The clinical presentation and genotype of protein C deficiency with double mutations of the protein C gene.

BACKGROUND: Severe protein C (PC) deficiency is a rare heritable thrombophilia leading to thromboembolic events during the neonatal period. It remains unclear how individuals with complete PC gene (PROC) defects develop or escape neonatal stroke or purpura fulminans (PF). PROCEDURE: We studied the onset of disease and the genotype of 22 PC-deficient patients with double mutations in PROC based on our cohort (n = 12) and the previous reports (n = 10) in Japan. RESULTS: Twenty-two patients in 20 unrelated families had 4 homozygous and 18 compound heterozygous mutations. Sixteen newborns presented with PF (n = 11, 69%), intracranial thromboembolism and hemorrhage (n = 13, 81%), or both (n = 8, 50%), with most showing a plasma PC activity of <10%. Six others first developed overt thromboembolism when they were over 15 years of age, showing a median PC activity of 31% (range: 19-52%). Fifteen of the 22 patients (68%) had the five major mutations (G423VfsX82, V339M, R211W, M406I, and F181V) or two others (E68K and K193del) that have been reported in Japan. Three of the six late-onset cases, but none of the 16 neonatal cases, had the K193del mutation, which has been reported to be the most common variant of Chinese thrombophilia. A novel mutation of A309V was determined in a family of two patients with late onset. CONCLUSIONS: The genotype of double-PROC mutants might show less diversity than heterozygous mutants in terms of the timing of the onset of thrombophilia (newborn onset or late onset).

Age-specific onset and distribution of the natural anticoagulant deficiency in pediatric thromboembolism.

BACKGROUND: The early diagnosis of inherited thrombophilia in children is challenging because of the rarity and hemostatic maturation. METHODS: We explored protein C (PC), protein S (PS), and antithrombin (AT) deficiencies in 306 thromboembolic patients aged ≤20 y using the screening of plasma activity and genetic analysis. RESULTS: Reduced activities were determined in 122 patients (40%). Low PC patients were most frequently found in the lowest age group (0-2 y, 45%), while low PS or low AT patients were found in the highest age group (16-20 y; PS: 30% and AT: 20%). Genetic study was completed in 62 patients having no other causes of thromboembolism. Mutations were determined in 18 patients (8 PC, 8 PS, and 2 AT genes). Six of eight patients with PC gene mutation were found in age 0-2 y (75%), while six of eight patients with PS gene mutation were in 7-20 y. Two AT gene-mutated patients were older than 4 y. Four PC-deficient and two PS-deficient patients carried compound heterozygous mutations. All but one PC gene-mutated patient suffered from intracranial thromboembolism, while PS/AT gene-mutated patients mostly developed extracranial venous thromboembolism. CONCLUSION: Stroke in low PC infants and deep vein thrombosis in low PS/AT school age children could be targeted for genetic screening of pediatric thrombophilias.

[Analysis of the Neuraminidase Amino Acid Sequences of Influenza A/H1N1pdm09, A/H3N2, and B Viruses Isolated from Influenza Patients in the 2013/14 Japanese Influenza Season].

