Clinical outcome is significantly better with spheroid-based autologous chondrocyte implantation manufactured with more stringent cell culture criteria.

Objective: Spherox (CO.DON AG) is an autologous chondrocyte implantation (ACI) product, consisting of spheroids of human autologous matrix-associated chondrocytes. The tendency of primary chondrocytes to dedifferentiate during cultivation and the high biologic variability caused by the autologous nature of the starting material makes it challenging to design a manufacturing process that performs consistently and delivers products that meet their intended function and the high quality criteria for cell-based ATMPs. The current study was submitted during the European authorization procedure, and addresses the requirement to justify the operational ranges of the manufacturing process using clinical data. Methods: In order to define the operational ranges, statistical correlation analyses were conducted between process parameters and clinical improvement data of 120 patients from Phase II and III treated with ACI (KOOS score, 1 year follow-up). Results: This approach identified cell culture time as a critical process parameter that negatively correlates with the product's efficacy. Subsequent analyses of the Phase III patients that were treated with chondrocyte spheroids that have been manufactured with shorter monolayer and spheroid cultivation times showed a higher average clinical improvement as well as a higher responder rate compared to the total group. In addition, retrospective analyses demonstrated superiority for the treatment with short-cultivated chondrocyte spheroids over micro-fracture treatment. Conclusion: These findings underscore the need to use clinical data to optimize the manufacturing process for autologous cell-based therapies. We expect that restricting the cultivation times during manufacturing minimizes the production of suboptimal batches, thus ensuring an efficacious product.

Lamellar body counts on gastric aspirates for prediction of respiratory distress syndrome.

AIM: To develop a rapid method for diagnosing lung maturity at birth with the purpose of administering surfactant early to infants with immature lungs and to spare infants with mature lungs from this treatment. METHODS: Lamellar body counts (LBC) on gastric aspirates from 191 newborns were counted in the platelet window in automatic blood cell counters. A preliminary study was performed on 108 aspirates from 2000 in infants with <32 weeks' gestation. Furthermore, 83 aspirates from 2004 to 2005 in infants with <30 weeks' gestation were analysed. RESULTS: Lamellar bodies in gastric aspirate were identified by electron microscopy. Seventy of the aspirates from 2004 to 2005 were analysed with a Sysmex XE-2100 (Sysmex, Holbaek, Naestved, Odense and Rigshospitalet, Denmark) counter. Twenty-four of these infants developed moderate to severe respiratory distress syndrome (RDS). The best cut-off value was 8000/µL with a sensitivity of 75% and a specificity of 72%. Forty-four of the 70 aspirates from 2004 to 2005 were analysed by Sysmex, Advia 120 and Cell-Dyn 4000. Thirteen other aspirates from 2004 to 05 were analysed by Sysmex and Coulter Counter LH755. Using Advia and Coulter the results were similar to Sysmex, but LBC obtained with Cell-Dyn were not correlated with the development of RDS. CONCLUSION: Lamellar body counts on gastric aspirate is a promising tool for prediction of development of RDS in infants of <30 weeks` gestation.

Prediction of respiratory distress syndrome by the microbubble stability test on gastric aspirates in newborns of less than 32 weeks' gestation.

AIM: There is a need for a rapid method to identify infants who will develop respiratory distress syndrome (RDS) soon after birth, to allow early treatment of affected infants with surfactant. The microbubble stability test (MST) may be one such method, but clinical experience is sparse. METHODS: The MST was performed on gastric aspirates from 188 infants with a mean gestational age of 29 (range 23-31) wk. RESULTS: 87 infants developed moderate to severe RDS, corresponding to a prevalence of 46%. The sensitivity, specificity and predictive values for identification of infants with moderate to severe RDS were determined for the average diameter of bubbles, the proportion of microbubbles with different diameters and the total number of microbubbles. The proportion of microbubbles with diameters <20 or 25 microm gave the best prediction, with a sensitivity of 78-79%, a specificity of 57-58%, a positive predictive value of 62% and a negative predictive value of 76%. Early treatment with nasal continuous positive airway pressure probably mitigated the development of RDS in some infants with a low-degree surfactant deficiency and this may explain the relatively low specificity. CONCLUSION: In infants of <32 wk gestation RDS can be predicted by computerized image analysis of the size distribution of microbubbles generated in gastric aspirates.

[Neonatal mortality and morbidity in gestational age shorter than 30 weeks for babies treated primarily in a central hospital. Follow-up studies of an unselected, consecutive patient group]. / Neonatal mortalitet og morbiditet ved svangerskabslaengde mindre end 30 fulde uger for børn behandlet primaert på et centralsygehus. Efterundersøgelse af en uselekteret, konsekutiv patientgruppe.

