Human plasma-derived immunoglobulin G fractionated by an aqueous two-phase system, caprylic acid precipitation, and membrane chromatography has a high purity level and is free of detectable in vitro thrombogenic activity.

BACKGROUND AND OBJECTIVES: Instituto Clodomiro Picado has developed an immunoglobulin G (IgG) plasma fractionation process combining a polyethylene glycol/phosphate aqueous two-phase system (ATPS), caprylic acid precipitation and anion-exchange membrane chromatography. We evaluated the purity and in vitro thrombogenicity of such IgG, in line with current international requirements. MATERIALS AND METHODS: Contributions of the different production steps to reduce thrombogenicity were assessed at 0·2 l-scale, and then the methodology was scaled-up to a 10 l-scale and final products (n = 3) were analysed. Purity, immunoglobulin composition, and subclass distribution were determined by electrophoretic and immunochemical methods. The in vitro thrombogenic potential was determined by a thrombin generation assay (TGA) using a Technothrombin fluorogenic substrate. Prekallikrein activator (PKA), plasmin, factor Xa, thrombin and thrombin-like activities were assessed using S-2302, S-2251, S-2222, S-2238 and S-2288 chromogenic substrates, respectively, and FXI by an ELISA. RESULTS: The thrombogenicity markers were reduced mostly during the ATPS step and were found to segregate mostly into the discarded liquid upper phase. The caprylic acid precipitation eliminated the residual procoagulant activity. The IgG preparations made from the 10 l-batches contained 100% gamma proteins, low residual IgA and undetectable IgM. The IgG subclass distribution was not substantially affected by the process. TGA and amidolytic activities revealed an undetectable in vitro thrombogenic risk and the absence of proteolytic enzymes in the final product. CONCLUSIONS: Fractionating human plasma by an ATPS combined with caprylic acid and membrane chromatography resulted in an IgG preparation of high purity and free of a detectable in vitro thrombogenic risk.

Dengue virus inactivation by minipool TnBP/Triton X-45 treatment of plasma and cryoprecipitate.

BACKGROUND AND OBJECTIVES: A minipool solvent/detergent (S/D; 1% TnBP/1% Triton X-45; 31°C) process was developed for viral inactivation of plasma and cryoprecipitate used for transfusion. The goal of this study was to determine the rate and extent of inactivation of dengue virus (DENV) during this process. MATERIALS AND METHODS: DENV-1 was propagated using C6/36 mosquito cells to an infectivity titre close to 9 log and spiked (10% v/v) into individual plasma and cryoprecipitate samples from two distinct donors. Samples were taken right after spiking and during viral inactivation treatment by 1% TnBP-1% Triton X-45 at 31°C. DENV-1 infectivity was assessed on Vero E6 cells by a focus-forming assay (FFA). Culture medium and complement-inactivated plasma were used as experimental controls. Experiments were done in duplicate. RESULTS: DENV-1 infectivity was 7·5 log in spiked plasma and 7·1 and 7·3 log in spiked cryoprecipitate. There was no loss of DENV-1 infectivity in the spiked materials, nor in the controls not subjected to S/D treatment. No infectivity was found in plasma and cryoprecipitate subjected to S/D treatment at the first time-point evaluated (10 min). CONCLUSION: DENV-1 was strongly inactivated in plasma and cryoprecipitate, respectively, within 10 min of 1% TnBP/1% Triton X-45 treatment at 31°C. These data provide a reassurance of the safety of such S/D-treated plasma and cryoprecipitate with regard to the risk of transmission of all DENV serotypes and other flaviviruses.

Impact of solvent/detergent treatment of plasma on transfusion-relevant bacteria.

BACKGROUND: A solvent/detergent (S/D) treatment in a medical device has been developed for pathogen reduction of plasma for transfusion. Impact of S/D on bacterial growth and on the capacity of complement to kill bacteria has been investigated in this study. STUDY DESIGN AND METHODS: A pool of apheresis plasma from four donors was spiked with eight transfusion-relevant bacteria. Plasma was treated with 1% tri(n-butyl) phosphate and 1% Triton X-45 at 31°C for 90 min and then extracted by oil at 31°C for 70 min. Decomplemented plasma and Phosphate Buffer Saline were used as controls. Bacterial count was determined in samples taken immediately after spiking, or after S/D and oil treatment. Similar experiments were conducted using three individual recovered plasma donations. Bacteria growth inhibition tests were performed using discs soaked with plasma samples whether containing the S/D agents or not. RESULTS: The mean reduction factors of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae due to complement during S/D treatment were >8·75, 4·71, and 4·18 log in pooled plasma and >7·42, 2·24 and >6·08 log in individual plasmas, respectively. Bacillus cereus and Bacillus subtilis were inactivated by S/D (>7·04 and 1·60 log in pooled, and >6·06 and 2·39 in individual plasmas, respectively). Staphylococcus aureus, Staphylococcus epidermidis and Enterobacter cloacae did not multiply during S/D treatment of plasma. Growth inhibition tests revealed an inhibition of three gram-negative bacteria by complement and all gram-positive by S/D. CONCLUSION: The S/D treatment of plasma does not alter the bactericidal activity of complement, and inactivates some gram-positive bacteria.

