Structural insights into the interaction between adenovirus C5 hexon and human lactoferrin.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.

Resistin, Elastase, and Lactoferrin as Potential Plasma Biomarkers of Pediatric Inflammatory Bowel Disease Based on Comprehensive Proteomic Screens.

Inflammatory bowel disease (IBD) is an immune-mediated chronic inflammation of the intestine, which can present in the form of ulcerative colitis (UC) or as Crohn's disease (CD). Biomarkers are needed for reliable diagnosis and disease monitoring in IBD, especially in pediatric patients. Plasma samples from a pediatric IBD cohort were interrogated using an aptamer-based screen of 1322 proteins. The elevated biomarkers identified using the aptamer screen were further validated by ELISA using an independent cohort of 76 pediatric plasma samples, drawn from 30 CD, 30 UC, and 16 healthy controls. Of the 1322 proteins screened in plasma from IBD patients, 129 proteins were significantly elevated when compared with healthy controls. Of these 15 proteins had a fold change greater than 2 and 28 proteins had a fold change >1.5. Neutrophil and extracellular vesicle signatures were detected among the elevated plasma biomarkers. When seven of these proteins were validated by ELISA, resistin was the only protein that was significantly higher in both UC and CD (p < 0.01), with receiver operating characteristic area under the curve value of 0.82 and 0.77, respectively, and the only protein that exhibited high sensitivity and specificity for both CD and UC. The next most discriminatory plasma proteins were elastase and lactoferrin, particularly for UC, with receiver operating characteristic area under the curve values of 0.74 and 0.69, respectively. We have identified circulating resistin, elastase, and lactoferrin as potential plasma biomarkers of IBD in pediatric patients using two independent diagnostic platforms and two independent patient cohorts.

Clearance of Neutrophils From ICH-Affected Brain by Macrophages Is Beneficial and Is Assisted by Lactoferrin and CD91.

BACKGROUND: Within hours after intracerebral hemorrhage (ICH) onset, masses of polymorphonuclear neutrophils (PMNs) infiltrate the ICH-affected brain. After degranulation involving controlled release of many toxic antimicrobial molecules, the PMNs undergo rapid apoptosis and then are removed by phagocytic microglia/macrophages (MΦ) through a process called efferocytosis. Effective removal of PMNs may limit secondary brain damage and inflammation; however, the molecular mechanisms governing these cleanup activities are not well understood. We propose that scavenger receptor CD91 on myeloid phagocytes especially in presence of CD91 ligand, LTF (lactoferrin, protein abundant in PMNs), plays an important role in clearance of dead apoptotic PMNs (ANs). METHODS: Mice/rats were subjected to an autologous blood injection model of ICH. Primary cultured microglia were used to assess phagocytosis of ANs. Immunohistochemistry was employed to assess CD91 expression and PMN infiltration. CD91 knockout mice selectively in myeloid phagocytes (Mac-CD91-KO) were used to establish the CD91/LTF function in phagocytosis and in reducing ICH-induced injury, as assessed using behavioral tests, hematoma resolution, and oxidative stress. RESULTS: Masses of PMNs are found in ICH-affected brain, and they contain LTF. MΦ at the outer border of hematoma are densely packed, expressing CD91 and phagocytosing ANs. Microglia deficient in CD91 demonstrate defective phagocytosis of ANs, and mice deficient in CD91 (Mac-CD91-KO) subjected to ICH injury have increased neurological dysfunction that is associated with impaired hematoma resolution (hemoglobin and iron clearance) and elevated oxidative stress. LTF that normally ameliorates ICH injury in CD91-proficient control mice shows reduced therapeutic effects in Mac-CD91-KO mice. CONCLUSIONS: Our study suggests that CD91 plays a beneficial role in improving ANs phagocytosis and ultimately post-ICH outcome and that the beneficial effect of LTF in ICH is in part dependent on presence of CD91 on MΦ.

AGA Clinical Practice Guideline on the Role of Biomarkers for the Management of Ulcerative Colitis.

