Waist circumference and waist-to-height ratio in Norwegian children 4-18 years of age: reference values and cut-off levels.

AIM: To establish reference values for waist circumference and waist-to-height ratio of Norwegian children. MATERIAL: Data were collected in 2003-2006 as part of a cross-sectional study, including 5725 children 4-18 years of age. Reference curves were fitted with the LMS method; appropriate cut-offs were selected using receiver operating characteristic analysis. RESULTS: Reference values for waist circumference and waist-to-height ratio are presented. Mean waist circumference increased with age for both genders. Boys had a higher waist circumference at almost all ages. Mean waist-to-height ratio decreased until early adolescence and thereafter increased slightly towards adult age. There was a strong positive correlation between waist circumference and BMI (r = 0.907, p < 0.01) and a moderate positive correlation between waist-to-height ratio and BMI (r = 0.397 p < 0.01). A waist circumference cut-off value of 1.0 SDS (85th percentile) gave a sensitivity of 79% and a specificity of 94% to detect overweight. A cut-off value of 1.6 SDS (95th percentile) gave a sensitivity of 94% and a specificity of 96% to detect obesity. CONCLUSION: This study presents the first reference values of waist circumference and waist-to-height ratio for Norwegian children 4-18 years, which also represent the first reference in Scandinavian schoolchildren. The 85th and 95th percentiles of waist circumference are proposed as appropriate cut-offs for central overweight and obesity.

Phagocytic capacity of normal and malignant rat glial cells in culture.

The phagocytic capacity of 4 continuous rat glioma cell lines (BT2C, BT4Cn, BT5c, and 9L) and normal BD IX fetal rat glial cells in culture has been studied. This was done by flow cytometric measurements of single cells from monolayer cultures having ingested fluorescent bacteria, zymosan particles, red blood cells, or fragments of normal glial cells. In addition, phagocytosis was studied in a three-dimensional culture system. The BT4Cn, BT5C, and 9L cell lines were tumorigenic and invasive both in vivo and in organ culture in vitro. In contrast, BT2C has shown variable tumorigenicity and does not seem to be invasive. The phagocytic capacity of the cell lines was compared to their destructive properties during invasion. Depending on the particle type, 30-40% of the normal glial cells were phagocytic. The fractions of phagocytic glioma cells were dependent on the particle type and the prey load. Of the invasive cell lines, BT5C showed high phagocytic activity both in monolayer and three-dimensional cultures. Two of the invasive cell lines (BT5C and 9L) had about the same fraction of phagocytic cells as normal glial cells. These 2 cell lines showed highly destructive growth during invasion. In contrast, the third invasive cell line (BT4Cn) had almost no phagocytic cells. The BT4Cn cells showed single-cell invasion with little destruction of target tissue. The noninvasive cell line (BT2C) showed low phagocytic activity, and almost no destruction was observed in the border zone between tumor cells and normal tissue. Phagocytosis seems to be an inherent property of both normal and malignant glial cells, although the fraction of phagocytic cells varies from one cell line to another. In organ culture high phagocytic capacity of invasive glioma cells seems to be related to destructive activity on the normal brain tissue during invasion.

Inhibition of phagocytosis by monoclonal antibodies to human myeloid differentiation antigens.

The influence of eight antimyeloid monoclonal antibodies on human leukocyte phagocytosis was investigated using flow cytometry. A granulocyte-specific monoclonal antibody, VIM-D5, inhibited the phagocytosis of both zymosan particles and Staphylococcus aureus in a dose-dependent fashion. In the presence of 5 micrograms/ml, the numbers of phagocyte-associated zymosan particles and bacteria were reduced by about 35% and 40%, respectively. Another monoclonal antibody, VIM-12, reacting with granulocytes, monocytes, and null lymphocytes, inhibited both granulocyte and monocyte phagocytosis of S. aureus. The inhibition was dose dependent, and in the presence of 10 micrograms/ml, the number of phagocyte-associated bacteria was reduced by about 40%. VIM-12 did not influence the phagocytosis of zymosan particles. Both VIM-D5 and VIM-12 inhibited the internalization phase of phagocytosis, whereas the attachment to the phagocyte surface was unaltered. The combined effect of VIM-D5 and VIM-12 was additive, amounting to about 70% reduction of phagocytosis of bacteria. The remaining six antimyeloid antibodies had no effect on leukocyte phagocytosis. The combined use of antimyeloid monoclonal antibodies and flow cytometry appears to be a promising tool for the study of phagocyte functions.

