NLRP12 promotes host resistance against Pseudomonas aeruginosa keratitis inflammatory responses through the negative regulation of NF-κB signaling.

OBJECTIVE: To investigate the role of NLRP12 in regulating Pseudomonas aeruginosa (P. aeruginosa) keratitis. MATERIALS AND METHODS: Real-Time-PCR and Western blot were performed to measure the NLRP12 level in corneas and bone marrow-derived macrophages (BMDMs) of C57BL/6 (B6) mice. B6 mice received a subconjunctival injection of lentivirus expressing active NLRP12 (NLRP12-lentivirus) or Ctl-lentivirus (as control), followed by infection of P. aeruginosa. The clinical score, slit lamp and bacterial plate count of mice were evaluated. In addition, myeloperoxidase (MPO) was detected to assess the infiltration of polymorphonuclear neutrophil (PMN). Cytokine levels were measured by Real Time-PCR and ELISA. Meanwhile, the bacterial burden was also evaluated. The activation of NF-κB signaling was determined by pIκBα/IκBα levels based on Western blot and NF-κB-dependent Luciferase activity on the basis of Luciferase assays using 293T cells. RESULTS: NLRP12 mRNA and protein levels were decreased in B6 corneas and BMDMs after P. aeruginosa infection. The over-expression of NLRP12 in B6 corneas significantly ameliorated the severity of corneal disease, bacterial burden, PMN infiltration and pro-inflammatory cytokine expression. In vitro analysis demonstrated that the up-regulation of NLRP12 suppressed pro-inflammatory cytokine production and enhanced bacterial clearance in RAW264.7 cells. The protein levels of pIκBα and IκBα were significantly decreased after NLRP12-lentivirus treatment compared with that of Ctl-lentivirus. NF-κB-dependent Luciferase activity was potently inhibited by NLRP12 infected with P. aeruginosa or cotransfected with the downstream signaling molecules including IKKα and IKKß in 293T cells. CONCLUSIONS: NLRP12 decreases the severity of P. aeruginosa keratitis, reduces corneal inflammation and bacterial burden through the down-regulation of the NF-κB signaling pathway.

MiRNA regulates OCT4 expression in breast cancer cells.

OBJECTIVE: Breast cancer cell infiltration, migration, and proliferation significantly affect its curative effect. Stemness gene octamer-binding transcription factor 4 (OCT4) upregulated in breast cancer tissue compared with normal control. MiRNA exhibits regulatory role in gene expression. This study adopted bioinformatics to predict the miRNA to regulate OCT4 gene and investigated its impact on breast cancer cell infiltration, migration, and proliferation. MATERIALS AND METHODS: MirBase database was analyzed to explore the potential miRNA in regulating OCT4 based on human OCT4 gene sequence. MiRNA mimics and inhibitor were synthetized and transfected to BS524 cells. qRT-PCR was applied to test miRNA and OCT4 mRNA expressions in cells at 12 h, 24 h, and 48 h after transfection. Western blot was selected to detect OCT4 protein expression. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was selected to determine cell proliferation. Scratch assay was adopted to evaluate cell migration. Transwell assay was used to analyze cell infiltration. RESULTS: MiR-145 may regulate OCT4 gene with score 82. OCT4 mRNA and protein increased at 12 h after transfection (p > 0.05). OCR4 gene significantly upregulated, cell proliferation, migration, and infiltration enhanced by miR-145 transfection compared with control (p < 0.05). OCT4 gene downregulated, while cell proliferation, infiltration, and migration markedly weakened in miR-145 inhibitor group compared with control (p < 0.05). CONCLUSIONS: MiR-145 affects breast cancer BS524 cell proliferation, infiltration, and migration via positively regulating OCT4 gene expression.

Exercise-induced bronchoconstriction among Malay schoolchildren.

