Successful management of severe asthma in a young boy with eosinophilic chronic rhinosinusitis who received omalizumab: a case report.

BACKGROUND: The incidence of chronic rhinosinusitis with nasal polyps has recently increased in Japan and other East Asian countries, and this disease is called eosinophilic chronic sinusitis (ECRS) in Japan. ECRS usually occurs in adults and is frequently accompanied by refractory bronchial asthma. However, its occurrence in children under 10 years of age is rare. Here, we present an unusual case of ECRS complicated by intractable asthma in an 8-year-old boy. CASE PRESENTATION: Oral administration of prednisolone (10 mg/day) initially relieved the ECRS and bronchial asthma, but both returned during prednisolone dose reduction. Because nasal cavity-opening surgery was ineffective, oral administration prednisolone at 10 mg/day was continued. Pancytopenia was observed 16 months after the start of treatment, and the patient was admitted to our hospital. He was diagnosed with refractory cytopenia in childhood, but gradually improved after cyclosporine treatment. Although the dose of cyclosporine was therapeutic for asthma, it did not alleviate the asthma attacks, and the patient's quality of life markedly decreased. We administered omalizumab even though its use was contraindicated by negative results in an inhalable antigen test. After the third administration of omalizumab, the asthma was better controlled and respiratory function improved; however, the nasal symptoms of ECRS persisted. Attempts to relieve these symptoms by increasing the therapeutic dose of omalizumab were only partially successful. We replaced omalizumab with mepolizumab; doing so slightly improved the sinusitis symptoms, but quality of life remained unsatisfactory. We repeated the nasal cavity-opening surgery. After surgery, the asthma and sinusitis were unchanged. CONCLUSIONS: Omalizumab effectively treated the severe combined asthma in a young patient, but its effect on sinusitis was insufficient. More cases and long-term follow-up data are needed to better evaluate the effectiveness of mepolizumab for treatment of ECRS.

A 3-year-old girl with Graves' disease with hypoglycemia following transient adrenal hyporesponsiveness.

A 3-year-old girl with Graves' disease developed a generalized convulsion as a result of hypoglycemia (25 mg/dL). At the time of the hypoglycemic seizure, her plasma adrenocorticotropin (ACTH) level (1460 pg/mL) was extremely high, but her serum cortisol level (28.4 microg/dL) was relatively low given the severe stress. The cortisol-releasing hormone (CRH) provocation test done after thyroid function had improved revealed normal ACTH and cortisol responses. Since there was no other cause of hypoglycemia, such as hyperinsulinemia, long-term starvation, suddenly advanced emaciation, or prolonged fasting, it was suspected that the transient adrenal hyporesponsiveness was the main cause of hypoglycemia.

Cleaved inflammatory lactoferrin peptides in parotid saliva of periodontitis patients.

Lactoferrin (Lf) is a member of the transferrin family of iron-binding anti-bacterial proteins, present in most exocrine secretions, such as saliva, and plays an important role in mucosal defense. In this study, we identified small Lf peptides with Con A low-affinity in the parotid saliva of chronic periodontitis patients by Con A two-dimensional immunoelectrophoresis, Con A affinity chromatography and Western blotting using anti-human Lf polyclonal Ab. N-terminal amino acid sequencing of the four Con A low-affinity Lf peptides confirmed them to be fragments of intact Lf. The detection ratio of the proteinase 3 (PR3)-like activity was elevated in the parotid saliva of periodontitis patients and was associated with the severity of clinical symptoms. PR3 protein was also detected in the parotid saliva of periodontitis patients, and PR3, but not human leukocyte elastase and cathepsin G, degraded intact Lf. Con A low-affinity saliva Lf peptides showed no anti-bacterial activity against Escherichia coli, and had a reduced iron-chelating capacity. Con A low-affinity saliva Lf peptides, PR3-treated Lf preparation and two of four synthetic polypeptides induced the production of interleukin IL-6, monocyte chemoattractant protein-1 and IL-8, and the activation of NF-kappaB in human oral epithelial HSC-2 cells. Furthermore, concentrations of the Lf peptides in the parotid saliva of periodontitis patients were increased with a correlation to the severity of clinical symptoms. These results suggest that Lf in the parotid saliva of periodontitis patients was degraded into small peptides by the PR3-like activity with the capability to induce inflammatory mediators.