Acute disseminated encephalomyelitis that meets modified McDonald criteria for dissemination in space is associated with a high probability of conversion to multiple sclerosis in Taiwanese patients.

BACKGROUND: Patients with acute disseminated encephalomyelitis (ADEM) may relapse and some may ultimately convert to multiple sclerosis (MS); however, no criteria that can predict MS conversion are available to date. Our aim was to describe the clinical and magnetic resonance imaging (MRI) features of patients with an initial ADEM attack and evaluate which MRI criteria can predict conversion to MS. METHODS: We retrospectively reviewed the records of 36 patients diagnosed with ADEM. We determined clinical signs/symptoms, examined the cerebrospinal fluid (CSF), and performed brain MRI scans and compared the findings between patients who did and did not convert to MS. RESULTS: Clinical signs/symptoms, and CSF analysis show no significant difference between the two groups. The rate of conversion to MS from ADEM in Taiwanese patients is low (11%) after a mean follow-up period of 28.36 months. Modified McDonald criteria were fulfilled in 19/36 patients: 21% (4/19) of those patients developed MS according to Poser criteria subsequently. Of the other patients (17/36) who did not fulfill these criteria, none converted to MS. (log rank test; P=0.027). CONCLUSIONS: It is difficult to predict from initial clinical presentations to address which patients with ADEM will convert to MS. Patients with ADEM whose brain MRI findings met the modified McDonald criteria may have clinically isolated syndrome because they have a significantly higher probability of conversion to MS. In contrast, patients whose brain MRI findings did not meeting these criteria may be considered as having classic ADEM because they have a lower probability of conversion to MS.

Pyridoxal phosphate is better than pyridoxine for controlling idiopathic intractable epilepsy.

AIM: To study the difference between pyridoxine (PN) and its active form, pyridoxal phosphate, (PLP) in control of idiopathic intractable epilepsy in children. METHODS: Among 574 children with active epilepsy, 94 (aged 8 months to 15 years) were diagnosed with idiopathic intractable epilepsy for more than six months. All received intravenous PLP 10 mg/kg, then 10 mg/kg/day in four divided doses. If seizures recurred within 24 hours, another dose of 40 mg/kg was given, followed by 50 mg/kg/day in four divided doses. For those patients whose seizures were totally controlled, PLP was replaced by the same dose of oral PN. If the seizure recurred, intravenous PLP was infused followed by oral PLP 50 mg/kg/day. RESULTS: Fifty seven patients had generalised seizures (of whom 13 had infantile spasms) and 37 had focal seizure. Eleven had dramatic and sustained responses to PLP; of these, five also responded to PN. Within six months of treatment with PLP or PN, five of the 11 patients were seizure free and had their previous antiepileptic medicine tapered off gradually. Two were controlled with pyridoxine and the other three needed PLP to maintain seizure freedom. The remaining six responders needed PLP exclusively for seizure control. Six of the 11 responders to PLP had infantile spasms (46%); four of them needed PLP exclusively. The other five responders were in the remaining 81 patients with other seizure type. CONCLUSIONS: PLP could replace PN in the treatment of intractable childhood epilepsy, particularly in the treatment of infantile spasms.

Heterocellular gap junctional communication between alveolar epithelial cells.

We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6 alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.

EMF genes interact with late-flowering genes in regulating floral initiation genes during shoot development in Arabidopsis thaliana.

