a 6-fold decrease in spleen weight with the migration of typical inflammatory cells in infected mice as a result of decreased inflammatory response. These results reveal that hLF protects against A. actinomycetemcomitans bacteremia, as indicated by rapid bacterial clearance and decreased host proinflammatory mediators.

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.

Lactoferrin (LF) is an iron-binding protein found in milk, mucosal secretions, and the secondary granules of neutrophils in which it is considered to be an important factor in the innate immune response against microbial infections. Moreover, LF deficiency in the secondary granules of neutrophils has long been speculated to contribute directly to the hypersusceptibility of specific granule deficiency (SGD) patients to severe, life-threatening bacterial infections. However, the exact physiological significance of LF in neutrophil-mediated host defense mechanisms remains controversial and has not yet been clearly established in vivo using relevant animal models. In this study, we used lactoferrin knockout (LFKO) mice to directly address the selective role of LF in the host defense response of neutrophils and to determine its contribution, if any, to the phenotype of SGD. Neutrophil maturation, migration, phagocytosis, granule release, and antimicrobial response to bacterial challenge were unaffected in LFKO mice. Interestingly, a stimulus-dependent defect in the oxidative burst response of LFKO neutrophils was observed in that normal activation was seen in response to opsonized bacteria whereas an impaired response was evident after phorbol myristate-13-acetate stimulation. Taken together, these results indicate that although LF deficiency alone is not a primary cause of the defects associated with SGD, this protein does play an immunomodulatory role in the oxidative burst response of neutrophils.

The mechanisms of hereditary deficiency of R binder, which originates in neutrophils and exocrine gland epithelium, are unknown and may be multiple. This led us to examine if defective R binder synthesis also involves proteins that colocalize with it in neutrophil-specific granules and exocrine epithelial cells and may be under common regulatory control. Stored plasma and saliva samples from five unrelated R binder-deficient patients and control subjects were assayed for R binder, lactoferrin, cationic antimicrobial protein-18, neutrophil gelatinase-associated lipocalin, gelatinase, lysozyme, and myeloperoxidase. One patient, patient A, had lactoferrin levels below the limits of detection in both plasma and saliva in addition to his R binder deficiency. Although his deficiency involved lactoferrin as well, he had no history of predisposition to infection. PCR amplification of his R binder gene promoter region and the beginning of the first exon revealed no DNA abnormalities. His son and the son of his equally deficient brother, both presumptive heterozygotes, had mild deficiency of both R binder and lactoferrin. The results show that R binder deficiency exists in at least two forms. One, presumably the less common of the two forms, is the new hereditary entity described here, which is characterized by deficiency of more than one specific granule protein in both plasma and saliva. Despite this more widely distributed absence of the proteins than is found in congenital specific granule deficiency, infection posed no clinical problem in the affected patient., (Copyright 2001 Wiley-Liss, Inc.)

We analyzed lactoferrin expression in 78 samples from patients with sporadic breast cancer and found 31/78 negative for mRNA expression. Similar results were obtained by immuno-histochemical localization of the lactoferrin protein. We did not find relationship between lactoferrin expression and clinical parameters. We investigated for the absent lactoferrin expression in some cases of breast cancer. In 68 of the samples analyzed, we found an inverse correlation between estrogen receptor expression and lactoferrin expression (P < 0,0001), thus indicating that regulation by the estrogen receptor is not the main element responsible for the expression of lactoferrin in breast cancer. Analysis of methylation of the lactoferrin genomic DNA extracted from the same patients revealed that the degree of methylation does not explain the observed absence of lactoferrin. The 937 bp lactoferrin promoter was investigated for possible mutations. By single-strand conformation polymorphism analysis one polymorphic site was found and characterized.