BACKGROUND & AIMS: Biomarkers are used frequently for noninvasive monitoring and treatment decision making in the management of patients with ulcerative colitis (UC). This American Gastroenterological Association (AGA) guideline is intended to support practitioners in decisions about the use of biomarkers for the management of UC. METHODS: A multidisciplinary panel of content experts and guideline methodologists used the Grading of Recommendations Assessment, Development and Evaluation framework to prioritize clinical questions, identify patient-centered outcomes, and conduct an evidence synthesis on the clinical performance of serum C-reactive protein (CRP), fecal calprotectin, and fecal lactoferrin as biomarkers of disease activity in patients with established UC in symptomatic remission or with active symptoms. The guideline panel used the Evidence-to-Decision framework to develop recommendations for the use of biomarkers for monitoring and management of UC and provided implementation considerations for clinical practice. RESULTS: The guideline panel made 7 conditional recommendations. In patients with UC in symptomatic remission, the panel suggests the use of a biomarker- and symptom-based monitoring strategy over a symptom-based monitoring strategy. For patients in symptomatic remission, the panel suggests using fecal calprotectin <150 µg/g, normal fecal lactoferrin, and/or normal CRP to rule out active inflammation and avoid routine endoscopic assessment of disease. In patients with UC with moderate to severe symptoms, the panel suggests using fecal calprotectin >150 µg/g, elevated fecal lactoferrin, or elevated CRP to inform treatment decisions and avoid routine endoscopic assessment of disease. However, in patients in symptomatic remission but elevated biomarkers, and in patients with moderate to severe symptoms with normal biomarkers, the panel suggests endoscopic assessment of disease to inform treatment decisions. In patients with UC with mild symptoms, the panel suggests endoscopic assessment of disease activity to inform treatment decisions. The panel identified the use of a biomarker-based monitoring strategy over an endoscopy-based monitoring strategy as a knowledge gap. The panel also proposed key implementation considerations for optimal use of biomarkers, and identified areas for future research. CONCLUSIONS: In patients with UC, noninvasive biomarkers, including fecal calprotectin, fecal lactoferrin, and serum CRP can inform disease monitoring and management.

Intracellularly delivered human lactoferrin functions as an activator of Na+/H+ exchanger 7.

Here, we report that human lactoferrin (hLF), known for its anticancer properties, induced intracellular activation of the Na+/H+ exchanger (NHE) 7 in human lung cancer PC-9 cells. Compared to non-fused hLF, the fusion of human serum albumin (HSA) with hLF (hLF-HSA) facilitated its internalization into PC-9 cells in a caveolae-mediated manner, thereby exhibiting enhanced anti-proliferative effects. Although hLF alone did not exhibit any discernible effects, hLF-HSA resulted in organelle alkalization as detected using an acidotropic pH indicator. hLF-HSA-induced elevation of organelle pH and inhibition of cancer growth were abolished by NHE7 siRNA. hLF-HSA upregulated NHE7. Thus, upon cellular uptake, hLF-HSA triggers proton leakage through the upregulation of NHE7. This process led to organelle alkalization, probably in the trans-Golgi network (TGN) as suggested by the localization of NHE7 in PC-9 cells, thereby suppressing lung cancer cell growth. Forcing the cellular uptake of hLF alone using a caveolae-mediated endocytosis activator led to an increase in organelle pH. Furthermore, cell entry of hLF also activated proton-loading NHE7, leading to organelle acidification in the pancreatic cancer cell line MIA PaCa-2. Therefore, the intracellularly delivered hLF functions as an activator of NHE7.

Phenotypic and genotypic assessment of iron acquisition in diverse bovine-associated non-aureus staphylococcal strains.