To investigate the mechanisms regulating the initiation of floral development in Arabidopsis, a construct containing beta-glucuronidase (GUS) gene driven by APETALA1 promoter (AP1::GUS) was introduced into emf fwa and emf ft double mutants. GUS activity was strongly detected on shoot meristem of emf1-1 single mutants harboring AP1::GUS construct just 5 d after germination. By contrast, GUS activity was undetectable on emf1-1 fwa-1, emf1-1 ft-1, emf2-1 fwa-1, emf2-3 fwa-1 and emf2-3 ft-1 double mutants harboring AP1::GUS construct 10 d after germination. GUS activity was only weakly detected on the apical meristem of 20-day-old emf1-1 fwa-1 and emf2-1 fwa-1 seedlings. During this time, only sessile leaves were produced. Further analysis indicated that AP1 was strongly expressed in 10-day-old emf1-1 and emf2-1 single mutants. Its expression was significantly reduced in all emf1-1 or emf2-1 late-flowering double mutants tested. Similar to AP1, the expression of LEAFY (LFY) was also high in emf1-1 and emf2-1 single mutants and reduced in emf1-1 or emf2-1 late-flowering double mutants. Our results indicate that the precocious expression of AP1 and LFY is dependent not only on the low EMF and FWA activities but also on the expression of most of the late-flowering genes such as FT, FCA, FE, CO and GI. These data also reveal that most late-flowering genes may function downstream of EMF or in pathways distinct from EMF to activate genes specified floral meristem identity during shoot maturation in Arabidopsis.

Recurrence of pneumococcal meningitis due to primary spontaneous cerebrospinal fluid fistulas.

The authors report a case of pneumococcal meningitis which recurred 3 times in a Taiwanese boy due to spontaneous cerebrospinal fluid (CSF) fistulas. The first time occurred at the age of 2 years, and the second episode presented as meningoencephalomyelitis at the age of 6 years 10 months. Studies including serum levels of immunoglobulin and complements, brain magnetic resonance imaging, and coronal cranial computed tomography (CT) were negative for a specific etiology. The third episode of meningitis developed 2 months after the second episode. Repeated immunological studies and high-resolution CT of paranasal sinuses and temporal bones were negative. Technetium-99m diethylenetriamine pentaacetic acid (Tc-99m-DTPA) radionuclide cisternography revealed abnormal retention of radioactivity over the right mastoid area. Neurosurgery was undertaken to seal the dural tear and pack the petrosal fissure. Two years after surgery, he has had no further CSF leak age or meningitis. Tracing back the history, there was no head injury, cranial surgery, brain tumor, or hydrocephalus, which might have created CSF fistulas. Primary spontaneous CSF fistulas constitute the most reasonable diagnosis. In cases of recurrent bacterial meningitis, underlying anatomic defects should be carefully evaluated if there is no immune defect.

FLD interacts with CO to affect both flowering time and floral initiation in Arabidopsis thaliana.

fld and co, both with significantly delayed flowering, are characterized as late-flowering mutations in Arabidopsis thaliana. Double mutants between fld-2 and co-3 were generated and the phenotypes characterized. Double mutants flower later than both single mutant parents, suggesting that there is an additive effect. In addition, the formation of flowers in double mutants was altered and showed a novel phenotype. Double mutant flowers contained a much longer stalk (pedicel). Sepals and petals were absent. Several leaf-like structures were produced in the position normally occupied by sepals and the organ numbers were reduced. The carpels were morphologically normal. The stamens produced were usually aborted in the early stage, thus, the flowers were sterile. The additive phenotype observed in double mutants provides evidence to support that these two genes, FLD and CO, are not only involved in rosette-to-inflorescence transition but also involved in the flower formation. This result also indicates that FLD and CO promote the reproductive program through two different pathways.

Late-flowering genes interact with early-flowering genes to regulate flowering time in Arabidopsis thaliana.

To investigate the genetic mechanisms regulating the transition from the vegetative to reproductive growth in Arabidopsis, double mutants between three different early-flowering mutants, early flowering 1-1, 2-1, 3-1, (elf 1-1, 2-1, 3-1) and five different late-flowering mutants, gi-1, ft-1, fwa-1, ld-1, and fca-9, were constructed and phenotypes analyzed. Double mutants in all combinations displayed the late-flowering phenotypes which resembled their respective late-flowering parents in both flowering time and the number of vegetative leaves produced. The results indicate that five late-flowering mutants are epistatic to all three early-flowering mutants tested here. This epistatic relationship suggests that ELF1, ELF2, and ELF3 genes function upstream of these five late-flowering genes no matter if they are functioning in autonomous or photoperiod pathways. These three early-flowering genes may negatively modify the activity of most late-flowering genes to influence the time of the vegetative-to-reproductive transition in Arabidopsis.

Phenotypic control of gap junctional communication by cultured alveolar epithelial cells.

