Plasma carnitine ester profile in homozygous and heterozygous OCTN2 deficiency.

The carnitine ester spectrum was studied using ESI tandem mass spectrometry in a 2.5-year-old male Roma child with homozygous deletion of 844C of the SLC22A5 gene, presenting with hepatopathy and cardiomyopathy. Besides the dramatic decrease of plasma free carnitine (1.38 vs 32.7 mumol/L in controls) all plasma carnitine esters were severely decreased in the proband: the total esters were 31.4% of the controls. In three heterozygous siblings the free carnitine level was 62.3% of the normal controls, while the levels of the individual carnitine esters ranged between 15.5% and 163% (average 70.9%). The heterozygous parents exhibited the same pattern. The proband was supplemented with 50 mg/kg per day of L-carnitine oral solution. After 2 months of treatment, his hepatomegaly, elevated transaminases and the pathological cardiac ultrasound parameters normalized. The plasma free carnitine rose to 12.8 mumol/L (39% of the controls). All of the carnitine esters also increased; however, the individual esters were still 8.5-169.7% of the controls (average 55.5%). After 13 months of treatment there was a further increase in free carnitine (15.9 mumol/L) as well as in the level of the individual esters, ranging between 16.1% and 140.3% of the controls (average 66.9%). The data presented here show that, besides the dramatic decrease of free carnitine, the carnitine ester metabolism is also affected in OCTN2 deficiency; the replenishment of the pools under treatment is slow. Despite an impressive clinical improvement, the carnitine metabolism can be still seriously affected.

Alterations of carbohydrate and lipoprotein metabolism in childhood obesity--impact of insulin resistance and acanthosis nigricans.

AIM: To study the prevalence of alterations of glucose and lipoprotein metabolism and the impact of acanthosis nigricans (AN) in childhood obesity. PATIENTS AND METHODS: 113 obese children, 57 with simple obesity (SO) and 58 with obesity and AN (OAN). Oral glucose tolerance test was performed, serum glucose, insulin and lipoprotein parameters were determined, and insulin resistance/sensitivity indices were calculated. RESULTS: Insulin resistance, basal and reactive hyperinsulinemia, impaired glucose tolerance (IGT) and dyslipidemia were found to be frequent conditions in children with OS as well as OAN. Reactive insulinemia was more pronounced in OAN than in SO, and insulin resistance was more frequent when AN was more prominent. Triglycerides were higher and HDL-C was lower, and atherogenic dyslipidemia was more frequent in OAN compared to SO. CONCLUSION: Children with obesity form a risk population. AN is a factor which can be used in metabolic risk factor clustering estimation in childhood obesity.

Alterations of glucoregulation in childhood obesity--association with insulin resistance and hyperinsulinemia.

AIM: To study the prevalence of alterations of glucoregulation in childhood obesity. PARTICIPANTS: 250 obese children. Oral glucose tolerance test was performed, serum glucose and insulin were determined, and HOMA-IR was calculated. RESULTS: Impaired fasting glucose (IFG) was found in 1.2% according to World Health Organisation criteria and 4.4% according to the criteria of the International Diabetes Federation. Impaired glucose tolerance (IGT) was found in 13.6%, type 2 diabetes mellitus (DM2) in 2.4%. Frequency of fasting glucose (FG) above 7.0 mmol/l was 1.2%. Basal hyperinsulinemia was increased in 70%, reactive hyperinsulinemia in 88%, frequency of elevated HOMA-IR was 78%. 120' insulin was increased in all cases with abnormal FG, IGT and DM2, HOMA-IR was elevated in 79% of patients with IGT and all patients with abnormal FG and DM2. Significant positive correlations were demonstrated between body mass index and insulin levels. CONCLUSION: Our data show that hyperinsulinemia can successfully compensate for insulin resistance in the majority of the obese children. Since IFG is less frequent than IGT there is a need for performing OGTT to demonstrate abnormality of glucoregulation in obese children.

