The protein tyrosine kinase JAK2 is activated in neutrophils from patients with severe congenital neutropenia.

Severe congenital neutropenia (SCN; or Kostmann syndrome) is an autosomal recessive disorder characterized by a maturation arrest of myelopoiesis at the level of promyelocytes. Myeloid precursor cells from patients with SCN require pharmacological dosages of recombinant human granulocyte colony-stimulating factor (r-metHuG-CSF; Filgrastim; Amgen, Thousand Oaks, CA) to differentiate to normal neutrophils. Thus, it is hypothesized that the underlying defect responsible for SCN is based on an abnormal G-CSF-induced signal transduction pathway. Because JAK2, a nonreceptor tyrosine kinase, is involved in the signaling pathway of G-CSF, we examined the expression and activity of JAK2 in neutrophils from SCN patients during r-metHuG-CSF treatment. The immunoprecipitated JAK2 protein showed increased tyrosine phosphorylation in neutrophils from SCN patients as compared with that in neutrophils from healthy donors, suggesting that this kinase is activated. In vitro kinase assays of immunoprecipitated JAK2 confirmed that neutrophils from SCN patients show an increased autophosphorylation of JAK2 in comparison with that of neutrophils from healthy volunteers. These findings suggest that JAK2 is activated in SCN patients.

Influence of modified natural or synthetic surfactant preparations on growth of bacteria causing infections in the neonatal period.

Connatal bacterial pneumonia is common in neonates. Animal studies and initial clinical reports indicate that surfactant dysfunction is involved in the pathophysiology of severe neonatal pneumonia. Since respiratory distress syndrome and connatal pneumonia may be difficult to differentiate in the first hours of life, neonates with respiratory failure due to bacterial infections might receive surfactant. Under such conditions surfactant components might be catabolized by bacteria and promote bacterial growth. We therefore investigated the influence of three modified natural (Curosurf, Alveofact, and Survanta) and two synthetic (Exosurf and Pumactant) surfactant preparations on the growth of bacteria frequently cultured from blood or tracheal aspirate fluid in the first days of life. Group B streptococci (GBS), Staphyloccocus aureus, and Escherichia coli were incubated in a nutrient-free medium (normal saline) for 5 h at 37 degrees C, together with different surfactants at concentrations of 0, 1, 10, and 20 mg/ml. With the exception of E. coli, incubation in saline alone led to a variable decrease in CFU. In the presence of Alveofact, Exosurf, and Pumactant the decline in bacterial numbers was less marked than in saline alone. Curosurf was bactericidal in a dose-dependent fashion for GBS and had a strong negative impact on the growth of a GBS subtype that lacked the polysaccharide capsule. In contrast, Survanta (10 and 20 mg/ml) significantly promoted the growth of E. coli, indicating that surfactant components may actually serve as nutrients. We conclude that bacterial growth in different surfactant preparations is influenced by microbial species and the composition and dose of the surfactant. Further studies are necessary to elucidate the mechanisms behind our findings and to evaluate the effects of surfactant on bacterial growth in vivo.

Direct and phagocyte-mediated lipid peroxidation of lung surfactant by group B streptococci.

In newborn infants, group B streptococci (GBS) often cause pneumonia, with polymorphonuclear leukocytes (PMN) migrating into the lungs. Because surfactant therapy may be needed in such patients, we evaluated the interaction between GBS or GBS-stimulated PMN and a surfactant preparation (Curosurf) in vitro. The superoxide production of GBS strains or GBS-activated PMN was measured, using the nitroblue tetrazolium (NBT) test and the subsequent lipid peroxidation (LPO) as the content of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE). The growth of GBS in surfactant was determined and related to the LPO. Finally, the effect of LPO on surfactant activity, caused by GBS-stimulated PMN, was assessed by measuring dynamic surface tension in a pulsating bubble surfactometer. Curosurf diminished the NBT reduction by both live GBS and GBS-stimulated PMN. Surfactant was peroxidized by reactive oxygen species (ROS) from both GBS and GBS-stimulated PMN in a time-dependent manner. Vitamin E significantly reduced the peroxidation level of surfactant in both cases. Surfactant peroxidation was associated with a reduction in the number of live bacteria. The biophysical activity of Curosurf was impaired by GBS-stimulated PMN, as reflected by increased minimum surface tension during cyclic compression. These findings indicate that Curosurf undergoes LPO by ROS produced by GBS and/or PMN. We speculate that exogenous surfactant preparations should be supplemented with vitamin E or another antioxidant, when given to infants with GBS pneumonia.

