Pediatric peripheral blood progenitor cell collection: haemonetics MCS 3P versus COBE Spectra versus Fresenius AS104

BACKGROUND: An increasing number of apheresis machines are becoming available for peripheral blood progenitor cell (PBPC) collection in children. STUDY DESIGN AND METHODS: At the Children's Hospital of Florence (Italy), three apheresis machines were evaluated: MCS 3P (Haemonetics) (10 procedures in 4 patients, aged 10–12 years, weight 23.5-64 kg), Spectra, (COBE) (8 procedures in 3 patients, aged 4–17 years, weight 19–59 kg), and AS104 (Fresenius) (24 procedures in 9 patients, aged 2–16 years, weight 13.6-60 kg). For PBPC quantitative analysis, CD34 cytofluorimetry was employed. Relevant variables analyzed included efficiency of CD34+ cell extraction and enrichment, mononuclear cell purity and red cell contamination of the apheresis components, and platelet count decreases after leukapheresis. RESULTS: No significant differences in CD34+ cell-extraction abilities were found. However, the AS104 provided consistently purer leukapheresis components in terms of mononuclear cell and CD34+ cell enrichment (441 +/− 59%, vs. 240 +/− 35% and 290 +/− 42% for MCS 3P and Spectra, respectively). Postapheresis platelet counts dropped the least with the AS104. The smallest patient who underwent apheresis with MCS 3P (the only machine working on discontinuous flow and hence with greater volume shifts) weighed 23.5 kg and tolerated the procedure well, with no signs of hemodynamic instability. No significant complications were observed. CONCLUSION: All machines seem to have comparable PBPC extraction efficiency, but the AS104 seems to give the component with the greatest PBPC enrichment. This feature might be relevant for further ex vivo cell processing (CD34+ cell selection, expansion, and so on).     

One-year expression from high-capacity adenoviral vectors in the brains of animals with pre-existing anti-adenoviral immunity: clinical implications.

The main challenge of gene therapy is to provide long-term, efficient transgene expression. Long-term transgene expression from first generation adenoviral vectors (Advs) delivered to the central nervous system (CNS) is elicited in animals not previously exposed to adenovirus (Ad). However, upon systemic immunization against Ad, transgene expression from a first generation Adv is abolished. High-capacity Advs (HC-Advs) provide sustained very long-term transgene expression in the brain, even in animals pre-immunized against Ad. In this study, we tested the hypothesis that a HC-Adv in the brain would allow for long-term transgene expression, for up to 1 year, in the brain of mice immunized against Ad prior to delivery of the vector to the striatum. In na?ve animals, the expression of beta-galactosidase from Adv or HC-Adv was sustained for 1 year. In animals immunized prior to vector delivery, expression from a first generation Adv was abolished. These results point to a very long-term HC-Adv-mediated transgene expression in the brain, even in animals that had been immunized systemically against Ad before the delivery of HC-Adv into the brain. This study therefore indicates the utility of HC-Adv as a powerful gene therapy vector for chronic neurological disorders, even in patients who had been pre-exposed to Ad prior to gene therapy.     

Neprilysin levels in plasma and synovial fluid of juvenile idiopathic arthritis patients

Objective Neprilysin (neutral endopeptidase, 3:4:24:11, CD10) (NEP) is a Zn metallopeptidase linked to controlling inflammation through the degradation of neuropeptides involved in neurogenic inflammation of chronic rheumatic diseases. The aim of our study was to evaluate circulating activity and cellular expression of NEP in the plasma of 58 children with juvenile idiopathic arthritis (JIA) and 52 controls. In 20 subjects requiring local steroid injection, NEP was measured in synovial fluid.Methods Plasma and synovial NEP were evaluated using a fluorimetric technique. Neprilysin, expressed as the antigen CD10, was determined on circulating and synovial fluid cells as mean fluorescence intensity (MFI) and as percentage of positive cells by two-color immunofluorescence.Results Circulating NEP levels were lower in JIA patients than in controls (42.0±16.6 vs 76.5±24 pmol/ml per min, P<0.001), while synovial fluid NEP values were higher than circulating levels (241.4±86.2 vs 40±15.3 pmol/ml per min, P<0.001). In monocytes, the percentage of CD10-positive circulating cells and the MFI in JIA were lower than in controls (11.6±5.2% vs 41.4±13%, P<0.001 and 18.1±7.5 vs 31.2±5.4, P<0.05, respectively). On synovial monocytes, the percentage of CD10-positive cells and the MFI were higher than on circulating monocytes (35.2±14.6% vs 9.1±2.4%, P<0.001 and 66.4±5.4 vs 22.8±14.7, P<0.001, respectively).Conclusions The downregulation of CD10 expression in monocytes and the reduction in NEP activity may be linked to the enzymes role in the control of peptides involved in the inflammation. The increased levels of NEP, MFI, and CD10-positive monocytes in synovial fluid, even though in plasma, might reflect a reactive effort to control synovial proliferation.     