Survival of group B streptococcus type III in mononuclear phagocytes: differential regulation of bacterial killing in cord macrophages by human recombinant gamma interferon and granulocyte-macrophage colony-stimulating factor.

Phagocytic and killing capacities of resident and cytokine-activated human macrophages against group B Streptococcus (GBS) type III were studied. Evidence is presented that monocyte-derived macrophages from cord and adults ingest serum-opsonized GBS but that killing of bacteria was negligible in resident cells. Treatment of adult macrophages with recombinant human gamma interferon (rhIFN-gamma; 100 U/ml) or recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 200 U/ml) resulted in significant increases of killing of GBS (P < 0.01 for each). The killing capacity of cord macrophages treated with rhGM-CSF was also enhanced compared to that of untreated cells (P < 0.01). However, treatment with rhIFN-gamma resulted in only a moderate increase in the capacity of cord macrophages to kill GBS (P > 0.1). These results mirrored the effect of rhIFN-gamma on candidacidal capacities of cord and adult macrophages, reported earlier from our laboratory. These data indicate differential modulation of neonatal macrophages by rhGM-CSF and rhIFN-gamma. We suggest that administration of rhGM-CSF to neonates with invasive GBS disease may enhance host resistance to these bacteria.

Rapid recruitment of late endosomes and lysosomes in mouse macrophages ingesting Candida albicans.

Candida albicans is an important opportunistic pathogen, whose interaction with cells of the immune system, in particular macrophages (MO), is poorly understood. In order to learn more about the nature of the infectious mechanism, internalisation of Candida albicans was studied in mouse MO by confocal immunofluorescence and electron microscopy in comparison with latex beads of similar size, which were coated with mannosyl-lipoarabinomannan (ManLAM) to target the MO mannose receptor (MR). Uptake of Candida yeasts had characteristics of phagocytosis, required intact actin filaments, and depended on the activity of protein kinase C (PKC). Candida phagosomes rapidly attracted lysosome-associated membrane protein (Lamp)-rich vacuoles, indicative of fusion with late endosomes and lysosomes. Rapid recruitment of late endosomes and lysosomes could be observed regardless of heat-inactivation or serum-opsonisation of Candida, but did not follow binding of the mannosylated-beads to MO, which suggest that this phenotype is not MR-specific. The yeasts developed germ tubes within phagolysosomes, distended their membranes and escaped, destroying the non-activated MO. The filamentous form of Candida could penetrate intact MO even when phagocytosis was blocked, and also attracted Lamp-rich organelles. Inhibition of lysosomal acidification and associated lysosomal fusion reduced germ tube formation of Candida within the phagolysosomes. These data suggest that rapid recruitment of late endocytic/lysosomal compartments by internalizing C. albicans favours survival and virulence of this pathogen.

A functional soluble form of the murine mannose receptor is produced by macrophages in vitro and is present in mouse serum.

A soluble form of the mannose receptor (sMR) has been found in conditioned medium of primary macrophages in vitro and in mouse serum. sMR was released as a single species, had a smaller size than the cell-associated form, and accumulated in macrophage-conditioned medium, in a cytokine-regulated manner, to levels comparable with those found for cell-associated mannose receptor. Pulse-chase experiments showed that sMR production in culture occurred by constitutive cleavage of pre-existing full-length protein. A binding assay was developed to determine the sugar specificity of sMR and its ability to interact with pathogens and particulate antigens (i.e. Candida albicans and zymosan). Protease inhibitor studies suggested that sMR was produced by cleavage of an intact mannose receptor by a matrix metalloprotease or ADAM metalloprotease. A role for sMR in the immune response is proposed based on its binding properties, regulation by cytokines, and the previous discovery of putative ligands for the cysteine-rich domain of the mannose receptor in lymph nodes and spleen.

Augmentation of human macrophage candidacidal capacity by recombinant human myeloperoxidase and granulocyte-macrophage colony-stimulating factor.