The dimer of hemoregulatory peptide (HP5B) stimulates mouse and human myelopoiesis in vitro.

A synthetic analogue of a pentapeptide associated with mature granulocytes has been described earlier and shown to suppress myelopoietic colony formation in vitro in concentrations from 10(-13) to 10(-6) M. By oxidation of the peptide, a dimer will rapidly occur by formation of disulfide bridges between cysteine residues. We here demonstrate that this dimer has the opposite effects of the monomer. For both mouse and human granulocyte-macrophage colony-forming units (CFU-GM), a dose-dependent enhancement of colony formation was observed in the dose range 10(-16) to 10(-5) M, where a saturation level was reached above 10(-8) M. At low doses of colony-stimulating activity (CSA) and in the linear stimulating phase, an up to ten times increase of colony formation was seen, whereas at higher doses the effect was less pronounced. Also at the plateau level of CSA stimulation an increased colony yield was seen. All types of colonies were stimulated. The dimer itself had no colony-stimulating factor activity and was not toxic to bone marrow cells in suspension cultures up to 24 h. Upon reduction of the dimer by use of sulfhydryl compounds, inhibitory effects on CFU-GM were restored. The peptide had no effect on the phagocytic process in human granulocytes, including attachment and internalization of bacteria or Zymosan particles. The monomerdimer equilibrium of hemoregulatory peptide may constitute a new mechanism for proliferative regulation of myelopoietic cells.

Reduced CD4 cell counts in blood do not reflect CD4 cell depletion in tonsillar tissue in asymptomatic HIV-1 infection.

OBJECTIVE: To investigate whether the loss of CD4 cells seen in peripheral circulation of HIV-1-positive individuals reflects a similar depletion of CD4 cells from lymphoid tissue. DESIGN: CD4 and CD8 cells in tonsillar mononuclear cell suspensions were quantified relative to tonsillar B cells, as these were thought to remain numerically unchanged in the course of HIV infection. Results were related to the CD4 cell counts in blood and to the clinical status of the patients. METHODS: Blood samples and tonsillar tissue were obtained from 13 HIV-1-seropositive individuals and six seronegative controls. B cells and T-cell subsets in mononuclear cells were quantified using a three-colour flow cytometry protocol. Histological sections were morphologically classified and B-cell areas were quantified by morphometry. RESULTS: The B-cell fraction was confirmed to be relatively unchanged in asymptomatic HIV-1-seropositive individuals compared with controls. The tonsillar CD4 : B-cell ratios in asymptomatic individuals was similar to those seen in controls, whereas the CD4 : B-cell ratios in symptomatic HIV-1-infected individuals were greatly reduced. The tonsillar CD4 : CD8 cell ratios in HIV-1-infected individuals were much lower than those seen in controls, in the asymptomatic group due to a considerable expansion of the tonsillar CD8 cell subset, and in the symptomatic group also due to a loss of CD4 cells. CONCLUSIONS: We found no evidence of CD4 cell depletion in tonsillar tissue in asymptomatic HIV-1-infected individuals despite low CD4 cell counts in blood. Loss of CD4 cells from this lymphoid tissue seems to occur as a late-stage phenomenon correlated with the onset of clinical symptoms.

Priming of human polymorphonuclear neutrophilic leukocytes by insulin-like growth factor I: increased phagocytic capacity, complement receptor expression, degranulation, and oxidative burst.