We examined phenotype-specific changes in gap junction protein [connexin (Cx)] expression and function by cultured rat alveolar type II cells. Type II cells cultured on extracellular matrix in medium containing keratinocyte growth factor (KGF) and 2% fetal bovine serum (FBS; KGF/2) retained expression of surfactant protein C and the 180-kDa lamellar body membrane protein (lbm180). These markers were lost when cells were cultured in medium containing 10% FBS (MEM/10). With RT-PCR, cells cultured in MEM/10 showed transient increases in Cx43 and Cx46 mRNA expression, whereas Cx32 and Cx26 decreased and Cx30.3 and Cx37 were unchanged. Transient changes in Cx32, Cx43, and Cx46 protein expression were confirmed by immunoblot. In contrast, cells cultured in KGF/2 retained expression of Cx32 and showed increased expression of Cx30.3 and Cx46 mRNAs, compared with that in day 0 cells. With immunofluorescence microscopy, Cx32 and Cx43 were at the plasma membrane of cells grown in KGF/2, whereas Cx46 was exclusively intracellular. Type II cells cultured in MEM/10 showed approximately 3- to 4-fold more intercellular transfer of microinjected lucifer yellow through gap junctions than cells grown in 2% FBS. Thus type II cells dynamically alter gap junctional communication, and distinct alveolar epithelial cell phenotypes express different connexins.

Non-typhoid Salmonella subdural empyema in children: report of two cases.

Subdural empyema caused by Salmonella in childhood is an uncommon condition. The predisposing factors for this condition are not clearly established, especially in young children. Here we present two cases of subdural empyema caused by non-typhoidal Salmonella. Both of the patients suffered prolonged fever without local signs of infection on admission. Subdural empyema was subsequently detected by brain echo and brain computerized tomography (CT) scan in both cases. Cerebrospinal fluid (CSF) study was not done in case one due to prominent mass effect on brain CT; in case two the CSF analysis showed pleocytosis, but CSF bacterial culture was negative. Neither enteritis nor obvious meningeal sign was noted. Both cases responded well to surgical drainage and systemic antibiotics treatment.

FLD interacts with genes that affect different developmental phase transitions to regulate Arabidopsis shoot development.

A new fld mutant allele, fld-2, which significantly delayed flowering, was isolated and characterized in Arabidopsis thaliana. Even under long-day conditions after more than 100 days in the greenhouse, the majority of fld-2 mutant plants had not bolted. In addition, mutant inflorescences produced more than 10 co-florescences that were subtended by a high number of rosette-like leaves before giving rise to flowers. The late-flowering phenotype of the fld-2 mutation could be partially overcome by both vernalization and GA treatment but it was not influenced by 5-azaC treatment. Phenotypic analyses of double mutants indicated that fld-2 is epistatic to early flowering mutants elf1, elf2 and elf3. In addition, fld-2 could enhance vegetative characteristics in embryonic flower 1 (emf1) mutants by causing many small sessile leaves in fld-2 emf1 double mutants. The relief of the terminal flower 1 (tfl1) mutant phenotype in fld-2 tfl1 double mutants, and the enhancement of leafy (lfy) and apetala1 (ap1) mutant phenotypes in fld-2 lfy and fld-2 ap1 double mutants, suggest that FLD is also likely to be involved in the floral transition. Our results strongly suggest that the FLD gene plays a key role in regulating the reproductive competence of the shoot and results in different developmental phase transitions in Arabidopsis.

Benign isolated abducens nerve palsy in Mycoplasma pneumoniae infection.

Benign acquired isolated abducens nerve palsy in children is a rare condition. The palsy may be due to a postinfectious process; Epstein-Barr virus and cytomegalovirus have been documented etiologies. This is a case report of a boy 26 months of age with benign acquired isolated bilateral abducens nerve palsy. It may be the first report of Mycoplasma pneumoniae as the cause of benign isolated abducens nerve palsy in children.

Ultrasonographic study of the sternocleidomastoid muscle in the management of congenital muscular torticollis.