Previous studies on neutrophils in patients with the myelodysplastic syndromes (MDS) have indicated deficiencies in the contents of primary and secondary granules. However, the granule membrane remains virtually unstudied despite its essential role in the dynamic function of the cytoplasmic granules. In this study, we examined the membrane glycoproteins of primary and secondary granules of peripheral blood and/or bone marrow neutrophils using the monoclonal antibody H36/71 to CD15 glycoproteins. In addition, myeloperoxidase activity and antigen, elastase and lactoferrin were also studied using cytochemical and immunocytochemical stains. A total of 216 patients were included. Deficiencies of granule membrane glycoproteins were the most common, detected in 49%, followed by myeloperoxidase activity (17%), elastase (16%), myeloperoxidase antigen (9%), and lactoferrin (8%). Multiple deficiencies always included granule membrane deficiency. We conclude that granule membrane defects are common in MDS, may provide a common mechanism for multiple granule deficiencies, and may prove to be an additional abnormality associated with granulocyte dysfunction.

To test whether the presence of airway inflammatory markers differentiated babies with hyaline membrane disease (HMD) who recovered (n = 18) from those in whom bronchopulmonary dysplasia (BPD) developed (n = 18), tracheal aspirate samples from 36 newborn infants with HMD who underwent intubation were collected during days 1 to 28 of life and analyzed for the mucosal antimicrobial proteins lactoferrin and lysozyme. For babies with HMD in whom BPD developed, lactoferrin concentrations were decreased during the first 4 days of life (7 +/- 3, 14 +/- 3, 18 +/- 3, and 18 +/- 3 micrograms/ml, respectively) in comparison with those in babies with HMD who recovered (23 +/- 8, 29 +/- 6, 41 +/- 9, and 81 +/- 19 micrograms/ml); group differences reached statistical significance on days 3 and 4 (p less than 0.05). Lysozyme levels in the secretions of babies with BPD were also lower on day 3 (31 +/- 5 micrograms/ml) than in those of babies who recovered (54 +/- 7.5 micrograms/ml). For babies with BPD whose endotracheal tube remained in place beyond day 4, lysozyme levels on days 5 to 12 were significantly lower for those classified as having severe BPD than for those with mild to moderate BPD. Because lysozyme and lactoferrin are products of serous cells found in submucous glands, it seems possible that the relative immaturity of submucous glands may influence the development of BPD.

We have studied a young male with lactoferrin deficiency and a bleeding tendency responsive to cryoprecipitate. This child has had increased bleeding following surgical procedures and a variably prolonged template bleeding time. The patient has a normal platelet count, normal in vitro platelet ATP secretion and aggregation in response to a variety of agonists, and normal concentration of plasma-von Willebrand factor ristocetin cofactor activity and antigen. Analysis of plasma-vWf multimers by agarose gel electrophoresis consistently demonstrated a subtle decrease in the largest vWf multimers. In contrast, analysis of the patient's platelet-vWf revealed normal vWf:Ag, decreased vWf ristocetin cofactor activity, and a striking absence of the high and intermediate size molecular weight vWf multimers. Analysis of surface bound platelet-vWf demonstrated normal amounts on the surface of unstimulated platelets, but after thrombin stimulation the platelet-vWf surface expression did not increase. This lack of increased platelet-vWf surface expression resulted from decreased binding of secreted platelet-vWf to be surface of stimulated platelets. These data suggest that the patient's bleeding tendency may be related to a defect in his platelet-vWf structure and/or mobilization. This case represents a unique demonstration of an abnormality of platelet-vWf in the presence of normal plasma-vWf, and supports the data indicating an important role for platelet-vWf in primary hemostasis.