Insulin-like growth factor I (IGF-I) is a GH-dependent peptide regulating mammalian growth that seems to be of importance for the normal development and function of the immune system. Polymorphonuclear neutrophilic leukocytes (PMNLs) are terminally differentiated phagocytes essential for host defense, and in the present study, recombinant human IGF-I was shown to be a powerful primer of mature human PMNLs. IGF-I augmented the PMNL phagocytosis of both immunoglobulin G-opsonized Staphylococcus aureus and complement-opsonized Candida albicans. In addition, the growth factor increased PMNL complement receptor expression [complement receptors 1 (CD35) and 3 (CD11b)] and primed the cells to stronger f-met-leu-phe-induced degranulation of both specific and azurophilic granules [markers: CD11b, CD35 and CD67 (specific granules); CD63 (azurophilic granules)]. In contrast, IGF-I did not alter the PMNL surface expression of Fc gamma RI (CD64), Fc gamma RII (CDw32), or Fc gamma RIII (CD16). PMNLs exposed to IGF-I increased their f-met-leu-phe and phorbol myristate acetate-induced oxidative burst, as evaluated by hydrogen peroxide production, whereas IGF-I did not influence PMNL actin polymerization. The priming of PMNLs by IGF-I was dependent on time and concentration, and saturating amounts of a monoclonal antibody to the IGF-I receptor blocked the priming of PMNLs by this peptide. These experiments demonstrate that IGF-I can selectively stimulate mature PMNL functions, providing further evidence for the interaction between the immune and the endocrine systems.

Signal transduction in human monocytes and granulocytes through the PI-linked antigen CD14.

A possible role for the PI-linked CD14 molecule in human monocyte and granulocyte signal mediation was investigated. Using flow cytometry and the fluorescent indicators Fluo-3 and dihydrorhodamine-123 it was shown that crosslinking of the CD14 molecule induces an increase in monocyte and granulocyte cytoplasmic calcium concentration and monocyte H2O2 production. These responses were found to be independent of IgG Fc receptors and suggest an intrinsic signal mediating capacity of the CD14 molecule.

Flow cytometric assay for combined measurement of phagocytosis and intracellular killing of Candida albicans.

A rapid quantitative flow cytometric (FCM) assay for the combined kinetic measurement of phagocytosis and intracellular killing of fluorescein-isothiocyanate (FITC) labelled Candida albicans is described. The fraction of phagocytosing leukocytes and the numbers of attached or internalized Candida albicans per phagocytosing leukocyte were quantified by FCM, using trypan blue and a fluorescence quenching technique. Results obtained by microscopy agreed with the FCM estimates of phagocytosis. Dead, but not live, Candida albicans stained by propidium iodide (PI). Thus, both viable and intracellularly killed fungi could be discriminated and measured by FCM. Phagocyte killing determined by the FCM assay correlated with killing measured by a standard microbiological test and by methylene blue staining. The method allows rapid and accurate testing of opsonic and phagocytic functions under both experimental and clinical conditions.

Flow cytometric assay for the measurement of serum opsonins to Neisseria meningitidis serogroup B, serotype 15.

A flow cytometric phagocytosis assay has been developed for the measurement of human serum opsonins to serogroup B meningococci. Live bacteria and bacteria inactivated by heat, formalin or ethanol were labelled with fluorescein-isothiocyanate (FITC). The bacteria were opsonized with sera from patients with group B meningococcal disease and sera from healthy controls, and phagocytosis determined by combined measurements of FITC-fluorescence and forward angle light scatter. Optimal sensitivity was obtained using viable bacteria, 5% serum, 20 bacteria per leukocyte capable of phagocytosis, 7.5 min opsonization time, 5 min phagocytosis time, 37 degrees C, and continuous agitation during opsonization and phagocytosis. The opsonic activity of sera from convalescent patients was markedly higher than that of sera from patients with acute illness. Only minor day-to-day and interindividual variations were observed. The flow cytometric phagocytosis technique is a rapid and reproducible method for the measurement of serum opsonins to meningococci.

Correlates of apoptosis of CD4+ and CD8+ T cells in tonsillar tissue in HIV type 1 infection.