Although the role of iron in bacterial infections has been well described for Staphylococcus (S.) aureus, iron acquisition in (bovine-associated) non-aureus staphylococci and mammaliicocci (NASM) remains insufficiently mapped. This study aimed at elucidating differences between four diverse bovine NASM field strains from two species, namely S. chromogenes and S. equorum, in regards to iron uptake (with ferritin and lactoferrin as an iron source) and siderophore production (staphyloferrin A and staphyloferrin B) by investigating the relationship between the genetic basis of iron acquisition through whole genome sequencing (WGS) with their observed phenotypic behavior. The four field strains were isolated in a previous study from composite cow milk (CCM) and bulk tank milk (BTM) in a Flemish dairy herd. Additionally, two well-studied S. chromogenes isolates originating from a persistent intramammary infection and from a teat apex were included for comparative purpose in all assays. Significant differences between species and strains were identified. In our phenotypical iron acquisition assay, while lactoferrin had no effect on growth recovery for all strains in iron deficient media, we found that ferritin served as an effective source for growth recovery in iron-deficient media for S. chromogenes CCM and BTM strains. This finding was further corroborated by analyzing potential ferritin iron acquisition genes using whole-genome sequencing data, which showed that all S. chromogenes strains contained hits for all three proposed ferritin reductive pathway genes. Furthermore, a qualitative assay indicated siderophore production by all strains, except for S. equorum. This lack of siderophore production in S. equorum was supported by a quantitative assay, which revealed significantly lower or negligible siderophore amounts compared to S. aureus and S. chromogenes. The WGS analysis showed that all tested strains, except for S. equorum, possessed complete staphyloferrin A (SA)-synthesis and export operons, which likely explains the phenotypic absence of siderophore production in S. equorum strains. While analyzing the staphyloferrin A and staphyloferrin B operon landscapes for all strains, we noticed some differences in the proteins responsible for iron acquisition between different species. However, within strains of the same species, the siderophore-related proteins remained conserved. Our findings contribute valuable insights into the genetic elements associated with bovine NASM pathogenesis.

Predictive value of fecal calprotectin and lactoferrin levels for negative outcomes in Clostridioides difficile infection.

PURPOSE: We investigated the role of fecal calprotectin (FC) and lactoferrin (FL) as predictive biomarkers in Clostridioides difficile infection (CDI). METHODS: We assembled a prospective cohort including all patients with a laboratory-confirmed CDI diagnosis between January and December 2017. FL and FC levels were measured at diagnosis by commercial ELISA and EIA kits. We investigated the diagnostic accuracy of FC and FL to predict CDI recurrence and severity (study outcomes) and explored optimal cut-off values in addition to those proposed by the manufacturers (200 µg/g and 7.2 µg/mL, respectively). RESULTS: We included 170 CDI cases (152 first episodes and 18 recurrences). The rates of recurrence (first episodes only) and severity (entire cohort) were 9.2% (14/152) and 46.5% (79/170). Both FL and FC levels were significantly higher in patients who developed study outcomes. Optimal cut-off values for FC and FL to predict CDI recurrence were 1052 µg/g and 6.0 µg/mL. The optimal cut-off value for FC yielded higher specificity (60.9%) and positive predictive value (PPV) (16.9%) than that proposed by the manufacturer. Regarding CDI severity, the optimal cut-off value for FC (439 µg/g) also provided higher specificity (43.9%) and PPV (54.1%) than that of the manufacturer, whereas the optimal cut-off value for FL (4.6 µg/mL) resulted in an improvement of PPV (57.5%). CONCLUSION: By modifying the thresholds for assay positivity, the measurement of FC and FL at diagnosis is useful to predict recurrence and severity in CDI. Adding these biomarkers to current clinical scores may help to individualize CDI management.

Characterization of recombinant human lactoferrin expressed in Komagataella phaffii.

This work presents a thorough characterization of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed in a yeast system at an industrial scale for the first time. Proteomic analysis confirmed that its amino acid sequence is identical to that of native human LF. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642 and they were predominantly oligomannose structures having five to nine mannoses. Helaina rhLF's protein secondary structure was nearly identical to that of human milk lactoferrin (hmLF), as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7 ± 0.9 °C and 91.9 ± 0.5 °C, consistently mirroring denaturation temperatures observed for apo- and holo-hmLF. Overall, Helaina rhLF differed from hmLF in the N-glycans they possessed; nevertheless, the characterization results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.

Hierarchical Micro- and Mesoporous Zeolitic Imidazolate Framework-8-Based Enzyme Hybrid for Combination Antimicrobial by Lysozyme and Lactoferrin.

