Isolation of large numbers of enriched human megakaryocytes from liquid cultures of normal peripheral blood progenitor cells

Investigations linking human megakaryocyte development and cell biology have been hindered by an inability to obtain large, relatively pure megakaryocyte cell preparations from in vitro stem cell cultures. We report here that such preparations can be generated from liquid cultures of normal human peripheral blood mononuclear cells stimulated by a serum source of megakaryocyte colony stimulating activity (Meg- CSA, the 0% to 60% ammonium sulfate protein fraction of aplastic canine serum). Adherent-depleted peripheral blood mononuclear cells are suspended at 5 x 10(5) to 10(6) cells/mL in supplemented liquid culture medium, platelet-poor human plasma 20% (vol/vol) and 1 to 2 mg/mL serum Meg-CSA protein. After 12 to 14 days of incubation, megakaryocytes constitute 3.0 +/- 2.9% (mean +/- SD, n = 8) of the unseparated cultured cell population. Megakaryocytes can be enriched by counterflow centrifugal elutriation to a purity of 58 +/- 14% (+/- SD) with a recovery of 13 +/- 7% and a viability of 67 +/- 19%. This algorithm results in the average isolation of approximately 3 x 10(5) enriched megakaryocytes from a 100-mL starting volume of peripheral blood. Cultured megakaryocytes exhibit normal light and ultrastructural morphology by Wright-Giemsa staining and electron microscopic analysis. After a 12-day culture interval, enriched megakaryocyte preparations exhibit morphologic stage distributions that are similar to normal human marrow. Stage distributions move rightward with culture duration indicating partial synchrony of megakaryocyte maturation. On cytospin preparations, megakaryocyte diameter averages 30.2 +/- 1.5 microns and increases with maturation stage. Flow cytometric analyses demonstrate the expression of platelet glycoproteins (GP) Ib and IIb/IIIa by the cultured megakaryocytes. The modal ploidy of the enriched cells at day 12 of culture is 16N and most remaining megakaryocytes are 8N or 32N. Liquid culture of serum Meg-CSA-stimulated human peripheral blood mononuclear cells represents a valuable investigative tool that should permit studies of human megakaryocyte biology that have not been possible in the past.     

Oral health survey of 5-12-year-old children of National Guard employees in Riyadh, Saudi Arabia

Objectives. To assess the oral health status, preventive practices and mutans streptococci (MS) levels among the children of National Guard personnel living in Riyadh, Saudi Arabia. Design. Cross‐sectional study of schoolchildren. Setting. Dental Clinic of the National Guard Hospital in Riyadh, Saudi Arabia. Sample and methods. A sample of 272 5–12‐year‐old children, 154 males and 118 females (95% Bedouin), were selected from approximately 35 National Guard schools in the Riyadh area. An examination was performed in the dental clinic in the National Guard hospital. Oral hygiene was assessed using the Simplified Debris Index (DI‐S); Gingival Index (GI) was used to measure gingival health; dental fluorosis was recorded according to the criteria of Dean. Dental caries (dmft, dmfs, DMFT and DMFS) was recorded according to the recommendations of Haugejorden. Concentration of MS in saline rinse samples was assessed by routine laboratory methods and expressed as colony forming units (CFU) per ml (log₁₀). Results. There was a high level of dental caries (mean dmft = 3·8 ± 3·2; mean dmfs = 21·5 ± 15·7; mean DMFT = 2·0 ± 1·9; mean DMFS = 3·1 ± 3·7). Only 0·7% of the children had no caries experience (dmft + DMFT). MS levels ranged from 0 to 7·5×10⁵ CFU per ml (mean MS = 4·10 ± 0·90 log₁₀ CFU per ml). A significant relationship between MS and caries experience was observed (P = 0·003). Mild fluorosis was observed in 14% of the children. Oral hygiene scores indicated that most of the examined tooth surfaces had detectable plaque (mean DI‐S = 1·78). Gingivitis was present in 100% of the children and was considered moderate to severe in 14% (mean GI = 1·18). Conclusions. The study revealed a high level of oral diseases and poor oral hygiene in the study population and a need for therapeutic and preventive measures.     

Clinically meaningful and lasting HbA<Subscript>1c</Subscript> improvement rarely occurs after 5 years of type 1 diabetes: an argument for early,targeted and aggressive intervention following diagnosis

Aims/hypothesis

Our objectives were to explore whether the phenomenon of HbA_1c ‘tracking’ occurs in individuals with type 1 diabetes, how long after diagnosis does tracking take to stabilise, and whether there is an effect of sex and age at diagnosis on tracking.

Methods

A total of 4525 individuals diagnosed with type 1 diabetes between 1 January 1995 and 1 May 2015 were identified from The Health Improvement Network (THIN) database. Mixed models were applied to assess the variability of HbA_1c levels over time with random effects on general practices (primary care units) and individuals within practices.

Results

4525 individuals diagnosed with type 1 diabetes were identified in THIN over the study period. The greatest difference in mean HbA_1c measurement (?7.0 [95% CI ?8.0, ?6.1] mmol/mol [0.6%]) was seen when comparing measurements made immediately after diagnosis (0–1 year since diagnosis) with those at 10 or more years (the reference category). The mean difference in HbA_1c for the successive periods compared with 10 or more years after diagnosis declined and was no longer statistically significant after 5 years. In the stratified analysis using sex and age group there was considerable heterogeneity with adult onset type 1 diabetes appearing to track earlier and at a lower mean HbA_1c.

Conclusions/interpretation

In individuals with type 1 diabetes, glycaemic control measured by HbA_1c settles onto a long-term ‘track’ and this occurs on average by 5 years following diagnosis. Age at diagnosis modifies both the rate at which individuals settle into their track and the absolute HbA_1c tracking level for the next 10 years.

     

Period between Completion of Radiation Therapy and Prosthetic Rehabilitation in Edentulous Patients: A Retrospective Study

PURPOSE: The primary purposes of this study were: (1) to describe the number and types of complications patients had before and after insertion of a removable prosthesis (i.e., denture) following radiation therapy to the head and neck and (2) to investigate whether the time between radiation therapy and denture insertion might contribute to those complications. MATERIALS AND METHODS: This research evaluated edentulous patients and those who were rendered edentulous as a result of their cancer treatment. After obtaining institutional approval following HIPAA regulations, a total of 349 charts were identified: 152 patients from Houston Veterans Administration Medical Center (HVAMC) and 197 patients from M. D. Anderson Cancer Center (MDACC). A total of 190 patients met the inclusion criteria with data available for review. RESULTS: No significant differences were found in any of the comparisons made, except when comparing complications that occurred after the dentures were inserted and the amount of time it took for prosthetic rehabilitation. The majority of patients had no complications. The patients who received their dentures in 180 days or less had the same number of complications when compared with those patients who received their dentures in 181 to 365 days and those who had to wait longer than a year for prosthetic rehabilitation. Patients with more pre-insertion complications tended to have delayed prosthetic rehabilitation. Those patients who had complications both before and after denture insertion tended to have bilateral dosing of their radiation treatment. Patients who had received radiation therapy were 1.7 times more likely to have post-prosthesis insertion complications. The majority of patients who experienced complications before and after denture insertion had greater than 5000 cGy. CONCLUSIONS: The numbers of complications reviewed in this retrospective analysis were considerably fewer than the number expected. There appears to be no difference in the number of pre- and post-insertion complications as a function of the time delay from oral surgical procedure to start of radiation treatment (10 to 21 days vs. 22 days or more).