Background: The interaction of the non-deletional α+-thalassaemia mutations Haemoglobin Constant Spring and Haemoglobin Quong Sze with the Southeast Asian double α-globin gene deletion results in non-deletional Haemoglobin H disease. Accurate detection of non-deletional Haemoglobin H disease, which is associated with severe phenotypes, is necessary as these mutations have been confirmed in the Malaysian population. Methods: DNA from two families with Haemoglobin H disease was extracted from EDTAanticoagulated whole blood and subjected to molecular analysis for α-thalassaemia. A duplex polymerase chain reaction was used to detect the Southeast Asian α-globin gene deletion. Polymerase chain reaction-restriction fragment length polymorphism analysis was then carried out to determine the presence of Haemoglobin Constant Spring and Haemoglobin Quong Sze. A combine- amplification refractory mutation system protocol was optimised and implemented for the rapid and specific molecular characterisation of Haemoglobin Constant Spring and Haemoglobin Quong Sze in a single polymerase chain reaction. Results and Conclusions: The combine- amplification refractory mutation system for Haemoglobin Constant Spring and Haemoglobin Quong Sze, together with the duplex polymerase chain reaction, provides accurate pre- and postnatal diagnosis of non-deletional Haemoglobin H disease and allows detailed genotype analyses using minimal quantities of DNA.

The molecular basis of variable phenotypes in P-thalassaemia patients with identical genotypes has been associated with co-inheritance of alpha-thalassaemia and persistence of HbF production in adult life. The Xmn I restriction site at -158 position of the Ggamma-gene is associated with increased expression of the Ggamma-globin gene and higher production of HbF This study aims to determine the frequency of the digammaferent genotypes of the Ggamma Xmn I polymorphism in P-thalassaemia patients in two ethnic groups in Malaysia. Molecular characterisation and frequency of the Ggamma Xmn I polymorphism were studied in fifty-eight Chinese and forty-nine beta-thalassaemia Malay patients by Xmn I digestion after DNA amplification of a 650 bp sequence. The in-house developed technique did not require further purification or concentration of amplified DNA before restriction enzyme digestion. The cheaper Seakem LE agarose was used instead of Nusieve agarose and distinct well separated bands were observed. Genotyping showed that the most frequent genotype observed in the Malaysian Chinese was homozygosity for the absence of the Xmn I site (-/-) (89.7%). In the Malays, heterozygosity of the Xmn I site (+/-) was most common (63.3%). Homozygosity for the Xmn I site (+/+) was absent in the Chinese, but was confirmed in 8.2% of the Malays. The ratio of the (+) allele (presence of the Xmn I site) to the (-) allele (absence of the Xmn I site)) was higher in the Malays (0.66) compared to the Chinese (0.05). The (+/-) and (+/+) genotypes are more commonly observed in the Malays than the Chinese in Malaysia.
Chinese People ; Thalassemia ; With frequency ; Malaysia ; seconds

Background: The cause of end-organ damage and acute respiratory distress syndrome (ARDS) in coronavirus disease 2019 (COVID-19) patients is postulated to be connected to the uncontrolled increase of pro-inflammatory cytokines. The upregulation of many cytokines is dependent on signaling through the Janus kinase 1 (JAK-1) and JAK-2 pathways. Ruxolitinib, a JAK-1 and JAK-2 inhibitor, is documented to have potent anti-inflammatory activity by targeting several cytokines and growth factors with proposed efficacy in the cytokine storm observed in severe COVID-19 patients; therefore, this study examines the efficacy and tolerability of ruxolitinib for adult COVID-19 patients. Materials and Methods: This review was conducted using preferred reporting items for aystematic reviews and meta-analyses (PRISMA) methodology. Six reviewers analyzed 1,120 results. Seven studies were selected and validated. A quantitative meta-analysis was further performed to evaluate clinical improvement at day 28, mortality at day 28, and oxygen requirements comparing treatment and standard of care groups. Results: 168 individuals were involved in the studies selected: 122 in cohort studies, 4 in case reports, and 41 in randomized controlled studies. The ruxolitinib group had a higher likelihood of clinical improvement by the 28th day of treatment when assessed with the standard of care (SOC) group (odds ratio [OR]: 1.48; 95% confidence interval [CI]: 0.53 -4.16; P = 0.45; I2 = 0%). The SOC group was at a higher risk of experiencing serious adverse events (OR: 0.17; 95% CI: 0.03 - 1.13; P = 0.07). Notably the SOC group had a higher likelihood of death (OR: 0.51; 95% CI: 0.11-2.29; P = 0.07; I2= 0%). Conclusion Prior studies on ruxolitinib have demonstrated it is able to decrease inflammatory markers. In recent studies on COVID-19, treatment with ruxolitinib decreased the time on mechanical ventilation, hospitalization time, and the need for vasopressor support. Additionally, ruxolitinib showed decreased mortality and demonstrated improvement in lung congestion as evidenced by computerized tomography imaging. These findings warrant further clinical investigation into Ruxolitinib as a potential treatment approach for severe COVID-19.