Pathogenic bacteria have consistently posed a formidable challenge to human health, creating the critical need for effective antibacterial solutions. In response, enzyme-metal-organic framework (MOF) composites have emerged as a promising class of antibacterial agents. This study focuses on the development of an enzyme-MOF composite based on HZIF-8, incorporating the advantages of simple synthesis, ZIF-8 antibacterial properties, lysozyme hydrolysis, and high biological safety. Through a one-pot method, core-shell nanoparticles (HZIF-8) were synthesized. This structure enables efficient immobilization of lysozyme and lactoferrin within the HZIF-8, resulting in the formation of the lysozyme-lactoferrin@HZIF-8 (LYZ-LF@HZIF-8) composite. Upon exposure to light irradiation, HZIF-8 itself possessed antibacterial properties. Lysozyme initiated the degradation of bacterial peptidoglycan and lactoferrin synergistically enhanced the antibacterial effect of lysozyme. All of the above ultimately contributed to comprehensive antibacterial activity. Antibacterial assessments demonstrated the efficacy of the LYZ-LF@HZIF-8 composite, effectively eradicating Staphylococcus aureus at a cell density of 1.5 × 106 CFU/mL with a low dosage of 200 µg/mL and completely inactivating Escherichia coli at 400 µg/mL with the same cell density. The enzyme-MOF composite exhibited significant and durable antibacterial efficacy, with no apparent cytotoxicity in vitro, thereby unveiling expansive prospects for applications in the medical and food industries.

Bovine lactoferrin inhibits inflammatory response and apoptosis in lipopolysaccharide-induced acute lung injury by targeting the PPAR-γ pathway.

BACKGROUND: Lactoferrin (LF) is an iron-binding multifunctional cationic glycoprotein. Previous studies have demonstrated that LF may be a potential drug for treating acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). In this study, we explored the anti-inflammatory effect and mechanism of bovine lactoferrin (bLF) in ALI using the RNA sequencing (RNA-seq) technology and transcriptome analysis. METHODS AND RESULTS: Based on the differentially expressed genes (DEGs) obtained from RNA-seq of the Lung from mouse model, the bioinformatics workflow was implemented using the BGISEQ-500 platform. The protein-protein interaction (PPI) network was obtained using STRING, and the hub gene was screened using Cytoscape. To verify the results of transcriptome analysis, the effects of bLF on Lipopolysaccharide (LPS)-induced BEAS-2B cells and its anti-reactive oxygen species (ROS), anti-inflammatory, and antiapoptotic effects were studied via Cell Counting Kit-8 (CCK-8) test, active oxygen detection test, ELISA, and western blot assay. Transcriptome analysis revealed that two hub gene modules of DEGs were screened via PPI analysis using the STRING and MCODE plug-ins of Cytoscape. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that these core modules are enriched in the PPAR (peroxisome proliferator-activated receptor) and AMPK (AMP-activated protein kinase) signaling pathways. Through cell experiments, our study shows that bLF can inhibit ROS, inflammatory reaction, and LPS-induced BEAS-2B cell apoptosis, which are significantly antagonized by the PPAR-γ inhibitor GW9662. CONCLUSION: This study has suggested that the PPAR-γ pathway is the critical target of bLF in anti-inflammatory reactions and apoptosis of ALI, which provides a direction for further research.

A common regulatory haplotype doubles lactoferrin concentration in milk.