Inflammatory effect of cleaved bovine lactoferrin by elastase on staphylococcal mastitis.

Elastase activity and concanavalin A (Con A) low affinity bovine lactoferrin (bLf) molecule were detected in mammary gland secretions (MGSs) from mammary glands (MGs) with clinical staphylococcal mastitis. Changes in clinical symptoms correlated with increases in both elastase activity and the concentration of Con A low-affinity Lf in MGSs from mastitic MGs. Bovine Lf treated with elastase (elastase-Lf) showed various small bLf molecules and the same image on Con A two-dimensional immunoelectrophoresis as low Con A affinity bLf in MGSs. We confirmed the presence of four common bLf peptides for the elastase-bLf and low Con A affinity bLf molecules in mastitic MGSs, and synthesized four peptides. Strong mRNA expression of interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) was induced in bovine mammary epithelial cells on stimulation with low Con A affinity bLf, elastase-bLf, and GQRDLLFKDSAL, a synthesis bLf peptide based on nuclear factor kappa B (NFkappaB) activation. These results suggest that bLf was cleaved by elastase, and that this cleavage changed the physical function of Lf. Our results indicate that elastase induced production of low Con A affinity bLf, including the bLf peptide GQRDLLFKDSAL, and had an inflammatory effect on staphylococcal mastitis.

Differential regulation of intestinal lipid metabolism-related genes in obesity-resistant A/J vs. obesity-prone C57BL/6J mice.

The effects of high-fat (HF) feeding on gene expression in the small intestine were examined using obesity-resistant A/J mice and obesity-prone C57BL/6J (B6) mice. Both strains of mice were maintained on low-fat (LF; 5% fat) or HF (30% fat) diets for 2 wk. Quantitative reverse transcription-PCR analysis revealed that lipid metabolism-related genes, including carnitine palmitoyltransferase (CPT) I, liver fatty acid binding protein, pyruvate dehydrogenase kinase-4, and NADP(+)-dependent cytosolic malic enzyme, were upregulated by HF feeding in both strains of mice. The upregulated gene expression levels were higher in A/J mice than in B6 mice, suggesting more active lipid metabolism in the small intestine of A/J mice. The prominent upregulation of the lipid metabolism-related genes were specific to the small intestine; the expression levels were little or unchanged in the liver, muscle, and white adipose tissue. The increase by HF feeding and predominant expression of the intestinal lipid metabolism-related genes in A/J mice were reflected in the enzyme activities; malic enzyme, CPT, and beta-oxidation activities were increased by HF feeding, and the upregulated malic enzyme and CPT activities were significantly higher in obesity-resistant A/J mice compared with those in obesity-prone B6 mice. These findings suggest that intestinal lipid metabolism is associated with susceptibility to obesity.

Effect of combination therapy with lactoferrin and antibiotics against staphylococcal mastitis on drying cows.

We examined combination therapy with both lactoferrin (Lf) and antibiotics on clinical mastitis due to Staphylococcus aureus (S.aureus) on drying cows. The clinical symptoms of mastitic quarters were cured 81% of combination therapeutic quarters at 7 days post injection (dpi). Moreover, most of mammary gland secretions (MGSs) in combination therapeutic quarters were normal at 7 days after parturition. In the quarters with combination therapy, S.aureus counts, Lf concentrations and content rate of concanavalin A (Con A) low-affinity Lf decreased and were lower than in the quarters treated with Lf or antibiotics alone. The mRNA expression of tumor necrosis factor alpha (TNFalpha) of the quarters with combination therapy also decreased and was lower than that of the Lf or antibiotics treated. The mRNA expression of pro-inflammatory cytokines and chemokines in bovine mammary gland epithelial lined cells (BMEC) stimulated with Lf were lower than those of Con A low-affinity Lf stimulated BMEC. Moreover, Lf showed an inhibitory effect to the induction of pro-inflammatory cytokine mRNA expression when co-stimulated with Lf and Con A low-affinity Lf. Nuclear factor kappa B (NFkappaB) activation was also induced with Con A low-affinity Lf, and the inhibitory effects of Lf were also confirmed on BMEC co-stimulated with Lf and Con A low-affinity Lf. These results indicated that the efficacy of combination therapy with antibiotics and Lf caused antibacterial effect of antibiotics and inhibition of pro-inflammatory cytokine and chemokine production with Lf via the inhibition of NFkappaB activation.