Background: The cause of end-organ damage and acute respiratory distress syndrome (ARDS) in coronavirus disease 2019 (COVID-19) patients is postulated to be connected to the uncontrolled increase of pro-inflammatory cytokines. The upregulation of many cytokines is dependent on signaling through the Janus kinase 1 (JAK-1) and JAK-2 pathways. Ruxolitinib, a JAK-1 and JAK-2 inhibitor, is documented to have potent anti-inflammatory activity by targeting several cytokines and growth factors with proposed efficacy in the cytokine storm observed in severe COVID-19 patients; therefore, this study examines the efficacy and tolerability of ruxolitinib for adult COVID-19 patients. Materials and Methods: This review was conducted using preferred reporting items for aystematic reviews and meta-analyses (PRISMA) methodology. Six reviewers analyzed 1,120 results. Seven studies were selected and validated. A quantitative meta-analysis was further performed to evaluate clinical improvement at day 28, mortality at day 28, and oxygen requirements comparing treatment and standard of care groups. Results: 168 individuals were involved in the studies selected: 122 in cohort studies, 4 in case reports, and 41 in randomized controlled studies. The ruxolitinib group had a higher likelihood of clinical improvement by the 28th day of treatment when assessed with the standard of care (SOC) group (odds ratio [OR]: 1.48; 95% confidence interval [CI]: 0.53 -4.16; P = 0.45; I2 = 0%). The SOC group was at a higher risk of experiencing serious adverse events (OR: 0.17; 95% CI: 0.03 - 1.13; P = 0.07). Notably the SOC group had a higher likelihood of death (OR: 0.51; 95% CI: 0.11-2.29; P = 0.07; I2= 0%). Conclusion Prior studies on ruxolitinib have demonstrated it is able to decrease inflammatory markers. In recent studies on COVID-19, treatment with ruxolitinib decreased the time on mechanical ventilation, hospitalization time, and the need for vasopressor support. Additionally, ruxolitinib showed decreased mortality and demonstrated improvement in lung congestion as evidenced by computerized tomography imaging. These findings warrant further clinical investigation into Ruxolitinib as a potential treatment approach for severe COVID-19.

The infusion and persistence in a transplant recipient of donor-derived bone marrow cells (DBMC) of multi-lineage can lead to a state of permanent chimerism. In solid vascular organ transplantation, the donor bone marrow lineage cells can even be derived from the transplant organ, and these cells can be detected in very small numbers in the recipient. This has been called microchimerism. Much controversy has developed with respect to the function of chimeric cells in organ transplantation. One idea is that the occurrence of these donor cells found in microchimerism in the recipient are coincidental and have no long-term beneficial effect on engraftment. A second and opposing view, is that these donor cells have immunoregulatory function that affect both the acute and chronic phases of the recipient anti-donor responses. It follows that detecting quantitative changes in chimerism might serve as an indication of the donor-specific alloimmune or regulatory response that could occur in concert with or independent of other adaptive immune responses. The latter, including autoimmune native disease, need to be controlled in the transplant organ. The safety and immune tolerance potential of DBMC infusion with deceased and living donor renal transplants was evaluated in a non-randomized trial at this center and compared with non-infused controls given identical immunosuppression. Overall DBMC infusions were well tolerated by the recipients. There were no complications from the infusion (s), no episodes of graft-vs-host disease (GVHD) and no increase infections or other complications. In the deceased DBMC- kidney trial, actuarial graft survival at 5 years was superior especially when graft survival was censored for recipient death. Acute rejections were significant reduced in patients given two DBMC infusions, and chronic rejection was dramatically reduced in all DBMC treated patients. The most interesting finding was that the degree of microchimerism slowly increased over the years the DBMC group that had exhibited no rejection episodes. In the DBMC-living related trial, the incidence of acute rejection did not differ between groups. However, DBMC chimerism in recipient iliac crest marrow had increased more rapidly than might be predicted from results previously seen in the cadaver group, despite four times fewer DBMC infused, with the generation of T- regulartory cells in-vitro assays.
*Bone Marrow Transplantation ; Humans ; *Kidney Transplantation ; *Living Donors ; *Tissue Donors ; *Transplantation Chimera ; *Transplantation Tolerance