BACKGROUND: Congenital muscular torticollis (CMT) in infancy is caused by the fibrotic change of the sternocleidomastoid muscle (SCMM). The etiology and management strategies remain controversial. METHODS: One hundred ninety-seven infants and children aged 1 month to 16 years who had CMT were examined by real-time ultrasonography of the SCMM between June 1995 and September 1996 in a prospective and longitudinal study. A total of 362 examinations were performed. There were 122 boys and 75 girls. RESULTS: The right side was involved in 117 patients (59.3%), the left side in 79 patients (40.1%), and both sides in one patient. The sonographic findings were homogeneous or heterogeneous (patchy) hyperechoic lesion within the SCMM, and all were diagnostic. The ultrasonographic appearance of the SCMM in this study has a close resemblance to the clinical course of CMT. The extent of fibrosis as represented by the cross section of lesion to muscle ratio (L/M ratio) decreased from 83.6% at 2 months to 59.9% at 9 months of age and further decreased to 40% beyond 1 year of age. This consistent decrease in fibrosis was caused by the increased normal muscle volume at the periphery and by the regenerated muscle fibers within the lesion. In this series of 197 patients, 32 (16.2%) eventually underwent surgery to release the SCMM because of persistent head tilt, chin deviation and limited range of neck motion beyond 1 year of age. The L/M ratio of the operative group was 62.7 +/- 16.0% compared with an L/M ratio of 54.5 +/- 14.2% (P = .035) for the nonoperative group at 1 year of age. The extent of fibrotic change in the cross section of the muscle was a significant factor in determining prognosis. In the longitudinal section, the fibrotic change was limited to only the lower third of the SCMM in 27 patients, and all of them recovered without operation. In 95 patients, the fibrotic lesion was limited to the middle and lower third or middle third only, and only six (6.3%) underwent operation. However, in 75 cases the entire length of muscle was involved, and 26 (34.7%) required surgical release of the contracted muscle. Whole-length muscle involvement was also important for predicting recovery without operative intervention. CONCLUSIONS: Ultrasonographic study of the SCMM is not only a valuable diagnostic tool but can also serve as a useful guideline for the treatment of infants who have congenital muscular torticollis.

Role of cavum septum pellucidum in akinetic mutism of hydrocephalic children.

Two children 9 and 13 years of age with histories of aqueductal stenosis developed akinetic mutism after multiple shunt revisions. The girl had spontaneous relief of her akinetic mutism 1 month later, after parenteral antibiotics and multiple shunt revisions. The akinetic mutism of the boy was resistant to antiparkinsonian therapy before megadosage of bromocriptine (120 mg/day). Both children had a prominent cavum septum pellucidum (CSP), which has rarely been involved in the pathogenesis of akinetic mutism. Because of the rapid shrinkage of the girl's CSP after shunt revision, but not that of the boy even after stereotactic fenestration of the wall of his CSP, their eventual outcomes were different. Wide CSP (WCSP) may play an important role in the occurrence of akinetic mutism in hydrocephalic patients.

Bacterial tracheitis: a case report.

An 18-month-old girl was brought to the emergency room of Chang Gung Children's Hospital with inspiratory stridor, suprasternal retractions and imminent respiratory failure. Despite orotracheal intubation, persistent poor air-entry was noted. Flexible bronchoscopy via the endotracheal tube showed a copious amount of mucopurulent secretions in the tracheobronchial tree without any foreign bodies. With vigorous suctioning and antibiotic treatment, she had a rapid recovery. Tracheal aspirates showed a growth of Haemophilus influenzae. Cefamandole was used with good response. In conclusion, although bacterial tracheitis is an uncommon obstructive upper airway disease in children, using a bronchoscope to diagnose and to guide specific therapy can decrease the morbidity and mortality.

Late-onset pyridoxine-dependent seizures: report of two cases.

We report two cases of late-onset pyridoxine-dependent seizures with age onset at 14 and 9 months, respectively. Their intractable seizures were refractory to diazepam, phenobarbital, phenytoin, carbamazepine, valproic acid, and adrenocorticotropic hormone, but stopped soon after intravenous administration of pyridoxine. The psychomotor development was retarded in both cases. Pyridoxine dependency is one of the few treatable intractable seizures in infancy. It should always be kept in mind, even in patients with intractable seizures beyond the neonatal period, regardless of previous patterns of electroencephalography and seizure types and responsiveness to the conventional anticonvulsants.

Cerebral cryptococcosis in a child.

Reported is one case of cerebral cryptococcosis in a 12-year-old girl. The diagnosis was confirmed by the detection of Cryptococcus neoformans with both India ink preparation of the cerebrospinal fluid and Sabouraud's media culture. Clinical presentation included progressive severe headache, vomiting, left eye pain, diplopia, dizziness and unstable gait. Fever was absent as a symptom. Initial brain magnetic resonance imaging revealed a focal lesion over the right cerebellar hemisphere with better demonstration than contrast-enhanced computed tomography. The patient was treated with amphotericin B and 5-flucytosine with good final outcome. Early diagnosis and proper therapy are necessary in order to decrease the motality of cerebral cryptococcosis.