The immunopathogenesis of human immunodeficiency virus type 1 (HIV-1) infection has been associated with increased death by apoptosis of T cell subsets. In the present study, we have examined correlates of apoptosis of CD4+, CD8S+CD28+, and CD8+CD28- T cells in tonsillar lymphoid tissue in persons with HIV-1. Single-cell suspensions of tonsillar lymphocytes were analyzed by flow cytometry to determine the fraction of cells showing typical characteristics of apoptosis as well as the expression of activation markers within the live and the apoptotic cell populations. The proportion of cells carrying infectious provirus was quantified by limiting dilution analysis. Compared with uninfected controls, apoptosis of both CD4+ and CD8+ T cells was enhanced in HIV-1 infection and was higher among CD8+ than among CD4+ T cells. Apoptosis of CD28-cells was more prevalent than apoptosis of CD28+ cells for both CD4+ and CD8+ T cells. Occurrence of apoptosis of CD4+ T cells correlated with provirus levels and proportional expression of the activation marker HLA-DR. Apoptosis of CD8+CD28+ cells correlated with expression of the activation markers CD69 and HLA-DR while apoptosis within CD8+CD28- cells did not correlate with any of the studied parameters. Although apoptosis was much more prevalent among CD8+ than CD4+ T cells, CD8+ T cells still accumulated in tonsillar lymphoid tissue in persons with HIV-1. Our data may be interpreted to suggest that apoptosis of CD4+, CD8+CD28+, and CD8+CD28- cells in tonsillar tissue is regulated by different mechanisms and the results are of importance to our understanding of the immunopathogenesis of HIV-1 infection.

Increased neutrophil insulin-like growth factor-I (IGF-I) receptor expression and IGF-I-induced functional capacity in patients with untreated Laron syndrome.

Insulin-like growth factor-I (IGF-I) is a growth hormone-dependent peptide with growth and immunoregulatory properties, and in Laron syndrome growth hormone insensitivity induces impaired production of IGF-I. In the present study we have determined the neutrophil expression of IGF-I receptors (IGF-I-Rs), as well as the IGF-I-induced priming of neutrophil functional capacity, in two children with Laron syndrome treated with recombinant human IGF-I, and in age-matched controls. Before treatment, the patient neutrophil expression of IGF-I-Rs was significantly increased. However, with replacement therapy the neutrophil IGF-I-R expression was downregulated to levels similar to those of the controls within one month. In the patients, the phagocytic capacity and oxidative burst of unprimed neutrophils were normal and similar to controls before the start of treatment. Moreover, IGF-I efficiently primed both patient and control neutrophils to increase their phagocytic capacity and oxidative burst in vitro. However, before therapy, the priming response to IGF-I was significantly stronger in the neutrophils in the patients than in the controls. The present data support earlier studies by us and others demonstrating that IGF-I is a potent regulator of mature neutrophil function, but also suggest that these leukocytes may function normally in the presence of very low levels of IGF-I in vivo.

Two cell kinetic methods studied on the rat corneal epithelium.

A stathmokinetic method (using Colcemid) and the [3H]thymidine technique (pulse labelling with tritiated thymidine, [3H]TdR) have been evaluated in the rat corneal epithelium. The dose is not of critical importance for the Colcemid method, thus indicating an all or nothing effect within the dose range studied. A one point estimate is sufficient to calculate the mitotic rate (MR), and in the rat corneal epithelium a 4 h accumulation period is recommended. After administration of [3H]TdR there is an increasing response with increasing dose, followed by a levelling off at higher doses. It seems reasonable to use the lowest maximal effective dose. The labelling index (LI) can be reliably registered 1 h after administration of the drug. For each of the drugs we found corresponding results after topical application and intraperitoneal injection. Hence, topical application of small doses of both Colcemid and [3H]TdR makes interesting in vivo experiments on larger animals and even on human beings possible. Due to the extreme regularity of the corneal epithelium this part of the eye is an interesting organ for cell kinetic studies and provides an excellent tool for evaluating cell kinetic methods.

Cell kinetics of the rat corneal epithelium.