BACKGROUND: Bovine lactoferrin (Lf) is an iron absorbing whey protein with antibacterial, antiviral, and antifungal activity. Lactoferrin is economically valuable and has an extremely variable concentration in milk, partly driven by environmental influences such as milking frequency, involution, or mastitis. A significant genetic influence has also been previously observed to regulate lactoferrin content in milk. Here, we conducted genetic mapping of lactoferrin protein concentration in conjunction with RNA-seq, ChIP-seq, and ATAC-seq data to pinpoint candidate causative variants that regulate lactoferrin concentrations in milk. RESULTS: We identified a highly-significant lactoferrin protein quantitative trait locus (pQTL), as well as a cis lactotransferrin (LTF) expression QTL (cis-eQTL) mapping to the LTF locus. Using ChIP-seq and ATAC-seq datasets representing lactating mammary tissue samples, we also report a number of regions where the openness of chromatin is under genetic influence. Several of these also show highly significant QTL with genetic signatures similar to those highlighted through pQTL and eQTL analysis. By performing correlation analysis between these QTL, we revealed an ATAC-seq peak in the putative promotor region of LTF, that highlights a set of 115 high-frequency variants that are potentially responsible for these effects. One of the 115 variants (rs110000337), which maps within the ATAC-seq peak, was predicted to alter binding sites of transcription factors known to be involved in lactation-related pathways. CONCLUSIONS: Here, we report a regulatory haplotype of 115 variants with conspicuously large impacts on milk lactoferrin concentration. These findings could enable the selection of animals for high-producing specialist herds.

Lactoferrin mediates epithelial-mesenchymal transformation by regulating the PI3K/AKT/mTOR pathway to inhibit nasopharyngeal carcinoma metastasis.

Nasopharyngeal carcinoma (NPC) is a common malignant tumor of the head and neck. Epithelial-mesenchymal transition (EMT) is a major player in regulating NPC transfer. There is increasing evidence that lactotransferrin (LTF) is an important regulator of EMT conversion. However, the potential role and mechanisms of LTF in regulating NPC cell EMT remain unclear. In this study, quantitative real-time PCR (qRT‒PCR) and Western blotting were applied to measure the expression of LTF in NPC cells. Subsequently, the influences of LTF on the proliferation, migration and invasion of NPC cells were verified by functional acquisition experiments. Finally, Western blotting was used to analyze the effects of EMT-related proteins and phosphoinositol 3-kinase (PI3K)/Akt/mammalian rapamycin target (mTOR) signaling pathways. The data of this study indicate that LTF was underexpressed in human NPC cells, and upregulation of LTF could restrain NPC cell proliferation, invasion, migration and EMT transformation. Moreover, the effects of LTF on NPC cell metastasis and EMT are partly determined by the PI3K/AKT/mTOR pathway. This study suggests that LTF is a potential biomarker of NPC and that LTF-mediated EMT progression plays a tumor-suppressive role in the progression of NPC metastasis.

Morphological cell profiling of SARS-CoV-2 infection identifies drug repurposing candidates for COVID-19.

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.

Complexation of bovine lactoferrin with selected phenolic acids via noncovalent interactions: Binding mechanism and altered functionality.

Noncovalent interactions of 4 selected phenolic acids, including gallic acid (GA), caffeic acid (CA), chlorogenic acid (CGA), and rosmarinic acid (RA) with lactoferrin (LF) were investigated. Compound combined with LF in the binding constant of CA > GA > RA > CGA, driven by van der Waals and hydrogen bonding for GA, and hydrophobic forces for others. Conformation of LF was affected at secondary and ternary structure levels. Molecular docking indicated that GA and CA located in the same site near the iron of the C-lobe, whereas RA and CGA bound to the C2 and N-lobe, respectively. Significantly enhanced antioxidant activity of complexes was found compared with pure LF, as demonstrated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis(2-ethylbenzothiazoline-6-sulfonate) (ABTS), and ferric reducing antioxidant power (FRAP) models. Caffeic acid, CGA, and RA significantly decreased the emulsifying stability index and improved foam ability of LF, and the effect of CA and RA was the most remarkable, respectively.

Schizochytrium sp. and lactoferrin supplementation alleviates Escherichia coli K99-induced diarrhea in preweaning dairy calves.