Connexin43 hemichannels mediate secondary cellular damage spread from the trauma zone to distal zones in astrocyte monolayers

The mechanism of secondary damage spread after brain trauma remains unsolved. In this work, we redirected the attention to astrocytic communication pathways. Using an in vitro trauma model that consists of a scratch injury applied to an astrocyte monolayer, we found a significant and transient induction of connexin43 (Cx43) hemichannel activity in regions distal from the injury, which was maximal ～1 h after scratch. Two connexin hemichannel blockers, La³⁺ and the peptide Gap26, abolished the increased activity, which was also absent in Cx43 KO astrocytes. In addition, the scratch‐induced increase of hemichannel activity was prevented by inhibition of P2 purinergic receptors. Changes in hemichannel activity took place with a particular spatial distribution, with cells located at ～17 mm away from the scratch presenting the highest activity (dye uptake). In contrast, the functional state of gap junction channels (dye coupling) was not significantly affected. Cx43 hemichannel activity was also enhanced by the acute extracellular application of 60 mM K⁺. The increase in hemichannel activity was associated with an increment in apoptotic cells at 24 h after scratch that was totally prevented by Gap26 peptide. These findings suggest that Cx43 hemichannels could be a new approach to prevent or reduce the secondary cell damage of brain trauma. GLIA 2015;63:1185–1199     

The effect of food intake on insulin receptor in man

Insulin binding to circulating monocytes was studied in 22 normal volunteers before and 1, 3 and 5 h after a 1400 Kcal meal. Results indicate that 3 h after food intake there is an increase in the specific cell binding fraction (P less than 0.001) with a change in receptor affinity. Data emerging from the present study demonstrate that there are rapid changes in insulin receptor properties during the day. These changes probably play a role in the regulation of the hormonal and metabolic pattern in normal subjects.     

Botulism: a laboratory investigation on biological and food samples from cases and outbreaks in Brazil (1982-2001)

Laboratory investigation of botulism from 1982 to 2001 confirmed the occurrence of eight positive outbreaks/cases of botulism in Brazil. From those, type A botulism was observed in seven of them. Biological material of one case (serum and feces) was positive in the first step of the bioassay, but the amount of sample was not sufficient for typification. One of the outbreaks that occurred in 2001 was negative for botulinum toxin in samples of serum, gastric washing and feces, collected eight days before the onset of the symptoms in the affected person who was clinically diagnosed as presenting the disease. Other two cases presenting compatible clinical diagnoses presented negative results. However, in those cases, the collection of samples was (1) after antiserum administration or (2) later than eight days of the onset of symptoms. Investigation was performed by mouse bioassay, as described in the Compendium of Methods for the Microbiological Examination of Foods (compiled by American Public Health Association--APHA)11, using specific antiserum from Centers for Disease Control (CDC), USA.     

A comparative study of the language comprehension of rural American Indian and rural Caucasian kindergarten age children

The purpose of the study was to investigate the level of language comprehension which rural American Caucasian and Indian children have upon entering kindergarten and after 20 weeks of school. The results indicated that the rural Caucasian children scored no differently than the national norms for the Test of Auditory Comprehension of Language either at the beginning or after 20 weeks of school, although significant gains were observed over this period. The rural Indian children scored significantly lower both at the beginning and after 20 weeks of school. Significant growth was also noted during this period. Implications centering on more appropriate language instruction at an earlier point in the Indian child's school experience were discussed.