Therapeutic dentin regeneration remains difficult to achieve, and a majority of the attention has been given to anabolic strategies to promote dentinogenesis directly, whereas, the available literature is insufficient to understand the role of inflammation and inflammatory complement system on dentinogenesis. The aim of this study is to determine the role of complement C5a receptor (C5aR) in regulating dental pulp stem cells (DPSCs) differentiation and in vivo dentin regeneration. Human DPSCs were subjected to odontogenic differentiation in osteogenic media treated with the C5aR agonist and C5aR antagonist. In vivo dentin formation was evaluated using the dentin injury/pulp-capping model of the C5a-deficient and wild-type mice. In vitro results demonstrate that C5aR inhibition caused a substantial reduction in odontogenic DPSCs differentiation markers such as DMP-1 and DSPP, while the C5aR activation increased these key odontogenic genes compared to control. A reparative dentin formation using the C5a-deficient mice shows that dentin regeneration is significantly reduced in the C5a-deficient mice. These data suggest a positive role of C5aR in the odontogenic DPSCs differentiation and tertiary/reparative dentin formation. This study addresses a novel regulatory pathway and a therapeutic approach for improving the efficiency of dentin regeneration in affected teeth.
Animals ; Cell Differentiation/physiology* ; Cells, Cultured ; Complement C5a/metabolism* ; Dental Pulp/physiology* ; Dentin ; Mice ; Receptor, Anaphylatoxin C5a ; Stem Cells

Our previous studies have demonstrated that Fam20C promotes differentiation and mineralization of odontoblasts, ameloblasts, osteoblasts and osteocytes during tooth and bone development. Ablation of the Fam20C gene inhibits bone and tooth growth by increasing fibroblast growth factor 23 in serum and causing hypophosphatemia in conditional knockout mice. However, control and regulation of the expression of Fam20C are still unknown. In this study, we generated a transgenic reporter model which expresses green fluorescence protein (GFP) driven by the Fam20C promoter. Recombineering was used to insert a 16 kb fragment of the mouse Fam20C gene (containing the 15 kb promoter and 1.1 kb of exon 1) into a pBluescript SK vector with the topaz variant of GFP and a bovine growth hormone polyadenylation sequence. GFP expression was subsequently evaluated by histomorphometry on cryosections from E14 to adult mice. Fluorescence was evident in the bone and teeth as early as E17.5. The GFP signal was maintained stably in odontoblasts and osteoblasts until 4 weeks after birth. The expression of GFP was significantly reduced in teeth, alveolar bone and muscle by 8 weeks of age. We also observed colocalization of the GFP signal with the Fam20C antibody in postnatal 1- and 7-day-old animals. Successful generation of Fam20C-GFP transgenic mice will provide a unique model for studying Fam20C gene expression and the biological function of this gene during odontogenesis and osteogenesis.
Animals ; Calcium-Binding Proteins ; genetics ; Extracellular Matrix Proteins ; genetics ; Green Fluorescent Proteins ; genetics ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Odontogenesis ; genetics ; Osteogenesis ; genetics