A new diagnostic indicator using concanavalin a low-affinity lactoferrin levels in mammary gland secretion in mastitic drying cows.

We examined the effective diagnostic indicator using the concanavalin A (Con A) low-affinity lactoferrin (Lf) to mastitic drying cows. The concentrations of both Lf and Con A low-affinity Lf in mammary gland secretions (MGSs) were lower than normal MGSs at the early and middle dry periods and colostrums. On the other hand, the levels of Con A low-affinity Lf in MGSs increased following the appearance of mastitis symptoms, and decreased when the mastitic symptoms were cured. Moreover, IgG1 concentrations of colostrums decrease on the quarters where a high level of Con A low-affinity Lf was determined after the onset of dry period. These results suggest that this method could be used as a useful indicator to mastitic drying cows.

Small molecule lactoferrin with an inflammatory effect but no apparent antibacterial activity in mastitic mammary gland secretion.

We have identified various lactoferrin (Lf) molecules in mastitic mammary gland secretions (MGSs), and these Lf molecules were examined for their physiological function in MG. These Lf molecules were isolated by Con A affinity chromatography, and then analyzed by various electrophoresis methods and N-terminal amino acid sequencing. The low Con A affinity Lf was found to have low molecular peptides as compared with the 86 kDa of the high Con A affinity Lf, which is usually detected in healthy MGSs. The N-terminal amino acid sequence of each of the small molecular Lfs were confirmed as fragments of 86 kDa Lf. This low Con A affinity Lf stimulated spleen adherent cells to produce more O(2)(-) than 86 kDa Lf. Furthermore, the low Con A affinity Lf showed low antibacterial activity against E. coli and S. aureus, and had decreased iron-binding capacity in comparison with 86 kDa Lf. Moreover, the 86 kDa Lf could stimulate bovine T cells or macrophages to produce IFN-gamma, IL-4, IL-6, and IL-1alpha. However low Con A affinity Lf induced the production of TNFalpha, but not physiological T cell or macrophage cytokines. It was also found that when the healthy MGs of dry cows were injected with the low Con A affinity Lf, there was an increase in polymorphonuclear cells together with TNFalpha, MCP-1, and IL-8 production. These results suggested that low Con A affinity Lf in mastitic MGSs differed from 86 kDa Lf in physiological characteristics, and, that it induced an inflammatory reaction in MGs.

Ingestion of a tea rich in catechins leads to a reduction in body fat and malondialdehyde-modified LDL in men.

BACKGROUND: Catechins, the major component of green tea extract, have various physiologic effects. There are few studies, however, on the effects of catechins on body fat reduction in humans. It has been reported that the body mass index (BMI) correlates with the amount of malondialdehyde and thiobarbituric acid-reactive substances in the blood. OBJECTIVE: We investigated the effect of catechins on body fat reduction and the relation between oxidized LDL and body fat variables. DESIGN: After a 2-wk diet run-in period, healthy Japanese men were divided into 2 groups with similar BMI and waist circumference distributions. A 12-wk double-blind study was performed in which the subjects ingested 1 bottle oolong tea/d containing 690 mg catechins (green tea extract group; n = 17) or 1 bottle oolong tea/d containing 22 mg catechins (control group; n = 18). RESULTS: Body weight, BMI, waist circumference, body fat mass, and subcutaneous fat area were significantly lower in the green tea extract group than in the control group. Changes in the concentrations of malondialdehyde-modified LDL were positively associated with changes in body fat mass and total fat area in the green tea extract group. CONCLUSION: Daily consumption of tea containing 690 mg catechins for 12 wk reduced body fat, which suggests that the ingestion of catechins might be useful in the prevention and improvement of lifestyle-related diseases, mainly obesity.

