Cyst formation following disruption of intracellular calcium signaling

Mutations in polycystin 1 and 2 (PC1 and PC2) cause the common genetic kidney disorder autosomal dominant polycystic kidney disease (ADPKD). It is unknown how these mutations result in renal cysts, but dysregulation of calcium (Ca²⁺) signaling is a known consequence of PC2 mutations. PC2 functions as a Ca²⁺-activated Ca²⁺ channel of the endoplasmic reticulum. We hypothesize that Ca²⁺ signaling through PC2, or other intracellular Ca²⁺ channels such as the inositol 1,4,5-trisphosphate receptor (InsP3R), is necessary to maintain renal epithelial cell function and that disruption of the Ca²⁺ signaling leads to renal cyst development. The cell line LLC-PK1 has traditionally been used for studying PKD-causing mutations and Ca²⁺ signaling in 2D culture systems. We demonstrate that this cell line can be used in long-term (8 wk) 3D tissue culture systems. In 2D systems, knockdown of InsP3R results in decreased Ca²⁺ transient signals that are rescued by overexpression of PC2. In 3D systems, knockdown of either PC2 or InsP3R leads to cyst formation, but knockdown of InsP3R type 1 (InsP3R1) generated the largest cysts. InsP3R1 and InsP3R3 are differentially localized in both mouse and human kidney, suggesting that regional disruption of Ca²⁺ signaling contributes to cystogenesis. All cysts had intact cilia 2 wk after starting 3D culture, but the cells with InsP3R1 knockdown lost cilia as the cysts grew. Studies combining 2D and 3D cell culture systems will assist in understanding how mutations in PC2 that confer altered Ca²⁺ signaling lead to ADPKD cysts.The commonly occurring genetic kidney disorder, autosomal dominant polycystic kidney disease (ADPKD), is the result of mutations in polycystin 1 or 2 (PC1 or PC2). The progressive cyst formation within all segments of the nephron that defines the disorder leads to renal failure requiring treatment by dialysis and/or organ transplantation (1–3). Altered Ca²⁺ signaling is one of several pathways that have been implicated in the disease (4, 5). A major limitation toward elucidating the role of Ca²⁺ signaling in cyst formation has been the lack of easily manipulated, physiologically relevant experimental methodologies.In the past, ADPKD research has relied largely upon data from mouse models and cells maintained in 2D cell culture. Mouse models have played a significant role in understanding the biology of cyst formation but are unable to fully recapitulate the physiology of disease progression in humans due to the inherent differences between the species including life span, genetics, and environment. Two-dimensional cell culture has the ability to provide information on signaling pathways and response to therapies in a fast, high-throughput manner, but is incapable of replicating the inherent 3D nature of cyst formation. Advances in 3D tissue culture over the past 2 decades have improved the ability to model cyst development in vitro. However, previously published 3D tissue models of ADPKD have relied upon short-term culture of Madin-Darby canine kidney (MDCK) cells (6–12) or cells from patients (13–18) or PC1-null mice (19, 20; for review, see ref. 21). Recently, 3D tissues have been developed that incorporate mouse cells containing a shRNA-mediated knockdown of PC1 (9, 19). The benefits of this system include the use of a cell line, thus eliminating the need to isolate primary cells, and the use of cells with a stable genetic background.Ca²⁺ signaling underpins many cellular processes ranging from cell proliferation to cell death. Intracellular Ca²⁺ levels can be modified by opening of the inositol 1, 4, 5-trisphosphate receptor (InsP3R) or other intracellular Ca²⁺ release channels, including PC2. Over 99% of PC2 resides on the endoplasmic reticulum (22), where it is known to act as a modulator of the InsP3R and the ryanodine receptor (RyR) (23), with the remainder on the primary cilia. PC2 itself can function as a Ca²⁺-activated Ca²⁺ release channel (22, 24).Although it was demonstrated in 3D cultures that the knockdown of PC1 leads to cyst development (25), the effect of knocking down PC2 or other Ca²⁺-signaling proteins has not been shown. It has been hypothesized that the disruption of PC2, or the proteins that it interacts with, will result in cyst growth, as Ca²⁺ is a major signaling molecule (26, 27). Cells with decreased PC2 have been linked with decreased Ca²⁺ signaling (28), and overexpression of PC2 has been shown to act as an inhibitor of cell proliferation (29). Changing PC2 expression levels alters the uptake of Ca²⁺ into the endoplasmic reticulum, leading to liver cyst formation (30), but no direct link involving the release of Ca²⁺ from the endoplasmic reticulum has been implicated in renal cyst development. Similarly, changes in the expression of the InsP3R have been correlated with various disease conditions; for example, the InsP3R is upregulated in colorectal cancer (31), but downregulated in bile duct obstruction and cholestasis (32, 33).Here, we demonstrate that cyst formation can be followed for several weeks using a 3D culture system and that the disruption of intracellular Ca²⁺ signaling, through the knockdown of either InsP3R or PC2, leads to cyst development.     