BACKGROUND AND PURPOSE: Prediction of intracranial aneurysm growth risk can assist physicians in planning of follow-up imaging of conservatively managed unruptured intracranial aneurysms. We therefore aimed to externally validate the ELAPSS (Earlier subarachnoid hemorrhage, aneurysm Location, Age, Population, aneurysm Size and Shape) score for prediction of the risk of unruptured intracranial aneurysm growth. METHODS: From 11 international cohorts of patients ≥18 years with ≥1 unruptured intracranial aneurysm and ≥6 months of radiological follow-up, we collected data on the predictors of the ELAPSS score, and calculated 3- and 5-year absolute growth risks according to the score. Model performance was assessed in terms of calibration (predicted versus observed risk) and discrimination (c-statistic). RESULTS: We included 1,072 patients with a total of 1,452 aneurysms. During 4,268 aneurysm-years of follow-up, 199 (14%) aneurysms enlarged. Calibration was comparable to that of the development cohort with the overall observed risks within the range of the expected risks. The c-statistic was 0.69 (95% confidence interval [CI], 0.64 to 0.73) at 3 years, compared to 0.72 (95% CI, 0.68 to 0.76) in the development cohort. At 5 years, the c-statistic was 0.68 (95% CI, 0.64 to 0.72), compared to 0.72 (95% CI, 0.68 to 0.75) in the development cohort. CONCLUSIONS: The ELAPSS score showed accurate calibration for 3- and 5-year risks of aneurysm growth and modest discrimination in our external validation cohort. This indicates that the score is externally valid and could assist patients and physicians in predicting growth of unruptured intracranial aneurysms and plan follow-up imaging accordingly.
Aneurysm ; Calibration ; Cohort Studies ; Discrimination (Psychology) ; Follow-Up Studies ; Humans ; Intracranial Aneurysm ; Risk Factors ; Subarachnoid Hemorrhage

X-linked hypophosphatemia (XLH) represents the most common form of familial hypophosphatemia. Although significant advances have been made in the treatment of bone pathology, patients undergoing therapy continue to experience significantly decreased oral health-related quality of life. The following study addresses this persistent oral disease by further investigating the effect of DMP1 expression on the differentiation of XLH dental pulp cells. Dental pulp cells were isolated from the third molars of XLH and healthy controls and stable transduction of full-length human DMP1 were achieved. RNA sequencing was performed to evaluate the genetic changes following the induction of odontogenic differentiation. RNAseq data shows the upregulation of inhibitors of the canonical Wnt pathway in XLH cells, while constitutive expression of full-length DMP1 in XLH cells reversed this effect during odontogenic differentiation. These results imply that inhibition of the canonical Wnt pathway may contribute to the pathophysiology of XLH and suggest a new therapeutic strategy for the management of oral disease.
Humans ; Familial Hypophosphatemic Rickets ; Wnt Signaling Pathway ; Dental Pulp ; Quality of Life ; Cell Differentiation

X-linked hypophosphatemia (XLH) is a rare disease of elevated fibroblast growth factor 23 (FGF23) production that leads to hypophosphatemia and impaired mineralization of bone and teeth. The clinical manifestations of XLH include a high prevalence of dental abscesses and periodontal disease, likely driven by poorly formed structures of the dentoalveolar complex, including the alveolar bone, cementum, dentin, and periodontal ligament. Our previous studies have demonstrated that sclerostin antibody (Scl-Ab) treatment improves phosphate homeostasis, and increases long bone mass, strength, and mineralization in the Hyp mouse model of XLH. In the current study, we investigated whether Scl-Ab impacts the dentoalveolar structures of Hyp mice. Male and female wild-type and Hyp littermates were injected with 25 mg·kg^-1 of vehicle or Scl-Ab twice weekly beginning at 12 weeks of age and euthanized at 20 weeks of age. Scl-Ab increased alveolar bone mass in both male and female mice and alveolar tissue mineral density in the male mice. The positive effects of Scl-Ab were consistent with an increase in the fraction of active (nonphosphorylated) β-catenin, dentin matrix protein 1 (DMP1) and osteopontin stained alveolar osteocytes. Scl-Ab had no effect on the mass and mineralization of dentin, enamel, acellular or cellular cementum. There was a nonsignificant trend toward increased periodontal ligament (PDL) attachment fraction within the Hyp mice. Additional PDL fiber structural parameters were not affected by Scl-Ab. The current study demonstrates that Scl-Ab can improve alveolar bone in adult Hyp mice.
Mice ; Male ; Female ; Animals ; Familial Hypophosphatemic Rickets/metabolism* ; Bone and Bones/metabolism* ; Tooth/metabolism* ; Periodontal Ligament/metabolism*