OBJECTIVE: Exercise-induced bronchoconstriction (EIB) may be demonstrated in 60-70% of asthmatic children in temperate climates. In areas of high humidity it is postulated to be low. The aim of the study was to determine the prevalence of EIB in a population of schoolchildren with wheezing, living in the humid tropical climate of Kuala Lumpur, Malaysia. METHOD: We performed a cross-sectional study using the International Study of Asthma and Allergies in Childhood questionnaire to identify 7-12-year-old Malay children with asthma symptoms from a primary school in central Kuala Lumpur. Sixty-five of 76 children with 'ever wheeze' performed an exercise challenge test successfully in an uncontrolled environment. A random sample of 80 schoolchildren with no history of wheeze were similarly tested as controls. The relative humidity and temperature were recorded. A fall of > 15% was considered as clinically important. RESULTS: The prevalence of EIB in schoolchildren with 'ever wheeze' was 47.7%. The prevalence of EIB in children with 'current wheeze' was 51.6%. The prevalence of EIB in controls was 7.5%. The relative humidity during the study ranged from 41 to 90%. There was no significant relationship between different humidity levels and EIB (P = 0.58, regression analysis). CONCLUSION: This study demonstrates that EIB is present in asthmatic children despite the highly humid tropical environment.

Home oxygen therapy for children with chronic lung diseases.

UNLABELLED: Home oxygen therapy programme is new in Malaysia. This programme enables children with respiratory insufficiency to be discharged home early. MATERIALS AND METHODS: Long term oxygen therapy was initiated using an oxygen concentrator in patients who i) remained hypoxic while breathing room air, ii) experienced desaturations of more than 20% during sleep as seen in patients with severe laryngomalacia and obstructive sleep apnoea syndrome and iii) had pulmonary hypertension with or without polycythaemia. The median with first and third quartile values are presented for the quantitative variables. RESULTS: A total of 71 patients mainly children with bronchopulmonary dysplasia (BPD) (32) and bronchiolitis obliterans (12) were discharged home on this programme. The median age at which home oxygen was initiated in children with BPD was 5.0 (Q1: 2, Q3: 8) months. The median total duration of oxygen requirement for BPD was 8.0 (Q1: 5, Q3: 12) months. The median duration of home oxygen dependency was 3.5 (Q1: 3, Q3: 6) months. However children with bronchiolitis obliterans required longer duration of oxygen therapy compared to children with BPD i.e. median duration of 28 months (Q1: 14.5, Q3: 66). In other respiratory conditions the mean duration of supplemental oxygen varies some of which may be life long. CONCLUSIONS: This paper has shown the importance of home oxygen program in children with respiratory disorders. It has significantly shortened hospital stay and thus saves hospital costs and prevents prolonged separation from the family.

Prevalence of childhood asthma and allergy in an inner city Malaysian community: intra-observer reliability of two translated international questionnaires.

OBJECTIVES: (a) To examine the intra-observer reliability of the Malay language versions of two international respiratory questionnaires i.e. the International Study of Asthma and Allergy in Children (ISAAC) and the American Thoracic Society (ATS) questionnaires, and (b) using the more reliable of these questionnaires, to estimate the prevalence of asthma and allergy related symptoms in an ethnically homogenous inner city community in Kuala Lumpur. METHODS: The study was conducted among 7 to 12 year old school children of Malay ethnic origin living in an inner city area of Kuala Lumpur. The sample consisted of 787 children attending the only primary school in the area. The Malay versions of both questionnaires were administered twice, one month apart, and were completed by parents. Agreement between the first and second responses to the same questions were assessed by Cohen's kappa. Kappa values < 0.4 were indicative of poor intra-observer reliability, 0.4-0.59 moderate reliability, 0.6-0.79 good reliability and > 0.79 excellent reliability. RESULTS: 77.9% and 36.3% of parents responded to the first and second administrations of the questionnaires respectively. Kappa values of > 0.4 were obtained in 15/16 (93.8%) and 17/27 (63.0%) questions of the ISAAC and ATS questionnaires respectively. Excellent kappa values were obtained in 4/16 (25%) questions of the ISAAC questionnaire versus only 1/27 (3.7%) questions of the ATS questionnaire. From the ISAAC questionnaire, all questions on wheeze had good reliability while those on asthma had excellent reliability. Questions on allergic symptoms had poor to moderate reliability. In contrast, from the ATS questionnaire, questions on wheeze had moderate reliability while questions on asthma were excellently reliable. Questions on allergic symptoms had moderate to good reliability while those on cough, phlegm and bronchitis had poor reliability. According to the ISAAC questionnaire the prevalence of ever wheeze, wheeze in the last 12 months, ever asthma and wheeze with exercise in the last 12 months was 12.5%, 6.6%, 10.3% and 5.9% respectively. The prevalence of ever sneeze or runny nose, sneeze or runny nose in the last 12 months, watery eyes in the last 12 months and ever eczema was 15.2%, 11.1%, 4.4% and 8.5% respectively. CONCLUSIONS: The translated ISAAC questionnaire was more reliable than the translated ATS questionnaire. Asthma and related symptoms were common among Malay school children in inner city Kuala Lumpur.