BACKGROUND: Neuraminidase (NA) is an essential surface protein for influenza virus replication. NA inhibitors are commonly used for the treatment of influenza patients in Japan. Several mutations that reduce the effect of NA inhibitors have been reported. We sequenced the whole NA segment of isolated virus from influenza patients and investigated the relation between the NA amino acid sequence and the 50% inhibitory concentration (IC50) of four NA inhibitors. MATERIALS AND METHODS: A total of 20 viruses that showed high or low IC50 of NA inhibitors were selected from A/H1N1pdm09, A/H3N2, and B isolates from the viruses isolated from patients in the 2013-14 influenza season. Viral RNA was extracted and RT-PCR was done. The amplified genome was sequenced using a next generation sequencer", and the deduced amino acid sequences were analyzed. RESULTS: Two A/H1N1pdm09 viruses that showed very high IC50 for oseltamivir (150 nM and 130 nM) contained the H275Y mutation. Otherwise, no significant relation was found between the NA amino acids and the IC50 of the four NA inhibitors. There was no significant relation between the NA amino acids and the IC50 of the four NA inhibitors for A/H3N2 viruses. The B viruses that showed a high IC50 for oseltamivir and laninamivir shared some amino acids. The B viruses that showed a high IC50 of zanamivir and peramivir also shared some amino acids. They were different from the shared amino acids found for oseltamivir and laninamivir. CONCLUSION: The previously reported H275Y mutation that causes oseltamivir resistance was found in the two A/H1N1pdm09 viruses that showed a very high IC50 for oseltamivir. No additional NA amino acid sequences related to the IC50 of the four NA inhibitors was found. The meaning of the shared amino acids among B viruses that showed a high IC50 would be an interesting target for further investigation.

[Analysis of influenza A/H3N2 neuraminidase genes obtained from influenza patients in the 2011/12 and 2012/13 seasons in Japan].

BACKGROUND: Influenza virus has neuraminidase (NA), a surface protein with enzymatic activity that is essential for virus replication. Mutation may affect the effectiveness of NA inhibitors that are used for the treatment of influenza patients. In this study, we determined the NA gene sequences from the clinical isolates of influenza patients to examine the chronological genetic changes and the relation to drug susceptibility. METHODS: For 96 A/H3N2 virus isolates the 50% inhibitory concentration (IC50) (48 each from the 2011-12 and 12-13 influenza seasons) was measured. RT-PCR was done with extracted viral RNA, followed by nucleotide sequencing. RESULTS: One putative amino acid mutation, D151N, was found in an NA activity-related cite in five of ninety-six tested isolate. The mutation did not affect the IC50 value. The mutations identified at amino acid positions 387 and 400 were statistically correlated with an increased IC50 value, although the change was less than ten times, suggesting no significant difference in the clinical effectiveness. A small number .of isolates showed mutation in the T and/or B cell epitope region of NA. CONCLUSION: No mutation that affected the IC50 value or effectiveness of NAIs was detected. Antigenic mutations of NA, which influence the selection of epidemic strains, were not determined. Continuous observation will be necessary to further clarify the genetic features of NA.

Staphylococcal endocarditis as the first manifestation of heritable protein S deficiency in childhood.

A 12-year-old Japanese girl developed infective endocarditis and central nervous system disease. The previously healthy girl showed altered consciousness and abnormal behaviors along with the classical signs of septic emboli. Staphylococcus aureus was isolated from peripheral blood, but not, the pleocytotic cerebrospinal fluid. Diagnostic imaging studies revealed a vegetative structure in the morphologically normal heart, and multiple thromboembolisms in the brain and spleen. Low plasma activity of protein S (12%) and thrombophilic family history allowed the genetic study, demonstrating that she carried a heterozygous mutation of PROS1 (exon 13; 1689C > T, p.R474C). Surgical intervention of the thrombotic fibrous organization and subsequent anticoagulant therapy successfully managed the disease. There are no reports of infective endocarditis in childhood occurring as the first presentation of heritable thrombophilia. Protein S deficiency might be a risk factor for the development or exacerbation of infective endocarditis in children having no pre-existing heart disease.

Involvement of protein kinase D in uridine diphosphate-induced microglial macropinocytosis and phagocytosis.