Phagocyte myeloperoxidase (MPO) is believed to be particularly important in defense against candida infection. We reported earlier that monocytes, rich in MPO, killed Candida albicans at a significantly higher rate and extent than did monocyte-derived macrophages, known to lack MPO, and that C. albicans is less resistant to MPO-dependent oxidants than less pathogenic Candida species. We hypothesized, therefore, that the capacity of macrophages to kill C. albicans might be improved in the presence of MPO. In this study, we evaluated the ability of recombinant human MPO (rhMPO) to augment the killing of C. albicans by resident macrophages and macrophages activated by recombinant human granulocyte-macrophage colony-stimulating factor. Addition of rhMPO (concentration range, 0.8 to 6.4 U/ml) to suspensions of resident and activated macrophages and opsonized C. albicans resulted in concentration-dependent and significant increases in candida killing. This enhancement was particularly pronounced with activated macrophages, whether C. albicans was opsonized or unopsonized and ingested through the macrophage mannose receptor. rhMPO did not affect the killing of C. albicans by monocytes, nor did it affect phagocytosis of opsonized or unopsonized C. albicans. These results indicate that exogenous rhMPO can augment the candidacidal capacity of both resident and activated macrophages, with a more profound effect on activated cells. We suggest that rhMPO may be effective in the treatment of invasive candidiasis.

Characteristics of invasive candidiasis in gamma interferon- and interleukin-4-deficient mice: role of macrophages in host defense against Candida albicans.

Murine models of invasive candidiasis were used to study the in vivo importance of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) in host defense against Candida albicans and to characterize the tissue inflammatory reactions, with special reference to macrophages (Mphi). Knockout (KO) IFN-gamma-deficient (GKO) and IL-4-deficient (IL-4 KO) and C57BL/6 parental mouse strains were challenged intraperitoneally with 10(8) C. albicans blastoconidia. Survival of GKO mice was significantly lower (16.7%) than that of C57BL/6 control (55.5%) and IL-4 KO (61.1%) animals, but was not correlated with the extent of organ colonization. Immunohistological analysis with a panel of myeloid and lymphoid markers revealed multiple renal abscesses, myocarditis, hepatitis, meningoencephalitis, and pneumonia in each strain, with a dominant presence of Mphi. In the absence of IFN-gamma, C. albicans induced striking changes in the phenotype of alveolar Mphi and extensive perivascular lymphoid infiltrates in the lung. Impairment in nitric oxide production by peritoneal Mphi was shown only in GKO mice, and they produced Candida-specific immunoglobulin G (IgG), IgM, IgA, and IgG subclasses in lower titers. Our in vivo studies with KO mice elucidate a critical role for IFN-gamma, but not IL-4, in host defense against C. albicans.

Effect of granulocyte colony-stimulating factor on granulopoiesis of congenital neutropenic children.

We report on two patients with congenital neutropenia, who were treated with filgrastim (recombinant human granulocyte-colony stimulating factor, G-CSF). A poor growth of bone marrow colonies and low sensitivity of colony forming units to colony stimulating factor in vitro before treatment seemed to be associated with a requirement for higher doses of G-CSF to achieve good clinical response in vivo.

Impaired microbicidal capacity of mononuclear phagocytes from patients with type I Gaucher disease: partial correction by enzyme replacement therapy.

The higher susceptibility to serious bacterial infections in patients with Gaucher disease (GD) may be due in part to defective function of phagocytic cells. We studied five patients with GD (type I) and examined the ability of granulocytes and mononuclear phagocytes from these patients to phagocytose and kill Staphylococcus aureus and to generate superoxide anion (O2-) on stimulation with fully opsonized bacteria. Serum-opsonized staphylococci were ingested equally by phagocytic cells from patients and controls. In the presence of normal serum, the extent of killing of S aureus and the release of O2- by granulocytes over incubation periods of 60 minutes and 30 minutes, respectively, were also equivalent for patients and controls. However, we found that killing of viable bacteria and release of O2- by the patients' monocytes was significantly lower than that in cells from controls (P < .05 for both). The magnitude of differences in killing and O2- release between patients' cells and those from controls was even more profound with monocyte-derived macrophages. Enzyme augmentation with macrophage-targeted glucocerebrosidase preparation for 6 months at doses from 7.5 to 10 U/kg/wk resulted in significant increases of functional activities and O2- generation of monocytes and macrophages along with hematologic and hepatosplenic improvements. These data suggest that mononuclear phagocytes from GD patients are defective in their ability to kill bacteria and to generate reactive oxygen intermediates. Our data also suggest that enzyme substitution may improve functions of monocytes and macrophages in patients with GD that should make them more resistant to severe bacterial infection.