Asthma education: how much does it improve knowledge of childhood asthma amongst medical students and paramedics?

The objective of this study was to measure the knowledge of childhood asthma among medical students and paramedics. A previously validated questionnaire about childhood asthma was completed by 281 of 314, third and fifth year medical students at Universiti Kebangsaan Malaysia, Kuala Lumpur. Their knowledge of asthma was assessed during the first and last weeks of their paediatric rotation. A similar questionnaire was completed by 23 of 60 paramedics from various medical disciplines in Hospital Kuala Lumpur. They had attended a two-day seminar on respiratory diseases and their knowledge was assessed prior to and six weeks after the seminar. On the initial assessment the mean score for the final year medical students was 24.5, third year medical students 20.9 and paramedics 18.3. After intervention their mean scores increased significantly to 26.3 (p < 0.0001), 24.6 (p < 0.0001) and 21.3 (p < 0.0001). After intervention, the final year medical students improved significantly in all questions except in the management of acute asthma. Post intervention, third year medical students showed a significant increase in knowledge pertaining to symptomatology, pathophysiology, trigger factors and prophylactic drugs used in asthma management. Although the knowledge of paramedics improved post intervention, they had major deficiencies in knowledge about pathophysiology, trigger factors, preventive and acute asthma therapy, side effects of asthma treatment as well as clinical scenarios. Improvement after intervention was only seen in six of the 31 questions. This study demonstrated an increase in knowledge about childhood asthma among medical students and paramedics after a short intervention.

Prevalence, clinical predictors and diagnosis of gastro-oesophageal reflux in children with persistent respiratory symptoms.

OBJECTIVES: This was a cross sectional study conducted in the Paediatric Institute among infants and children with chronic respiratory symptoms with the following objectives: i) to determine the prevalence of gastro-oesophageal reflux in children with persistent respiratory symptoms, ii) to identify the clinical predictors of GOR (Gastro-oesophageal reflux) in children with persistent respiratory symptoms and iii) assess the validity of abdominal ultrasound, barium oesophagogram and chest radiograph in diagnosing GOR in these patients. MATERIALS AND METHODS: Forty-four patients were recruited over a period of six months. All the presenting symptoms were identified. The patients were subjected to chest radiograph, abdominal ultrasound, barium oesophagogram and 24-hour pH oesophageal monitoring. The predictive validity of clinical symptoms, chest radiograph, abdominal ultrasound and barium oesophagogram were assessed. Twenty-four hours oesophageal pH was the gold standard to diagnose GOR. RESULTS: The mean age of patients was 9.1 months (1-58 months). Thirty-one patients (70.5%) were confirmed to have GOR by pH study. Respiratory symptoms alone were not useful to predict GOR. Cough had the highest sensitivity of 51.6%. Stridor, wheeze and choking each had a specificity of 76%. Wheeze, vomiting, choking and stridor were identified to have high specificity (90-100%) in diagnosing GOR when any two symptoms were taken in combination. Collapse/consolidation was the commonest radiological abnormality but had low sensitivity (35.5%) and specificity (53.8%). However hyperinflation on chest radiograph had a specificity of 92.3% with positive predictive value of 80% in diagnosing GOR. Barium oesophagogram has low sensitivity (37.9%) and moderate specificity (75%) in diagnosing GOR in children with respiratory symptoms. Abdominal ultrasound was a valid mode of diagnosing GOR when there were three or more reflux episodes demonstrated during the screening period with a specificity of 90.9%. However the sensitivity was low i.e. 20-25%. The specificity increased to 90-100% when two positive tests were taken in combination (abdominal ultrasound and barium oesophagogram). However the sensitivity remained low (10-20%). Chest radiograph did not improve the predictive value when considered with the above tests. Combination of clinical symptoms were useful as clinical predictors of GOR. In the absence of a pH oesophageal monitoring, a combination of barium oesophagogram and ultrasound may be helpful in diagnosing GOR.