The clearance of tissue debris by microglia is a crucial component of maintaining brain homeostasis. Microglia continuously survey the brain parenchyma and utilize extracellular nucleotides to trigger the initiation of their dynamic responses. Extracellular uridine diphosphate (UDP), which leaks or is released from damaged neurons, has been reported to stimulate the phagocytotic activity of microglia through P2Y(6) receptor activation. However, the intracellular mechanisms underlying microglial P2Y(6) receptor signals have not been identified. In this study, we demonstrated that UDP stimulation induced immediate and long-lasting dynamic movements in the cell membrane. After 60 min of UDP stimulation, there was an upregulation in the number of large vacuoles formed in the cell that incorporate extracellular fluorescent-labeled dextran, which indicates microglial macropinocytosis. In addition, UDP-induced vacuole formation and continuous membrane motility were suppressed by the protein kinase D (PKD) inhibitors, Gö6976 and CID755673, unlike Gö6983, which is far less sensitive to PKD. The inhibition of PKD also reduced UDP-induced incorporation of fluorescent-labeled dextran and soluble ß-amyloid and phagocytosis of microspheres. UDP induced rapid phosphorylation and membrane translocation of PKD, which was abrogated by the inhibition of protein kinase C (PKC) with Gö6983. However, Gö6983 failed to suppress UDP-induced incorporation of microspheres. Finally, we found that inhibition of PKD by CID755673 significantly suppressed UDP-induced engulfment of IgG-opsonized microspheres. These data suggest that a PKC-independent function of PKD regulates UDP-induced membrane movement and contributes to the increased uptake of extracellular fluid and microspheres in microglia.

Involvement of vasodilator-stimulated phosphoprotein in UDP-induced microglial actin aggregation via PKC- and Rho-dependent pathways.

Microglia are major immunocompetent cells in the central nervous system and retain highly dynamic motility. The processes which allow these cells to move, such as chemotaxis and phagocytosis, are considered part of their functions and are closely related to purinergic signaling. Previously, we reported that the activation of the P2Y(6) receptor by UDP stimulation in microglia evoked dynamic cell motility which enhanced their phagocytic capacity, as reported by Koizumi et al. (Nature 446(7139):1091-1095, 2007). These responses require actin cytoskeletal rearrangement, which is seen after UDP stimulation. However, the intracellular signaling pathway has not been defined. In this study, we found that UDP in rat primary microglia rapidly induced the transient phosphorylation at Ser157 of vasodilator-stimulated phosphoprotein (VASP). VASP, one of actin binding protein, accumulated at the plasma membrane where filamentous (F)-actin aggregated in a time-dependent manner. The phosphorylation of VASP was suppressed by inhibition of PKC. UDP-induced local actin aggregations were also abrogated by PKC inhibitors. The Rho inhibitor CT04 and the expression of p115-RGS, which suppresses G(12/13) signaling, attenuated UDP-induced phosphorylation of VASP and actin aggregation. These results indicate that PKC- and Rho-dependent phosphorylation of VASP is involved in UDP-induced actin aggregation of microglia.

Activation of P2X7 receptors induces CCL3 production in microglial cells through transcription factor NFAT.

Microglia are implicated as a source of diverse proinflammatory factors in the CNS. Extracellular nucleotides are well known to be potent activators of glial cells and trigger the release of cytokines from microglia through purinergic receptors. However, little is known about the role of purinoceptors in microglial chemokine release. In this study, we found that high concentrations of ATP evoked release of CC-chemokine ligand 3 (CCL3)/macrophage inflammatory protein-1alpha from MG-5 cells, a mouse microglial cell line, and rapid up-regulation of CCL3 mRNA was elicited within 30 min of ATP stimulation. The release of CCL3 was also stimulated by 2'- and 3'-O-(4-benzoylbenzoyl) ATP, an agonist of P2X(7) receptors. Brilliant Blue G, an antagonist of P2X(7) receptors, strongly inhibited this ATP-induced CCL3 release. Similar pharmacological profile was observed in primary microglia. In MG-5 cells, ATP caused de-phosphorylation and nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT). ATP-induced NFAT de-phosphorylation was also dependent on P2X(7) receptor activation. Furthermore, ATP-induced CCL3 release and production were prevented by a selective inhibitor of NFAT. Taken together, the results of this study demonstrate an involvement of NFAT in the mechanism underlying P2X(7) receptor-mediated CCL3 release.