In the rat corneal epithelium the mitotic rate (MR) is almost equal throughout the epithelium in the morning (Haaskjold et al. 1988). The labelling index (LI) shows a marked reduction in the central cornea, which could suggest a lack of uptake of tritiated thymidine via the salvage pathway (Haaskjold et al. 1989). In the present study we have used [3H]deoxycytidine, and [3H]thymidine after prior treatment with a methotrexate regimen to elucidate this discrepancy. Deoxycytidine is incorporated into DNA independent of thymidine kinase, while methotrexate, which depletes the cells of reduced folates, makes the cells completely dependent on the salvage pathway. With both techniques the same pattern of labelling was observed, confirming that in the morning the ratio between the MR and the LI differs throughout the cornea. Based on previous observations, an analysis of the MR/LI ratio during 24 h was performed, showing that these parameters were strongly correlated. This suggests that there may be different circadian variations in the cell proliferation parameters throughout the corneal epithelium. The methotrexate regimen may be a useful tool to investigate the salvage pathway.

Phagocytosis by human leukocytes, phagosomal pH and degradation of seven species of bacteria measured by flow cytometry.

Phagocytosis by human leukocytes, phagosomal pH and degradation of seven species of bacteria were studied by a flow cytometric method. The percentage of phagocytosing leukocytes was similar for all bacterial strains examined, but Salmonella typhi and Neisseria meningitidis were more slowly phagocytosed than other bacteria. The phagosomal pH surrounding the different bacterial species 15 min after the start of phagocytosis were: Streptococcus pneumoniae 4.4; N. meningitidis 4.9; Str. pyogenes 5.1; Staphylococcus aureus 5.2; Escherichia coli 5.3; S. typhi 5.4; and Klebsiella pneumoniae 5.7. For longer incubation periods, the phagosomal pH remained nearly constant. Staph. aureus, E. coli and S. typhi were the most readily degraded of the species tested. The proteins of all bacteria were degraded more rapidly than their DNA as determined by measurements of the loss of fluorescein-isothiocyanate-fluorescence and ethidium bromide-fluorescence, respectively. The rate of degradation varied from one bacterial species to another. The degradation of proteins and DNA was maximal for bacteria residing in a phagosomal environment estimated to be between pH 5.2 and 5.4.

Early cell kinetic effects of a single dose of narrow-banded ultraviolet B irradiation on the rat corneal epithelium.

The right eyes of 40 rats were exposed to a single erythemogenic dose of ultraviolet B irradiation (UVB) at 297 nm. The irradiation was directed perpendicular to the center of the cornea. The left eyes served as controls. The animals were randomly assigned into 10 groups. The labeling index (LI) after pulse labeling with tritiated thymidine and the mitotic rate (MR) after Colcemid administration were registered in the corneal epithelium at predetermined intervals up to 96 h after the irradiation. A mathematical method was used to correlate corresponding corneal areas from the different animals. In the central cornea the LI was considerably reduced up to 36 h after the irradiation. The LI increased toward the peripheral cornea and reached normal values at the limbal area. The MR was also reduced up to 36 h. However, this reduction was over the entire epithelium. The block in cell proliferation was followed by increased proliferation.

Microbial colonization of large wounds.

This study determines the nature of microbial wound colonization in 28 patients with large burns admitted to the Burn Centre, Haukeland University Hospital, Bergen. Altogether, 748 swabs were taken in 141 sampling procedures. A total of 414 microbial isolates were detected and their resistance patterns to a variety of systemic antimicrobial agents determined. The most frequent isolates were coagulase-negative staphylococci (21.5 per cent) and Staphylococcus aureus (14 per cent), followed by Enterococcus species (11.3 per cent), Pseudomonas aeruginosa (10.9 per cent) and Candida species (9.7 per cent). Forty-one per cent of the enterococci and 36 per cent of the coagulase-negative staphylococci were resistant to the aminoglycosides routinely given in conjunction with surgery in our ward. Only four of the 89 strains of coagulase-negative staphylococci were insensitive to methicillin, and no Staph. aureus were methicillin resistant. The time-related changes of burn wound colonization showed that on admission and during the first week, staphylococci and alpha-haemolytic streptococci were dominant. During the next weeks, these bacteria were gradually superceded by enterococci, gram-negative opportunists (mainly Pseud. aeruginosa, Acinetobacter calcoaceticus and Escherichia coli) and Candida species. The nature of microbial wound colonization and how the flora changes with time should be taken into consideration by those treating thermally injured patients.

