Kokkona Kouzi-Koliakos

A novel high-yield volume-reduction method for the cryopreservation of UC blood units

BACKGROUND For the application of umbilical cord blood (UCB) units as hematopoietic grafts, a dose of 3.7 x 10(7) nucleated cells (NC)/kg body weight is required. NC can be lost during volume-reduction processing and during thawing. A novel modification of the double-processing protocol with the aim of minimizing NC loss is described and evaluated. METHODS One-hundred and fifty UCB were collected. The volume was reduced by a centrifugation step following double-processing in the presence of 2% HES 200/0.5. Pre- and post-processing cell counts and platelet parameters were measured with an automatic counter. The number of viable CD34+ hemopoietic stem cells was measured by flow cytometry. In 25 of the samples, colony-forming units (CFU) were also determined. The same samples were thawed 6 months after cryopreservation and re-evaluated. RESULTS The volume was reduced to 6 +/- 1.5 mL. The recovery of NC, MNC, CD34+ hemopoietic stem cells, RBC depletion and CFU following double-processing was 93.6 +/- 3.2%, 95.8 +/- 2.2%, 98.4 +/- 1.5%, 96.8 +/- 1.1% and 107.1 +/- 6.1% (for 25 samples), respectively. The post-thaw recoveries of NC, MNC, CD34+ hemopoietic stem cells and CFU (for 25 samples) were 78.6 +/- 5.4%, 90.8 +/- 4.4%, 96.4 +/- 2.5%, 89.1 +/- 4.1%, respectively. No post-thaw cell aggregation was observed. A significant (P<0.05) post-thaw loss of platelets and signs of platelet activation was observed. DISCUSSION The protocol uses non-expensive equipment and clinically approved materials and results in samples that can be used in patients with a mean weight of 32.7 kg.

Time and temperature before processing influence the recovery of umbilical cord blood hematopoietic progenitors

Most blood services test blood components intended for cellular therapy, such as peripheral blood progenitor cells (PBPCs) or marrow-derived PBPCs, for infectious agents by the same methods used to test blood components intended for transfusion. A recent study in Uganda, where human herpesvirus 8 (HHV-8) is endemic, demonstrated that HHV-8 can be transmitted by transfusion of blood collected from HHV-8–infected donors. At present, blood components for transfusion are not tested for HHV-8 because, like human T-lymphotropic virus type I, HHV-8 infectivity decreases during storage, and leukoreduction adds to the protective effect. These incidental protections, however, do not apply to blood products intended for cellular therapy. In fact, the risk to recipients of cellular therapy products is increased by the short storage time for these products and the likelihood that recipients will be immunocompromised and therefore at increased risk for developing Kaposi’s sarcoma. Although the spread of HHV-8 by blood transfusion may not appear to be significant in nonendemic industrialized countries, the risk of collecting and transfusing a cellular therapy product that is infective for HHV-8 may be significant. In a study of volunteer blood donors in France, the HHV-8 seroprevalence was 1.5 percent. A study in the United States estimated the HHV-8 seroprevalence to be 3.5 percent. For these reasons, we suggest that the increased risk of transmitting HHV-8 by cellular therapy products warrants consideration of specific laboratory testing to exclude donors who are potentially infective for HHV-8. Jean-Jacques Lefrère, MD, PhD Department of Blood-Transmissible Agents National Institute of Blood Transfusion, Paris and Laboratory of Hematology Amiens Hospital Amiens Nicolas Guillaume, PhD Laboratory of Hematology Amiens Hospital Amiens Syria Laperche, MD, PhD Department of Blood-Transmissible Agents National Institute of Blood Transfusion Paris, France e-mail: slaperche@ints.fr REFERENCES

Late Postoperative Opacification of Hydrogel Intraocular Lenses: Analysis of 13 Explanted Lenses