Calf diarrhea, a common disease mainly induced by Escherichia coli infection, is one of the main reasons for nonpredator losses. Hence, an effective nonantibacterial approach to prevent calf diarrhea has become an emerging requirement. This study evaluated the microalgae Schizochytrium sp. (SZ) and lactoferrin (LF) as a nutrient intervention approach against E. coli O101:K99-induced preweaning calve diarrhea. Fifty 1-d-old male Holstein calves were randomly divided into 5 groups (n = 10): (1) control, (2) blank (no supplement or challenge), (3) 1 g/d LF, (4) 20 g/d SZ, or (5) 1 g/d LF plus 20 g/d SZ (LFSZ). The experimental period lasted 14 d. On the morning of d 7, calves were challenged with 1 × 1011 cfu of E. coli O101:K99, and rectum feces were collected on 3, 12, 24, and 168 h postchallenge for the control, LF, SZ, and LFSZ groups. The rectal feces of the blank group were collected on d 14. Data were analyzed using the mixed procedure of SAS (version 9.4; SAS Institute Inc.). The E. coli K99 challenge decreased the average daily gain (ADG) and increased feed-to-gain ratio (F:G) and diarrhea frequency (control vs. blank). Compared with the control group, the LFSZ group had a higher ADG and lower F:G, and the LFSZ and SZ groups had lower diarrhea frequency compared with the control group. In addition, the LFSZ and SZ groups have no differences in diarrhea frequency compared with the blank group. Compared with the control group, the blank group had lower serum nitric oxide (NO), endothelin-1, d-lactic acid (D-LA), and lipopolysaccharide (LPS) concentrations, as well as serum IgG, IL-1ß, IL-6, IL-10, and TNF-α levels on d 7 and 14. On d 7, compared with the control group, all treatment groups had lower serum NO level, the SZ group had a lower serum D-LA concentration, and the LF and LFSZ groups had lower serum LPS concentration. On d 14, compared with the control group, the fecal microbiota of the blank group had lower Shannon, Simpson, Chao1, and ACE indexes, the LFSZ group had lower Shannon and Simpson indexes, the SZ and LFSZ groups had a higher Chao1 index, and all treatment groups had a higher ACE index. In fecal microbiota, Bifidobacterium and Actinobacteria were negatively associated with IL-10 and d-lactate, while Akkermansia was negatively associated with endothelin-1 and positively correlated with LPS, fecal scores, and d-lactate levels. Our results indicated that LF and SZ supplements could alleviate E. coli O101:K99-induced calf diarrhea individually or in combination. Supplementing 1 g/d LF and 20 g/d SZ could be a potential nutrient intervention approach to prevent bacterial diarrhea in calves.

Managing children with frequent respiratory infections and associated wheezing: a preliminary randomized study with a new multicomponent nasal spray.

BACKGROUND: Preschoolers frequently have respiratory infections (RIs), which may cause wheezing in some subjects. Type 2 polarization may favor increased susceptibility to RIs and associated wheezing. Non-pharmacological remedies are garnering increasing interest as possible add-on therapies. The present preliminary study investigated the efficacy and safety of a new multi-component nasal spray in preschoolers with frequent RIs and associated wheezing. METHODS: Some preschoolers with these characteristics randomly took this product, containing lactoferrin, dipotassium glycyrrhizinate, carboxymethyl-beta-glucan, and vitamins C and D3 (Saflovir), two sprays per nostril twice daily for 3 months. Other children were randomly treated only with standard therapy. Outcomes included the number of RIs and wheezing episodes, use of medications, and severity of clinical manifestations. RESULTS: Preschoolers treated add-on with this multicomponent product experienced fewer RIs and used fewer beta-2 agonists than untreated children (P = 0.01 and 0.029, respectively). CONCLUSIONS: This preliminary study demonstrated that a multicomponent product, administered add-on as a nasal spray, could reduce the incidence of RIs and use of symptomatic drugs for relieving wheezing in children.

A sustainable sensitive spectrofluorimetric approach for the determination of the multipotent protein lactoferrin in different pharmaceuticals and infant milk formula: Compliance with greenness metrics.