Interstitial lung disease in children--a report of four cases.

Interstitial lung disease (ILD) is very rare in children. In the majority of cases the aetiology is unknown. Very little is known about the clinical course of this condition in children. Prognosis may be influenced by sex, age of onset of symptoms, radiographic features, presence of right ventricular hypertrophy and histopathology. We report our experience in managing four children with interstitial lung disease. All these children presented in early infancy with cough, respiratory distress, cyanosis and failure to thrive. Three of these children had finger clubbing and right ventricular hypertrophy. All patients received oral steroids. Chloroquine was added in two patients who showed no response. A trial of oral cyclophosphamide was started in one patient who failed with both drugs. One child is oxygen independent while another is on home oxygen therapy. The other two patients eventually died.

Parental reactions to febrile seizures in Malaysian children.

The reactions of 117 parents to the febrile seizure experienced by their children; and their fears and worries were investigated. A standard questionnaire was used and clinical information was abstracted from the notes. In 88.9% of the cases, the adult present at the seizure was one of the parents usually the mother. Most of the parents (66.7%) did tepid sponging to bring the fever down but a third tried to open the clenched teeth of the child. The adults present placed the child supine in 62.9%, on the side in 9.5% and prone in 6.0%. Over half of the parents brought the child to a private clinic first before bringing to hospital. A fifth of the children were given antipyretics by the parent or the doctor and an anticonvulsant was given in 7.7% of cases. Interestingly, in 12% of children traditional treatment was given for the seizure. Three quarters of the parents knew that the febrile seizure was caused by high fever (which we have taken as the correct knowledge of febrile seizure). However "ghosts" and "spirits" were blamed as the cause of the seizure by 7% of parents. Factors significantly associated with correct knowledge were higher parental education and higher family income. The most common fear expressed was that the child might be dead or might die from the seizure (70.9%). Fear of death was associated with low paternal education. We concluded that the majority of our parents had reacted appropriately to a febrile seizure and their knowledge of the cause of febrile seizure was generally correct. Their fears and worries were similar to those elsewhere. However, traditional beliefs and practices may have to be taken into consideration during counselling.

Gastro-oesophageal reflux in children with severe respiratory symptoms--clinical spectrum and management.

Respiratory symptoms in children may be associated with underlying gastro-oesophageal reflux (GOR). We reviewed the case notes of 20 children who presented to us from June 1993 to June 1994 with respiratory symptoms and GOR. The patients consisted of 16 Malays, two Chinese and two Indians with equal number of males and females. Their age at diagnosis was less than one year in 17 patients. The earliest age at presentation was at the third day of life. All patients had major respiratory manifestations i.e. recurrent wheezing, recurrent cough and pneumonia. In addition, three patients had stridor and six patients had apparent life threatening episodes (ALTE). Fourteen patients required ventilation because of respiratory failure. Diagnosis of GOR was based on clinical grounds supported by barium oesophagogram in seven patients and ultrasound examination in 11 patients. Eight patients were fundoplicated because of ALTE and recurrent severe bronchospasm. On follow up, 14 patients had hyperactive airways requiring inhaled bronchodilator and steroid therapy.

An evaluation of the agreement between three peak flow meters: Wright's, Miniwright's, and Pocket, when used in children.

Three peak flow meters (PFM), Wright's, Miniwright's II (MPFM), and Pocket, were evaluated in 50 children in a pediatric outpatient clinic. It was found that there was close agreement between the Pocket and the MPFM as shown by an intraclass correlation (rho) of 0.95. Less agreement was noted between the MPFM and the Pocket, respectively, with the Wright's. It was also noted that the coefficient of correlation (r) was not a suitable test for agreement in this study. Both the Pocket and the MPFM also read higher than the Wright's. We conclude that children should be monitored using the same type of peak flow meter.