Neutrophil dysfunction after thermal injury: alteration of phagolysosomal acidification in patients with large burns.

The neutrophil phagolysosomal acidification during phagocytosis of Staphylococcus aureus was examined in six patients with large burns, using a flow cytometric technique allowing the simultaneous measurement of phagocytosis and phagolysosomal pH. The kinetics of neutrophil phagolysosomal acidification were altered during the first 20 days following injury, as the initial alkalinization of the phagolysosomes documented in control neutrophils could not be demonstrated in patient cells. Only at discharge and follow-up were the kinetics of phagolysosomal acidification normal. In addition, measurements of neutrophil maximal phagolysosomal acidification showed a lower pH in patient phagolysosomes than in the controls during the first 5 days of hospitalization. The changes of phagolysosomal acidification did not correlate with the alterations of neutrophil maturity or phagocytic capacity. The results demonstrate alterations of an oxygen-independent microbicidal mechanism in neutrophils from patients with large burns, which may contribute to the reduced capacity of neutrophil intracellular killing following thermal injury.

Impaired actin polymerization and depolymerization in neutrophils from patients with thermal injury.

Acquired neutrophil dysfunction is considered an important cause of increased susceptibility to infection in patients with burns. In the early postinjury phase, large amounts of circulating chemo-attractants, cytokines and endotoxins induce strong systemic activation of neutrophils which may impair their motile functions. Actin is the most prevalent component of the microfilament lattice that generates force for the neutrophil motile responses, and in the present study we examined the dynamics of actin polymerization and depolymerization in neutrophils from 11 patients with large burns. At admission, the amount of polymerized actin in unstimulated neutrophils was 39.9 per cent higher than that of parallel controls. In addition, there was a positive correlation between the amount of polymerized actin and the total body surface area (TBSA) burn. The time course of patient neutrophil actin polymerization in response to FMLP, C5a, (Ser-IL-8)72, (Ala-IL-8)77 and crosslinking of surface Fc gamma RII was similar to that of controls, and the maximal amount of neutrophil F-actin was demonstrated after 30 s stimulation. At the peak of actin polymerization, however, patient neutrophils contained 27.3, 24.0, 24.7 and 25.6 per cent more polymerized actin than control cells stimulated with FMLP, (Ser-IL,-8)77, (Ala-8)77 and Fc gamma RII crosslinking, respectively. However, the relative increase of neutrophil F-actin following stimulation was significantly lower in patients than in controls. Moreover, the rate of patient neutrophil actin depolymerization was 39.0, 23.5, 63.3 and 51.7 per cent lower than that of controls after stimulation with FMLP, C5a (Ser-IL-8)72 and Fc gamma RII crosslinking, respectively. At discharge, the dynamics of neutrophil actin polymerization and depolymerization were similar to that of controls. The results demonstrate that in neutrophils during the early postburn phase, there are increased basal levels of polymerized actin, a lower responsiveness to stimulation and a reduced rate of actin depolymerization. As periodic polymerization and depolymerization of actin is essential for all neutrophil motile responses, it is probable that the alterations observed may contribute significantly to the overall neutrophil dysfunction following thermal injury.

Effects of topical nasal steroids on human respiratory mucosa and human granulocytes in vitro.

Human respiratory mucosa and human granulocytes were exposed to topical nasal steroids in vitro. The preparations containing benzalkonium chloride and benzalkonium chloride alone destroyed the mucosa within 10 days. The same preparations also inhibited human neutrophil actin polymerization, degranulation and oxidative burst in vitro in a time and concentration dependent manner. Preparations without benzalkonium chloride, as well as the steroid compounds themselves, did not have these effects. It is concluded that benzalkonium chloride has toxic effects on human respiratory mucosa and human neutrophils in vitro.