The current study presents the first spectrofluorimetric approach for the estimation of lactoferrin, depending on the measurement of its native fluorescence at 337 nm after excitation at 230 nm, without the need for any hazardous chemicals or reagents. It was found that the fluorescence intensity versus concentration calibration plot was linear over the concentration range of 0.1-10.0 µg/mL with quantitation and detection limits of 0.082 and 0.027 µg/mL, respectively. The method was accordingly validated according to the ICH recommendations. The developed method was applied for the estimation of lactoferrin in different dosage forms, including capsules and sachets with high percent recoveries (97.84-102.53) and low %RSD values (<1.95). Lactoferrin is one of the key nutrients in milk powder and a significant nutritional fortifier. In order to assess the quality of milk powder, it is essential to rapidly and accurately quantify the lactoferrin content of the product. Therefore, the presented study was successfully applied for the selective estimation of lactoferrin in milk powder with acceptable percent recoveries (96.45-104.92) and %RSD values (≤3.607). Finally, the green profile of the method was estimated using two assessment tools: Green Analytical Procedure Index (GAPI) and Analytical GREEnness (AGREE), which demonstrated its excellent greenness.

Lactoferrin deficiency during lactation increases the risk of depressive-like behavior in adult mice.

BACKGROUND: Lactoferrin is an active protein in breast milk that plays an important role in the growth and development of infants and is implicated as a neuroprotective agent. The incidence of depression is currently increasing, and it is unclear whether the lack of lactoferrin during lactation affects the incidence of depressive-like behavior in adulthood. RESULTS: Lack of lactoferrin feeding during lactation affected the barrier and innate immune functions of the intestine, disrupted the intestinal microflora, and led to neuroimmune dysfunction and neurodevelopmental delay in the hippocampus. When exposed to external stimulation, adult lactoferrin feeding-deficient mice presented with worse depression-like symptoms; the mechanisms involved were activation of the LPS-TLR4 signalling pathway in the intestine and hippocampus, reduced BDNF-CREB signaling pathway in hippocampus, increased abundance of depression-related bacteria, and decreased abundance of beneficial bacteria. CONCLUSIONS: Overall, our findings reveal that lactoferrin feeding deficient during lactation can increase the risk of depressive-like behavior in adults. The mechanism is related to the regulatory effect of lactoferrin on the development of the "microbial-intestinal-brain" axis.

Concentrations of antimicrobial components in milk at dry off and postpartum and their relationships to new high somatic cell counts at quarter level in dairy cows.

We investigated the antimicrobial components in cow milk at dry off and postpartum and their contribution in preventing new high SCC at quarter level. Milk samples from 72 quarters of 19 lactating cows were collected at last milking before dry off and at 7 d after parturition. Milk yield of each cow was recorded and SCC, IgG, IgA, lactoferrin, lingual antimicrobial peptide (LAP), and S100A7 concentrations in each quarter milk sample were measured. The postpartum milk yield was significantly higher than that at dry off. The IgG, IgA and lactoferrin concentrations in milk at dry off were significantly higher than those at postpartum, whereas the LAP concentration was lower. Quarters with SCC < 300 000 cells/ml at both dry off and postpartum were classified as persistent low SCC (PL) whereas those that rose above that same threshold postpartum were classified as new high SCC (NH). At dry off, IgG and LAP concentrations in milk were significantly higher in PL than in NH. These results suggest that high LAP concentrations during the dry period may contribute toward the prevention of new high SCC.

Dose Range-Finding Toxicity Study in Rats With Recombinant Human Lactoferrin Produced in Komagataella phaffii.

The oral toxicity of recombinant human lactoferrin (rhLF, Helaina rhLF, Effera™) produced in Komagataella phaffii was investigated in adult Sprague Dawley rats by once daily oral gavage for 14 consecutive days. The study used groups of 3-6 rats/sex/dose. The vehicle control group received sodium citrate buffer, and the test groups received daily doses of 200, 1000, and 2000 mg of rhLF in sodium citrate buffer per kg body weight. Bovine LF at 2000 mg/kg body weight per day was used as a comparative control. Clinical observations, body weight, hematology, clinical chemistry, iron parameters, immunophenotyping, and gross examination at necropsy were used as criteria for detecting the effects of treatment in all groups and to help select dose levels for future toxicology studies. Quantitative LF levels were also analyzed as an indication of bioavailability. Overall, administration of Helaina rhLF by once daily oral gavage for 14 days was well tolerated in rats at levels up to 2000 mg/kg/day, or 57 × Helaina's intended commercial use in adults, and indicating that a high dose of 2000 mg/kg/day is appropriate for future definitive toxicology studies.