The weight of MMRV-related febrile convulsions among other clinical factors contributing to febrile convulsions in children

Background

It was previously demonstrated that MMRV vaccine causes a higher rate of febrile convulsions (FC) compared to the MMR vaccine. Additional risk factors for FC include age, familial tendency, day care attendance, viral diseases, complications at birth and developmental delay.

Objective

We evaluated the relative and attributable risk of FC for vaccinees’ age, ethnicity, low birth weight, preterm birth and MMRV vaccination in 10–24 months old children.

Methods

Data on medical history and vaccination were extracted from data warehouses of Clalit Health Services and Israel's Ministry of Health and linked on an individual record level for 90,294 MMR- and 8344 MMRV-vaccinees. A retrospective study design was used to reveal the risk factors associated with FC in study participants.

Results

During the second week after immunization, an elevated relative risk of FC was demonstrated in MMRV-recipients (adjusted RR = 2.16 (95%CI: 1.01; 4.64)). However, the cumulative incidence of FC during the entire 40-day observation period did not differ between the MMR and MMRV vaccinees. The MMRV-specific attributable risk of FC was not statistically significant at any point of observation period and was exceedingly low compared to other risk factors, equaling 5.3 FC cases per 10,000 vaccinees (95%CI: −1.4; 12.2).

Discussion

Our findings demonstrate that MMRV-associated FC in 10–24 months old contributes very marginally to the overall rate of FC in this population.

Conclusion

Given the low number of MMRV-specific FC cases, their transient nature and the benefit of vaccination, the overall benefit-risk of the vaccine can be considered favourable. Nonetheless, the option of separate immunization with MMR + V should be offered to parents, in order to maintain sufficient vaccine uptake in the population.     

Evaluation of a parenting program for children with behavioural problems: Signposts in Singapore

Abstract

Background The Signposts for Building Better Behaviour program, developed by the Parenting Research Centre, Victoria, Australia, was conducted at a public hospital facility in Singapore.

Method More than 1,000 parents completed the program, and filled in questionnaires about their child's behaviours.

Results Parents rated themselves in the questionnaires as being significantly less hassled, stressed, depressed, and anxious after attending the program. They were more confident and satisfied with managing their child, and rated their children's behaviours as having improved. Effect sizes ranged from 0.12 to 0.59. The findings were maintained 3 months after completion of the program.

Conclusions The study provides evidence of the cross-cultural applicability of the principles underlying the Signposts program. As there are long-term repercussions when children's behaviour problems are not dealt with appropriately, such behaviour management programs should be made more available to parents and caregivers.     