Febrile seizures in Malaysian children: epidemiology and clinical features.

A child with a febrile seizure is a common cause of admission in a general paediatric ward in Malaysia. We set out to look prospectively into the clinical and epidemiological features of these children. A total of 117 children were admitted into the study. The ratio of boys to girls were 1.5:1.0. The racial breakdown was 62.4% Malays, 26.5% Indians, 8.5% Chinese and 2.6% others. The first febrile seizure occurred before the age of three years in 92.9% of our patients. The highest number of febrile seizures was in the six to 12 months age group. The average length of seizure was 9.5 minutes and the majority were non-recurrent. Febrile seizures with complex features occurred in 33.3% of the children. Upper respiratory tract infection was the most common cause of fever in our patients. There was a family history of seizures (febrile or afebrile) in 26.5% of patients.

Parents' views of lumbar puncture in children with febrile seizures.

A study was carried out to ascertain the views of parents regarding the performance of lumbar punctures on their children admitted for febrile seizures. One hundred and seventeen (117) children with febrile seizures were recruited over nine months. Either one of the parents was interviewed a day after admission. In most cases, this was usually the mother. The ethnic groups of the patients were Malays (62.4%), Indians (26.5%), Chinese (8.5%) and others (2.6%). Lumbar punctures were requested by the doctor in attendance in only 28 (23.9%) patients This showed that the rate of request for lumbar punctures in febrile seizures was low. Parents of eight of them refused. The main reasons for the refusal were: fears that the child might be paralysed, advice from relatives and fear that the child might die from the procedure, or might find it too painful. All the parents who refused were Malays. A lumbar puncture was also more likely to be refused in a girl. Those who consented to lumbar puncture did so because they wanted the doctor to get to the diagnosis. Another reason given was that it might be therapeutic. Parents whose children did not require a lumbar puncture also thought that lumbar puncture may cause paralysis. The main sources of information on lumbar punctures for the parents were their relatives and/or friends. In only 85% of the cases were the reasons for the lumbar puncture explained to the parent. In 71.4% of the time the explanation was done by the medical officer, and in only 4.8% of the time was the consultant involved.(ABSTRACT TRUNCATED AT 250 WORDS)

A one-step monoclonal antibody typing procedure that simplifies HLA class I and class II typing.

We have developed monoclonal antibodies to most HLA specificities, making it possible for us to devise a simple, rapid, one-step microcytotoxicity test. The test is performed by adding 1 microliter of cells to 1 microliter of antibody-complement mixture predotted on the microtest tray. The reactions are read following a 1-hour incubation period (30 minutes in some instances). The analysis of reactions seen on testing 105 class I antibodies and 50 class II antibodies is shown. A comparison of typing by the standard NIH method and the new one-step procedure showed a > 96% concordance in the 500 T cells and 200 B cells we examined. Class I and class II typing could be performed using B cells, thus obviating the need to isolate both T and B cells for HLA typing.

A monoclonal antibody cross-reactive with human platelets, megakaryocytes, and common acute lymphocytic leukemia cells.

A cytotoxic monoclonal antibody, CALL1, produced against a human schwannoma tumor was found to react with human platelets, common acute lymphocytic cells (cALL), and lymphoblasts from lymphoid blast crisis of chronic myelocytic leukemia (CML). The hybridoma was repeatedly cloned, and the antibody was considered reactive to a single antigen by absorption tests demonstrating that platelets remove cALL activity and cALL cells absorb platelet activity from the antibody. In addition, chromatofocusing showed that the antibody against platelets and cALL had the same isoelectric point. The CALL1 antibody bound to megakaryocytes but inhibited neither myeloid (CFU-C) nor erythroid (BFU-E) colony formation from bone marrow stem cells. Immunoprecipitation and SDS-gel electrophoresis indicated that CALL1 reacts with a polypeptide of 26,000 daltons.