Combined treatment with surfactant and specific immunoglobulin reduces bacterial proliferation in experimental neonatal group B streptococcal pneumonia.

Neonates suffering from group B streptococcal (GBS) pneumonia often lack type-specific opsonizing antibodies. We studied the influence of combined intratracheal treatment with surfactant and a specific antibacterial polyclonal antibody (IgG fraction) on bacterial proliferation and lung function in an animal model of GBS pneumonia. Near-term newborn rabbits received an intratracheal injection of either the specific IgG antibody, nonspecific IgG, surfactant, a mixture of surfactant and the antibody, or 0.9% saline. At 30 min the rabbits were infected with a standard dose (10(8)) of the encapsulated GBS strain 090 Ia. After 5 h of mechanical ventilation the mean estimated increase in bacterial number in lung homogenate (log10 colonies/g) was 0.76 in the antibody group, 0.92 in the nonspecific IgG group, 0.55 in the surfactant group, and 1.29 in the saline group. A mean decrease in bacterial number (-0.05) was observed in the group that received combined treatment with surfactant and antibody (p < 0.05 versus all other groups). Lung-thorax compliance was significantly higher in both groups of surfactant-treated animals compared with saline or IgG treatment. We conclude that in experimental neonatal GBS pneumonia combined treatment with surfactant and a specific immunoglobulin against GBS reduced bacterial proliferation more effectively than either treatment alone.

Effects of perfluorocarbons and perfluorocarbons/surfactant emulsions on growth and viability of group B streptococci and Escherichia coli.

OBJECTIVE: Partial liquid ventilation with perfluorocarbons (PFC) might be used as a new ventilatory strategy to treat respiratory insufficiency in congenital pneumonia. The present study investigates for the first time effects of PFC on growth and viability of group B streptococci (GBS) and Escherichia coli, bacteria frequently causing congenital pneumonia. DESIGN: Prospective, in vitro study. SETTING: Research laboratory in a university. MATERIAL: Group B streptococci 090 Ia HD Colindale and E. coli K12, JM 101. INTERVENTIONS: E. coli (10(7)/mL) were grown in the absence or presence of different PFC (RM 101, PF 5080, FO 6167) for up to 6 hrs. To study bacterial viability, GBS (5 x 10(7)/mL) were incubated in saline with or without different PFC, PFC/surfactant emulsions, or surfactant (Curosurf) for up to 5 hrs. Every 2 hrs, the colony forming units were determined by plating different dilutions of bacteria on agar. MEASUREMENTS AND MAIN RESULTS: RM 101 or PF 5080 alone and in emulsions with surfactant had no effect on viability of GBS or growth of E. coli. For FO 6167, a previously described toxicity was found, even if 1 mL of GBS suspension was incubated with only 100 microL of FO 6167, verifying the experimental design that guarantees a PFC bacteria contact. The toxic effects were almost prevented by forming a PFC-in-surfactant emulsion but not by preincubation of GBS with surfactant and subsequent FO 6167 exposure. CONCLUSION: RM 101 and PF 5080 did not influence bacterial growth in vitro; direct effects on bacterial proliferation during partial liquid ventilation in congenital pneumonia seem, therefore, unlikely. Interestingly, we found that the known toxic effects of FO 6167 can be prevented by covering PFC with a surfactant film. Surfactant reduced the cytotoxic effects of FO 6167, probably by preventing a direct contact between FO 6167 and the bacterial cell wall.