PURPOSE We report the clinical, morphological, and ultrastructural findings of 13 consecutively explanted opacified Hydroview(R) (hydrogel) intraocular lenses (IOLs). Our purpose was to provide a comprehensive account on the possible factors involved in late postoperative opacification of these IOLs. PATIENTS AND METHODS Thirteen consecutive opacified hydrogel IOLs (Hydroview H 60 M, Bausch & Lomb) were explanted due to the significant visual impairment they caused. The IOLs underwent macroscopical examination, transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and electrophoresis for protein detection. Three unused control Hydroview IOLs served for comparison. RESULTS Macroscopical examination showed a diffuse or localized grey-whitish opacification within the IOL optic. TEM confirmed the presence of lesions inside the optic in all the explanted IOLs and revealed 3 patterns of deep deposits: a) diffuse, thick, granular, electron-dense ones; b) small, thin, lattice-like ones, with prominent electron-lucent areas; and c) elongated electron-dense formations surrounded by electron-lucent halos. SEM showed surface deposits on four IOLs. EDS revealed oxygen and carbon in all IOLs and documented calcium, phosphorus, silicon and/or iron in the deposits. Two of the patients with iron in their IOLs had eye surgery prior to their phacoemulsification. Iron correlated well with the second TEM pattern of deep lesions, whereas calcium with the third TEM pattern. No protein bands were detected on electrophoresis. Control lenses did not show any ultrastructural or chemical abnormality. CONCLUSIONS The present study supports the presence of chemical alterations inside the polymer of the optic in late postoperative opacification of Hydroview IOLs. This opacification does not follow a unique pathway but may present under different ultrastructular patterns depending on the responsible factors. Mechanical stress during surgery may initiate a sequence of events where ions such as calcium, phosphorus, silicon, and/or iron, participate in a biochemical cascade that leads to gradual alteration of the polymer network. Intraocular inflammation due to previous operation may be a factor inducing opacification through increase of iron-binding capacity in the aqueous humour. Calcification accounts only partially for the opacification noted in this type of IOL.

Intraperitoneal administration of bevacizumab intraoperatively does not affect abdominal wound healing in rats.

Background: Bevacizumab is a monoclonal antibody targeted at vascular endothelial growth factor (VEGF) to treat advanced colorectal cancer as well as other malignancies, but the ideal time point for its administration in patients scheduled for surgery is not well defined due to serious concerns regarding possible side effects on wound healing. Therefore, we conducted an experimental study in rats to clarify this issue. Methods: Four groups of 10 Wistar rats each underwent a 4-cm midline laparotomy and closure of the wound in 2 layers. In the treatment groups (A and B), bevacizumab (Avastin®) received a single dose of 5 mg/kg i.p., and an equal amount of saline was given to the control groups (C and D). Groups A and C were sacrificed on the 7th postoperative day, and groups B and D on the 14th postoperative day. Wounds were inspected by two independent observers upon sacrifice and results were recorded; wound tissues were sent for histology to assess the degree of fibrosis and measurement of tissue hydroxyproline levels. Serum levels of endothelin-1, C-reactive protein, pro-oxidant/antioxidant balance and carbonylated proteins were also determined. For statistical analysis, the Mann-Whitney U test was used. Results: Wound healing did not differ among groups both on the 7th and the 14th postoperative days, and there was also no significant difference regarding the degree of inflammation, fibroblast proliferation and collagen synthesis, as well as hydroxyproline and biochemical marker levels among the groups. Conclusions: Intraperitoneal bevacizumab administered intraoperatively does not significantly affect abdominal wound healing in rats.