Post-immunization pneumococcal antibody titers and IgG subclasses

IgG antibodies against pneumococcal polysaccharides are found predominantly within IgG subclass 2. We wished to evaluate retrospectively IgG subclasses and post-immunization pneumococcal antibody titers in children with recurrent respiratory infections. We examined total immunoglobulin levels and IgG subclasses, as well as pneumococcal antibody titers against serotypes 3, 7F, 9N, and 14 present 4–6 weeks after pneumococcal immunization in 56 children 2–18 years old. Titers >200 ng Ab N/ml to any of the 4 serotypes tested were arbitrarily considered protective. Four patients did not have protective antibody levels against any of the 4 serotypes tested following vaccination. Of those, 3 had normal IgG subclass levels and 1 had an IgG2 subclass deficiency. Of 3 additional patients with IgG2 deficiency, 2 had protective antibody levels to only 1 serotype and 1 had protective antibody levels to 2 serotypes. Furthermore, in 2 patients with undetectable IgG2 at the time of immunization, the response was only transient. We conclude that patients with IgG2 deficiency may not develop protective antibody levels to all pneumococcal serotypes and that some may have deficient memory for IgG anti-pneumococcal polysaccharide antibodies. Pediatr Pulmonol. 1996; 22:167–173. © 1996 Wiley-Liss, Inc.     

Degenerative lumbar spinal stenosis diagnostic value of the history and physical examination

Objective. To assess the value of the history and physical examination findings in the diagnosis of symptomatic degenerative lumbar spinal stenosis (LSS). Methods. The study was performed in 3 specialty clinics, and included patients with low back pain who were at least age 40. Findings from a standardized history and physical examination were compared with the diagnostic impression of expert attending clinicians. Imaging studies were available in 88% of those with LSS, and the findings further supported the diagnosis of LSS in each case. The sensitivity, specificity, and likelihood ratio associated with each history and physical examination finding were calculated in bivariate analyses, and independent correlates of LSS were identified with multivariate analyses. Results. Ninety-three patients were evaluated. History findings most strongly associated with the diagnosis of LSS (likelihood ratio ≥2) were greater age, severe lower-extremity pain, and absence of pain when seated. Physical examination findings most strongly associated with the diagnosis were wide-based gait, abnormal Romberg test result, thigh pain following 30 seconds of lumbar extension, and neuromuscular deficits. Independent correlates of LSS included advanced age (P = 0.0001), absence of pain when seated (P = 0.006), wide-based gait (P = 0.013), and thigh pain following 30 seconds of lumbar extension (P = 0.002). Conclusion. Specific history and physical examination findings are useful in the diagnosis of LSS and should be ascertained routinely in older patients with low back pain.     

The Oregon Child Absenteeism Due to Respiratory Disease Study (ORCHARDS): Rationale,objectives, and design

BackgroundInfluenza viruses pose significant disease burdens through seasonal outbreaks and unpredictable pandemics. Existing surveillance programs rely heavily on reporting of medically attended influenza (MAI). Continuously monitoring cause‐specific school absenteeism may identify local acceleration of seasonal influenza activity. The Oregon Child Absenteeism Due to Respiratory Disease Study (ORCHARDS; Oregon, WI) implements daily school‐based monitoring of influenza‐like illness‐specific student absenteeism (a‐ILI) in kindergarten through Grade 12 schools and assesses this approach for early detection of accelerated influenza and other respiratory pathogen transmission in schools and surrounding communities.MethodsStarting in September 2014, ORCHARDS combines automated reporting of daily absenteeism within six schools and home visits to school children with acute respiratory infection (ARI). Demographic, epidemiological, and symptom data are collected along with respiratory specimens. Specimens are tested for influenza and other respiratory viruses. Household members can opt into a supplementary household transmission study. Community comparisons are possible using a pre‐existing and highly effective influenza surveillance program, based on MAI at five family medicine clinics in the same geographical area.ResultsOver the first 5 years, a‐ILI occurred on 6634 (0.20%) of 3,260,461 student school days. Viral pathogens were detected in 64.5% of 1728 children with ARI who received a home visit. Influenza was the most commonly detected virus, noted in 23.3% of ill students.ConclusionORCHARDS uses a community‐based design to detect influenza trends over multiple seasons and to evaluate the utility of absenteeism for early detection of accelerated influenza and other respiratory pathogen transmission in schools and surrounding communities.