The iron-binding proteins lactoferrin (LF) and H-ferritin have been implicated in the negative regulation of myelopoiesis in vitro and in vivo. The present studies evaluated the functional activity of affinity-purified LF from polymorphonuclear neutrophils (PMN) of patients with chronic myelogenous leukemia (CML) and LF/H-ferritin-cell interactions in a nonleukemic patient with LF deficiency with normal levels of circulating blood leukocytes. Affinity-purified CML-PMN-LF was found to be qualitatively deficient as a suppressor of the release of colony-stimulating factors from mononuclear blood cells, adding to previous information from our group documenting defective LF-cell interactions in CML. LF was detected by immunoradiometric assay in PMN of the patient with LF deficiency, but at a much lower level than normal. This LF was found, however, to be active as a suppressor molecular against the patient's cells and normal donor cells. Patient cells were as responsive as normal cells to effects of purified milk LF. Decreased LF levels in this patient were associated with increased levels of monocyte H-ferritin inhibitory activity, consistent with the known suppressive effects in vitro of LF on H-ferritin release from monocytes. Patient marrow hematopoietic progenitor cells were as responsive as progenitors from normal donors to suppression by purified H-ferritin and prostaglandin E1. These results are consistent with a role of LF and H-ferritin in the control of myelopoiesis in this patient.

Previous studies have identified patients with susceptibility to bacterial infection associated with lactoferrin deficiency in dysmorphic neutrophils containing abnormal or no secondary granules and abnormal nuclear segmentation. We have investigated the subcellular distribution of vicinal glycol-containing complex carbohydrates in marrow and blood myeloid cells of such a patient using the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining method and have examined the response of these neutrophils to the degranulating agents N-formylmethionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA). As in normal specimens, immature primary granules were strongly PA-TCH-SP reactive; however, unlike normal specimens, masking of PA-TCH-SP reactivity did not occur in mature primary granules. Endoplasmic reticulum demonstrated moderately strong PA-TCH-SP staining, in contrast to absent staining of this organelle in normal promyelocytes and consistent with abnormal primary granule genesis. Small abnormal elongated granules (0.1-0.2 micron in diameter) were identified at the myelocyte state of development and were the predominant granule type in late neutrophils. These granules were identified as secondary granules on the basis of their PA-TCH-SP positivity and were differentiated from primary and tertiary granules on the basis of a lack of peroxidase, acid phosphatase, and sulfate staining. When the neutrophils were exposed to PMA, cell aggregation occurred, and the abnormal granules degranulated in a manner similar to the degranulation observed with normal secondary granules. Although PA-TCH-SP staining of the plasma membrane appeared normal, a decrease in FMLP receptors was demonstrated. Thus, a defect(s) is present in complex carbohydrate distribution and staining that involves primary and secondary granules and possibly the plasmalemma of neutrophils from this patient. This results in abnormal packaging of primary granules and synthesis of normal numbers of secondary granules that are qualitatively and morphologically abnormal, but can be recruited to degranulate with PMA.

Patients with specific granule deficiency (SGD) develop recurrent severe bacterial skin infections. Neutrophils from patients with SGD are deficient in lactoferrin (Lf), an antimicrobial protein commonly found in many mucosal secretions. Unstimulated and stimulated nasal secretions, saliva, and tears were collected from a patient with SGD and from normal control subjects and were analyzed for Lf. The secretions from the patient contained normal values of Lf, suggesting that the glands secrete Lf from a source other than neutrophils. Immunohistochemical staining of normal nasal mucosa demonstrated that Lf is localized within serous submucosal gland cells and that neutrophils are not normally observed in the nasal mucosa. These findings suggest that glandular tissues produce and locally secrete Lf by processes that are independent of neutrophil degranulation.

Neutrophil specific granule deficiency (SGD) is a congenital disorder associated with an impaired inflammatory response and a deficiency of several granule proteins. The underlying abnormality causing the deficiencies is unknown. We examined mRNA transcription and protein synthesis of two neutrophil granule proteins, lactoferrin and myeloperoxidase in SGD. Metabolically labeled SGD nucleated marrow cells produced normal amounts of myeloperoxidase, but there was no detectable synthesis of lactoferrin. Transcripts of the expected size for lactoferrin were detectable in the nucleated marrow cells of two SGD patients, but were markedly diminished in abundance when compared with normal nucleated marrow cell RNA. Because lactoferrin is secreted by the glandular epithelia of several tissues, we also assessed lactoferrin in the nasal secretions of one SGD patient by ELISA and immunoblotting. Nasal secretory lactoferrin was the same molecular weight as neutrophil lactoferrin and was secreted in normal amounts. From these data, we conclude that lactoferrin deficiency in SGD neutrophils is tissue specific and is secondary to an abnormality of RNA production. We speculate that the deficiency of several granule proteins is due to a common defect in regulation of transcription that is responsible for the abnormal myeloid differentiation seen in SGD patients.