Induction of nitric oxide production mediated by tumor necrosis factor alpha on staphylococcal enterotoxin C-stimulated bovine mammary gland cells.

Mammary gland (MG) secretions (MGS) derived from secretory cows infected with coagulase-negative staphylococci (CoNS) showed somatic cell counts and lactoferrin similar to levels found in the MGS of secretory cows infected with Staphylococcus aureus. However, nitrite and nitrate (NOx) and staphylococcal enterotoxin C (SEC) were found in MGS infected with S. aureus at much higher levels than in cows infected with CoNS. These results suggested that NOx could be intimately correlated with the production of SEC in secretory cows infected with S. aureus. Therefore, we examined the production of NOx and the expression of proinflammatory cytokines and microsomal cytochrome P450 (CYP450) after injection of SEC into the MGS of secretory cows. We were able to detect NOx and the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) on MG cells of SEC-injected MGS. It was also found that CYP450 in the MG cells from SEC-injected MGS was down-regulated by approximately one-third in comparison with the cells from phosphate-buffered saline-injected MGS. This in vitro system also showed that NOx could be induced in the culture of bovine macrophage-lined cells (FBM-17) with the supernatants of SEC-stimulated bovine peripheral blood lymphocytes (BoPBLs) but not in the culture of peripheral mononuclear cells with SEC-stimulated BoPBLs. The expression of the mRNA for both inducible nitric oxide synthase and TNF-alpha in FBM-17 was enhanced by culturing with the supernatant of SEC-stimulated BoPBLs, although CYP450 was down-regulated. These results indicate that the down-regulation of CYP450 was caused by the production of TNF-alpha in SEC-stimulating MG cells containing macrophages and via NOx production. Therefore, we suggest that NOx released from activated MG cells via the superantigenic activity of SEC caused oxidative damage to the MG in S. aureus-induced mastitis.

Anti-inflammatory effects of intramammary infusions of glycyrrhizin in lactating cows with mastitis caused by coagulase-negative staphylococci.

OBJECTIVE: To determine the anti-inflammatory effects of glycyrrhizin (GL) in lactating cows with mastitis attributable to naturally occurring infection with coagulase-negative staphylococci (CNS). ANIMALS: 12 lactating Holstein cows with mastitis attributable to infection with CNS and 2 healthy cows without mastitis. PROCEDURE: Clinical signs, number of bacteria in milk, somatic cell count (SCC) in milk, concentrations of alpha-lactalbumin and lactoferrin in milk, and concentration of histamine in milk were investigated before and after intramammary infusion of GL (6 cows) or antimicrobials (6 cows). Glands of 2 healthy cows were infused with staphylococcal enterotoxin; milk leukocytes were then harvested and incubated with various doses of GL. RESULTS: In cows infected with CNS that had a low bacterial concentration in milk, infusion of GL alone resulted in significant improvements in swelling, firmness of glands, and number of clots in milk, and it decreased the SCC, but not significantly. Percentage of neutrophils decreased significantly (to < 30%) by 2 days after infusion. Use of lactoferrin as a marker of inflammation in mammary glands revealed a decrease in concentrations, whereas use of alpha-lactalbumin as a marker of recovery for mammary glands revealed significant increases in concentrations in the GL-infused group. Accompanying these anti-inflammatory effects, a decrease in the concentration of histamine in milk was observed in the GL-infused group. Glycyrrhizin decreased histamine production by milk leukocytes in a concentration-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Infusion of GL may regulate intramammary inflammation through modulation of inflammatory mediators such as histamine.

Differential gene expression of cytokine and cell surface molecules in T cell subpopulation derived from mammary gland secretion of cows.