AIM: The aim of the study was to investigate the mortality and morbidity of very premature babies treated at a Danish county hospital in the mid-1990s. MATERIALS AND METHOD: The 62 survivors of 70 consecutive babies born in 1993-1997 were given a neuropaediatric follow-up examination at a corrected mean age of 49 (22-69) months. The mean gestational age was 27 (24-29) weeks and the mean birth weight was 1012 (550-1374) g. RESULTS: Eight babies (11%) died within 28 days of birth. Four (6%) had an intracerebral haemorrhage, grade 3-4; four (6%) had retinopathy of prematurity; and three (4%) had bronchopulmonary dysplasia at 36 weeks of gestation. Four of the survivors developed cerebral palsy. Two of these babies and a neurologically normal baby had visual impairments. None developed epilepsy. DISCUSSION: Thus, our babies had a low mortality and only a few complications at long-term follow-up. Early nasal continuous positive airway pressure and early supplementation with a surfactant seem to be essential for these positive results in our most premature babies.

Neonatal Salmonella meningitis: two case reports.

Two cases of neonatal Salmonella meningitis are reported. Both infants were seriously ill: one died 6 days after admittance to the hospital; the other required artificial ventilation for 4 days and recovered without sequelae. In both cases close relatives had shown signs of gastrointestinal infection just prior to the reported cases. The symptoms, the outcome and antibiotic treatment are discussed. In view of the increasing incidence of salmonella infections in Denmark and the serious prognosis of neonatal meningitis, it is important to consider the diagnosis in all cases of bacterial meningitis in infancy.

Growth hormone normalizes vertebral strength in ovariectomized rats.

Ten-month-old lactating breeders were ovariectomized and kept on a low calcium diet. One month after ovariectomy, the rats were dosed once daily for 6 months with either a low dose of growth hormone (GH) (0.05 mg), a high dose of GH (1 mg), estrogen plus gestagen (E-G) [17 beta-estradiol (0.1 mg); norethisterone-acetate (0.05 mg)], or a combination of GH and E-G. Mechanical competence, apparent dry density, apparent ash density and ash concentration were measured in the fourth lumbar vertebral body. The reduced mechanical strength, ash density and ash concentration found in the vertebral body of ovariectomized animals were prevented by the high dose of GH alone, or in combination with E-G. E-G alone partly prevented the decline in ash density and ash concentration, but did not affect the reduced mechanical strength seen after ovariectomy. Only minor effects were observed after treatment with the low dose of GH, alone or in combination with E-G. In conclusion this study shows that GH administration prevents the decline in mechanical strength and bone mineral density seen in rats after ovariectomy.

Studies on the renal kinetics of growth hormone (GH) and on the GH receptor and related effects in animals.

Growth hormone (GH) is filtered through the kidney, and may exert effects on renal function when presented via the circulation. Investigations on kidney-related aspects of GH are increasing in number. Using in vitro and in vivo approaches, the present study attempted to provide answers to a number of unresolved or debated issues. In vitro, we detected both GH and type 1 IGF receptors (R) in a porcine renal epithelial cell line. The saturation and down regulation kinetics of the GH-R indicate that it has the properties of a classical GH-R. Furthermore, the simultaneous presence of GH-R and IGF-R on a phenotypically homogeneous cell line suggests the presence of GH-induced auto-/paracrine IGF-1 bioactivity in the kidney. Experiments with isolated proximal rabbit tubules incubated with physiological concentrations of 125I-GH demonstrated a time-and dose-dependent increase in unlabelled GH-displaceable cell-associated radioactivity, lending support to the concept of GH mediating its renal effects via proximal tubular GH-R. Short term administration of GH to rats and humans elicited electrolyte and water retention that may cause edema in adults. In the present study, long term administration of GH to rats caused only a minor increase in serum phosphate levels, with no changes observed in the renal electrolyte clearance. During the first 4 days of GH treatment in rats, no change in plasma renin activity was detected and we were thus unable to confirm the hypothesis that the renin-angiotensin system is responsible for the early phase of GH-associated fluid retention. Pharmacokinetically, when GH was administered to rats with functional disconnection of the kidneys as a model of renal insufficiency, the whole body clearance of GH decreased by ca. two thirds, and was reflected by an increase in the mean residence time and AUCplasma for GH. The plasma half-life, however, was not significantly affected, suggesting that the volume of distribution (Vd) had decreased for the GH administered to the renally compromised animals. A renal contribution to the Vd was visualized as intense radioactive staining in the kidney region on whole body autoradiographs (WBA) of rats dosed with 125I-labelled hGH. The liver region was also intensely stained. Kidney-associated radioactivity was found to be related not only to glomerular filtration, but also to peritubular uptake, since the renal clearance of free GH was found to exceed the GFR.(ABSTRACT TRUNCATED AT 400 WORDS)