Neutrophils from a patient with lactoferrin deficiency were examined and the quantity and subcellular localization of protein markers were determined on Percoll density gradients. Distribution of azurophilic and specific granule markers was abnormal in that azurophilic granules were lighter than normal and appeared in the fraction of the gradient where normally the specific granules sediment. The specific granule membrane markers, cytochrome b-235 and its associated flavoprotein, were abnormally distributed in the gamma fraction, the site of the plasma membrane marker alkaline phosphatase. Thus, the b-cytochrome-flavoprotein complex had either been incorporated into the plasma membrane or was still present in the membranes of granules that were abnormally light and cosedimented with the plasma membranes. This is of particular interest in regard to the patient's respiratory burst oxidase function, since the b-cytochrome/flavoprotein complex normally translocates from the specific granules to the plasma membrane to constitute the active respiratory burst oxidase. The functional consequences of this abnormal distribution are discussed, as is the importance of characterizing both intragranular enzymatic markers and granule membrane proteins to define granular disorders.

Semiquantitative analysis of lactoferrin deficiency in neutrophil polymorphonuclear leucocytes in various haematological and non-haematological disease was carried out by scoring polymorphonuclear leucocytes stained for lactoferrin by the immunoperoxidase method. The staining patterns for lactoferrin were classified into four types (0-III) based on the intensity of reaction, and the sum of the ratings of 100 polymorphonuclear leucocytes was considered as "lactoferrin score" with a possible range of 0-300. As a result, significantly low lactoferrin-scores were frequently observed in acute leukaemias and the acute phase of chronic leukaemias. Of 35 cases with leukaemias, lactoferrin-negative polymorphonuclear leucocytes (type 0) were observed in the following cases: eight cases of acute myelogenous leukaemia (8/14), a case of chronic myelogenous leukaemia (1/10) in blast crisis, one of acute promyelocytic leukaemia (1/1), one of acute monocytic leukaemia (1/2), and a case of chronic myelomonocytic leukaemia (1/2) in a transitional phase to an acute myelomonocytic leukaemia. In two cases of acute myelogenous leukaemia, in which the majority of polymorphonuclear leucocytes were negative for lactoferrin, ultrastructural cytochemical study revealed total lack of specific granules in these polymorphonuclear leucocytes. This suggests that lactoferrin is localised in the specific granules of neutrophils as has been postulated previously by others.

The initiation of both the normal and pathologic responses of human neutrophils to surface stimulation and the ensuing biochemical and physiologic events are elucidated. This knowledge has contributed to an understanding of the controlling mechanisms that may account for impaired phagocytic function in several clinical disorders associated with recurrent bacterial infections.

Neutrophils from a boy suffering from recurrent infections were found to be totally deficient in specific granules when studied by electron microscopy. In contrast, myeloperoxidase-containing azurophil granules were increased in number. This deficiency of specific granules could be detected at the light-microscopic level using an immunocytochemical technique to demonstrate the absence of lactoferrin. Neutrophils also exhibited abnormal nuclear segmentation, nuclear clefts, an abnormally weak cytochemical reaction for alkaline phosphatase, and an increased number of mitochondria and ribosomes. Some granulocytic precursors were abnormal, and many of these cells were phagocytosed by macrophages in the bone marrow. Despite these multiple abnormalities and the history of severe pyogenic infection, the in vitro bactericidal capacity of the neutrophils was within normal limits, and normal degranulation of azurophil granules occurred following phagocytosis. The precise mechanism by which the deficiency of specific granules in this patient led to an enhanced in vivo susceptibility to infection therefore remains obscure. However, attention is drawn to the fact that in three previously described cases of specific granule deficiency a history of recurrent infections was present.