PROBLEM: As T cell subpopulations in the mammary gland secretion (MGS) of cows dynamically vary through the lactation cycle, their functional analysis is important to understand the mammary immune responses. METHOD OF STUDY: T cell subpopulations were positively selected from MGS during lactation period and non-lactation period (dry period) by a magnetic cell sorter. The messenger RNA (mRNA) expression of cytokine and cell surface molecules in the subpopulations stimulated with anti-CD3 was investigated using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: CD4+ T cells from MGS significantly expressed mRNA of interferon (IFN)-gamma, interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factor (TGF)-beta, tumor necrosis factor (TNF)-alpha, IL-4, CD40 ligand (CD40L), Fas ligand (FasL) and IL-2 receptor (IL-2R) during dry period, and mRNA of IFN-gamma, IL-2 and TGF-beta during lactation period. Their expression during lactation period was always less than that during dry period. CD8+ T cells from MGS substantially expressed mRNA of IFN-gamma, IL-2, GM-CSF, TGF-beta, TNF-alpha, FasL and IL-2R during dry period and mRNA of IFN-gamma, GM-CSF, TGF-beta, TNF-alpha and c-kit during lactation period. The TGF-beta, TNF-alpha, c-kit and IL-2R mRNA expression of T cells in MGS during lactation period mostly depended on gammadelta T cells. Interestingly, c-kit mRNA was exclusively expressed in gammadelta T cells. CONCLUSIONS: The cytokine expression of T cells in MGS of cows depended on the T cell subpopulations. The present findings suggested that the activation of gammadelta T cells via c-kit receptor participated in the suppressed expression of cytokine mRNA in T cells during lactation period.

Effects of bovine lactoferrin by the intramammary infusion in cows with staphylococcal mastitis during the early non-lactating period.

To evaluate the clinical effects of bovine lactoferrin on staphylococcal mastitis in Holstein cows during the early non-lactating period, 41 mammary quarters were selected randomly from 36 cows on 3 dairy farms. Twelve quarters were infused intramammarily with bovine lactoferrin. Twenty-nine quarters were infused with antibiotic as a control. In the bovine lactoferrin-infused group, 91.7% of mastitic quarters were cured at 7 days after calving, compared with 48.3% in the control group. Furthermore, the changes in mammary secretion induced by the infusion of bovine lactoferrin were investigated. Mean numbers of staphylococci in mammary gland secretions were significantly decreased in both 5 bovine lactoferrin-infused quarters and 5 antibiotic-infused control quarters (p<0.05). Unlike in the control quarters, the mean total cell concentration in the mammary gland secretions increased in bovine lactoferrin-infused quarters. Similar results were obtained in 6 healthy quarters which were infused with bovine lactoferrin. In these quarters, the cell population contained mainly phagocytes such as polymorphonuclear leukocytes and cells positive for CD11b which is known as a complement receptor. The mean concentration of C3 in mammary gland secretions was significantly increased in 5 mastitic quarters infused with bovine lactoferrin (p<0.05), but showed no significant change in 5 mastitic control quarters. These results suggested that bovine lactoferrin treatment for staphylococcal mastitis in the early non-lactating period might increase the rate of cure through the induction of innate immunity in the host.

Lactoferrin stimulates A Staphylococcus aureus killing activity of bovine phagocytes in the mammary gland.

Lactoferrin (Lf) may play a key role in the clearance of microorganisms from a host. To study in vitro the bactericidal mechanisms of Lf during nonlactating periods, we investigated whether the effects of Lf were influenced by bovine mammary gland secretory cells (MGSC) and fresh normal bovine serum (NBS) as a source of complement. Phagocytic killing tests demonstrated that a phagocytic mixture of unopsonized Staphylococcus aureus (S. aureus) and MGSC in the presence of Lf reduced bacterial growth, compared with that of unopsonized S. aureus and MGSC without Lf. The opsonization with Lf and fresh NBS together resulted in more than a 95% reduction in CFU. The activation of complement induced by Lf also resulted in increased deposition of C3 on S. aureus, and the phagocytic activity of MGSC was augmented by opsonization with Lf and fresh NBS. Inhibition of C3 deposition by Lf was not induced in the presence of Mg-EGTA, but was induced by the addition of bovine Lf antiserum. These results strongly suggest that Lf induces the activation of complement in fresh NBS mainly through an alternative pathway. The results demonstrated a Lf-dependent, antibody-independent and complement-mediated phagocytic killing of S. aureus, and implied that Lf was synergistically capable of activating both the alternative pathway of the bovine complement cascade and phagocytosis by phagocytes.