Resistance of different surfactant preparations to inactivation by meconium.

A disease similar to acute respiratory distress syndrome may occur in neonates after aspiration of meconium. The aim of the study was to compare the inhibitory effects of human meconium on the following surfactant preparations suspended at a concentration of 2.5 mg/mL: Curosurf, Alveofact, Survanta, Exosurf, Pumactant, rabbit natural surfactant from bronchoalveolar lavage, and two synthetic surfactants based on recombinant surfactant protein-C (Venticute) or a leucine/lysine polypeptide. Minimum surface tension, determined with a pulsating bubble surfactometer, was increased >10 mN/m at meconium concentrations >or=0.04 mg/mL for Curosurf, Alveofact, or Survanta, >or=0.32 mg/mL for recombinant surfactant protein-C, >or=1.25 mg/mL for leucine/lysine polypeptide, and >or=20 mg/mL for rabbit natural surfactant. The protein-free synthetic surfactants Exosurf and Pumactant did not reach minimum surface tension <10 mN/m even in the absence of meconium. We conclude that surfactant activity is inhibited by meconium in a dose-dependent manner. Recombinant surfactant protein-C and leucine/lysine polypeptide surfactant were more resistant to inhibition than the modified natural surfactants Curosurf, Alveofact, or Survanta but less resistant than natural lavage surfactant containing surfactant protein-A. We speculate that recombinant hydrophobic surfactant proteins or synthetic analogs of these proteins can be used for the design of new surfactant preparations that are relatively resistant to inactivation and therefore suitable for treatment of acute respiratory distress syndrome.

Clinical relevance of point mutations in the cytoplasmic domain of the granulocyte colony-stimulating factor receptor gene in patients with severe congenital neutropenia.

Recently, point mutations in the gene of the granulocyte colony-stimulating factor (G-CSF) receptor have been reported in two patients with severe congenital neutropenia who developed acute myeloid leukemia (AML). We investigated the frequency of these specific G-CSF receptor mutations in patients with congenital neutropenia undergoing treatment with r-metHuG-CSF (Filgrastim) and the clinical relevance of these mutations. Nucleotides 2306 to 2561 including the critical region (nucleotides 2384-2429) from the intracellular domain of the G-CSF receptor gene were amplified by reverse transcriptase-polymerase chain reaction. Detection of point mutations was performed with specific restriction enzyme analysis, as well as sequencing of PCR products. Both genomic DNA and cDNA from neutrophils and mononuclear cells were analyzed from 28 patients with severe congenital neutropenia. Four of 28 patients with congenital neutropenia displayed a point mutation in the tested cytoplasmic region of the G-CSF receptor gene. The point mutations replace a glutamine codon by a stop codon of the G-CSF receptor gene. Among these four congenital neutropenia patients with a mutated G-CSF receptor, two developed AML. All four patients were investigated regularly and no correlation between occurrence of G-CSF receptor mutation and time or dose of r-metHuG-CSF treatment was found. No point mutations in the G-CSF receptor critical domain could be detected in cells from the other 24 congenital neutropenia patients. Furthermore, we tested six family members of the two patients with AML including mothers and fathers, one sister, and one brother who suffers from congenital neutropenia, as well. All family members displayed a normal G-CSF receptor gene. After the acquisition of the G-CSF receptor mutations, the congenital neutropenia patients continued to respond to G-CSF therapy with an increase in absolute neutrophils in the peripheral blood. We conclude that the point mutations in the critical region of the intracellular part of the G-CSF receptor occur spontaneously and are not inherited. From our data, we suggest that the described G-CSF receptor point mutations do not alter the response to treatment with r-metHuG-CSF and are not the cause of severe congenital neutropenia.