A gene therapy induced emphysema model and the protective role of stem cells

BackgroundChronic obstructive pulmonary disease presents with two different phenotypes: chronic bronchitis and emphysema with parenchymal destruction. Decreased expression of vascular endothelial growth factor and increased endothelial cell apoptosis are considered major factors for emphysema. Stem cells have the ability of vascular regeneration and function as a repair mechanism for the damaged endothelial cells. Currently, minimally invasive interventional procedures such as placement of valves, bio-foam or coils are performed in order to improve the disturbed mechanical function in emphysema patients. However, these procedures cannot restore functional lung tissue. Additionally stem cell instillation into the parenchyma has been used in clinical studies aiming to improve overall respiratory function and quality of life.MethodsIn our current experiment we induced emphysema with a DDMC non-viral vector in BALBC mice and simultaneously instilled stem cells testing the hyposthesis that they might have a protective role against the development of emphysema. The mice were divided into four groups: a) control, b) 50.000 cells, c) 75.000 and d) 100.000 cells.ResultsLung pathological findings revealed that all treatment groups had less damage compared to the control group. Additionally, we observed that emphysema lesions were less around vessels in an area of 10μm.ConclusionsOur findings indicate that stem cell instillation can have a regenerative role if applied upon a tissue scaffold with vessel around.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_195

Addition of Adipose-Derived Stem Cells in Cord Blood Cultures Stimulates Their Pluripotent Differentiation

INTRODUCTION Adipose tissue is recognized as an important source of postnatal mesenchymal stem cells for generative medicine applications. Moreover, cord blood stem cells have been shown to contain pluripotent stem cells called unrestricted somatic stem cells (USSCs). However, this population is rare and cannot be generated from every cord blood sample. In this study, we have presented a new method of co-culture of adipose-derived stem cells (ADPCs) and cord blood stem cells that results in pluripotent differentiation. MATERIALS AND METHODS ADPCs were obtained from a piece of adipose tissue after treatment with 0.075% collagenase, which was subsequently inactivated with DMEM/10% FBS. The cellular pellet of centrifugation was plated at 5-7 x 10(6) cells/mL in T25 culture flasks in a low-glycose DMEM with 30% FCS. Cord blood stem cells were obtained by centrifugation following double-processing in the presence of 2% HES 200/0.5 and plated at 5-7 x 10(6) cells/mL in the same medium. To investigate the crucial role of ADPCs in pluripotent cord blood differentiation, we added a ADPCS as (1 x 10(4) cells/mL) to the cord blood cultures and analyzed the contribution of ADPCs using a microscope as well as with flow cytometry. RESULTS After only 3 days, adherent cells (USSC colonies) of fibroblastic morphology were detected in all co-cultured samples, whereas this was observed later or not at all in the non-co-cultured samples. The greater density of colonies in the co-coltured samples was another point. Hematopoietic CD45 cells were no longer detected after the first passage. Pluripotent stem cells were obtained from all co-cultured samples that contained stem cells positive for CD29, CD44, CD49e, CD90, CD105, CD51 Stro, and C-kit antibodies but negative for CD34, CD45, CD133, and glycophorin A. CONCLUSION Addition of ADPCs was crucial to generate pluripotent-derived stem cells from cord blood samples. This double culture may be a useful tool for a universal allogeneic stem cell source for tissue repair or regeneration.

A New Glucose 6-Phosphate Dehydrogenase Variant (G6PD Thessaloniki) in a Patient with Idiopathic Myelofibrosis

A new deficient glucose 6-phosphate dehydrogenase (G6PD) variant, G6PD Thessaloniki, which was found in the red blood cells of a 70-year-old woman who had idiopathic myelofibrosis, is described. G6PD Thessaloniki had a low Michaelis constant (Km) for G6P (20 microM), high Km for NADP (10.1 microM), normal pH optimum, reduced heat stability, decreased electrophoretic mobility (96-98% of the normal), increased 2-deoxy-G6P and decreased galactose 6-phosphate utilization. Several other enzymatic activities measured in the patients red blood cells were normal. Studies of red blood cell survival and glucose utilization gave evidence of haemolysis caused by defective glucose utilization by the pentose phosphate pathway. The only son of the patient had normal G6PD in his red blood cells. In an attempt to investigate the origin of G6PD Thessaloniki, heat stability tests of G6PD extracted from the patients skin have been performed.