Chronic neutropenia and defect in superoxide generation of granulocytes in two patients: enhancement of bactericidal capacity and respiratory burst activity by treatment with recombinant human granulocyte colony-stimulating factor.

We have identified two unrelated girls with chronic neutropenia [absolute neutrophil counts (ANC) 10-870 and 10-940/microL in patients 1 and 2, respectively] and severe defect in superoxide anion generation by granulocytes. Formyl-methionyl-leucyl-phenylalanine-induced superoxide release was 1.2 +/- 0.9 and 1.9 +/- 1.9% (mean +/- SEM, n = 3) of normal controls', mean value in patients 1 and 2, respectively. However, granulocytes from both patients released a normal amount of superoxide upon stimulation with phorbol myristate acetate. Patient 2 exhibited characteristic features of Duane syndrome, a rare disorder of eye movement. Treatment of the patients with recombinant granulocyte colony-stimulating factor led to significant clinical improvements and reduction of infectious complications and to increases in the ANC, to 400-2100/microL in patient 1 and to 500-3000/microL in patient 2. Treatment with 5 micrograms/kg/d resulted in increased intracellular killing of opsonized Staphylococcus aureus by granulocytes and an enhancement of superoxide release upon stimulation with formyl-methionyl-leucyl-phenylalanine in both patients up to 11.1 +/- 6.0 and 13.5 +/- 7.0% (mean +/- SEM, n = 5) of normal controls', mean value in patient 1 and patient 2, respectively. These data suggested that recombinant human granulocyte colony-stimulating factor treatment enhanced resistance to bacterial infection by stimulation of superoxide generation and increasing the bactericidal capacity of peripheral blood granulocytes.

Candidacidal mechanisms in the human neonate. Impaired IFN-gamma activation of macrophages in newborn infants.

We studied the interaction between Candida albicans and mononuclear phagocytes derived from cord blood. In the presence of normal serum, the extent of phagocytosis and killing of candida by monocyte-derived macrophages was equivalent for newborns and adults. In the absence of serum both phagocytosis and killing by macrophages were reduced by half, but cord and adult cells were still equivalent. Mannosylated BSA and mannan inhibited ingestion of unopsonized candida by macrophages, suggesting a role for the mannose receptor. Exposure of cord and adult macrophages to IFN-gamma (10-500 U/ml) gave quantitatively different results in Candida killing, as well as in release of superoxide anion (O2-). Maximal increase in these functions with adult macrophages was achieved with 100 U/ml IFN-gamma. No enhancement with cord macrophages could be detected after treatment with 100 U/ml, and at 500 U/ml there was still significantly lower killing and O2- release compared with adult cells. Defective up-regulation of O2- release was also present in cord monocytes exposed to IFN-gamma on day 0. Studies of the surface expression of IFN-gamma receptors using a "nonblocking" mAb against the IFN-gamma receptor revealed a comparable number of receptors on cord and adult monocytes. When blocking Abs were used, however, there was a three times higher number of positive cells in cord monocytes. Specific binding of 125I-IFN-gamma to cord monocytes and macrophages was also higher compared with adult cells. These data suggest that neonatal macrophages have a normal capacity to ingest and kill both opsonized and unopsonized Candida but cannot be fully activated by IFN-gamma, a finding that could not be attributed to lower expression of IFN-gamma receptors on the neonatal cells.