Guidelines for the collection, processing and storage of human bone marrow and peripheral stem cells for transplantation

SUMMARY. There are no current U.K. or international guidelines or regulations covering the production, processing and storage of haemopoietic cells such as to allow their engraftment following myeloablative therapy. This paper seeks to provide such guidelines. It enumerates how quality control and assurance can be applied to this area of transfusion medicine; procedural steps relating to bone marrow harvest on peripheral blood stem cell collection are outlined and recommended doses of nucleated cells suggested for both procedures. General specifications for identification, storage and transportation of bone marrow and peripheral blood stem cells are included and specific laboratory procedures related to the provision of haemopoietic cells for engraftment are outlined. Umbilical cord blood transplants and long-term bone marrow culture are alluded to but these are still in a research phase.     

Polymer Reaction Engineering VI Halifax,Canada May 21–26, 2006

Conference Reports: This section contains reports on topical conferences. Reports are usually written at the request of the editorial office, but unsolicited contributions are also welcome. Suggestions should be sent to the editorial office of the Macromolecular journals, preferably by E‐mail to macromol@wiley‐vch.de.     

Angiotensin converting enzyme insertion/deletion polymorphism does not influence postcardiac transplantation hypertension onset or progression.

BACKGROUND: The angiotensin converting enzyme insertion deletion polymorphism (ACE I/D) has been associated with much cardiovascular pathology, including posttransplantation hypertension. Hypertension is a significant cause of morbidity and mortality after cardiac transplantation. We investigated the influence of the ACE I/D polymorphism on posttransplantation hypertension. METHODS: A total of 211 heart transplant recipients and 154 corresponding donors were genotyped for the ACE I/D polymorphism by polymerase chain reaction. ACE enzymatic activity was measured by spectrophotometric kinetic analysis. Sitting systolic and diastolic blood pressures were recorded at 3 consecutive visits, and the mean was calculated. Clinical data, including demographics and medication, were collected for all recipients. Results were analyzed by the chi-square test and analysis of variance, taking a p value of <0.05 to be significant. RESULTS: A total of 41.7% of the subjects were hypertensive (diastolic blood pressure >90 mm Hg) at the time of the study, with 79.6% taking at least one antihypertensive agent. We found no difference between the number of antihypertensive agents, cyclosporin dose and level, renal function, or systolic blood pressure for the different recipient or donor genotypes. We also found no significant correlation between ACE enzymatic activity and systolic or diastolic blood pressure. CONCLUSIONS: Our study of 211 recipients and 154 corresponding donors is the largest investigation of this polymorphism in a cardiac transplantation population. We found no apparent relationship between the ACE genotype (of either donor or recipient) and systemic hypertension (absolute measurements and the number or dose of antihypertensive agents used).     

Mapping the rat somatosensory pathway from the anterior cruciate ligament nerve endings to the cerebrum

The anterior cruciate ligament (ACL) serves as the primary restraint to anterior tibial translation. In addition to this biomechanical function, the ACL appears to have a function in neuromuscular control. This hypothesis was formulated after the discovery of mechanoreceptors within the ACL. The full somatosensory pathway from the ACL to the cerebrum has yet to be elucidated. In order to map this sensory pathway, we conducted a viral trans-synaptic tracing experiment using the neurotropic pseudorabies virus (PRV). The pseudorabies virus was injected into the ACL of rats and allowed to replicate and spread trans-synaptically for 6-7 days. The brain and spinal cord of each sacrificed rat was then removed and processed immunohistochemically to detect the presence of PRV. PRV-immunoreactive neurons were found to be localized in several different regions from the spinal cord to the cerebrum. Four nuclei in the reticular formation of the brain stem demonstrated strong positive labeling: the mesencephalic reticular nucleus, magnocellular reticular nucleus, paragigantocellular reticular nucleus, and gigantocellular reticular nucleus. This finding suggests that the nerve endings of the rat ACL project into the cerebrum and that the reticular formation may play an important role in the afferent pathway of those nerve endings.     

The detection of intracytoplasmic interleukin-1 alpha, interleukin-1 beta and tumour necrosis factor alpha expression in human monocytes using two colour immunofluorescence flow cytometry.

Two colour flow cytometry was used to analyse in situ cytokine expression by human monocytes. Whole blood was cultured in siliconised glass bottles, with or without E. coli lipopolysaccharide (LPS), for various times, and the mononuclear cells (MNCs) then exposed to a variety of permeabilisation procedures prior to flow cytometric analysis. Paraformaldehyde (PF)/saponin fixation preserved cellular morphology, and caused a reproducible degree of permeabilisation (estimated by propidium iodide inclusion: mean 94%, range 86-99% (n = 33)). After fixation with 4% PF and permeabilisation with 1% saponin at 0 degrees C in PBS containing 20% human serum, MNCs were incubated with phycoerythrin(PE)-conjugated mouse anti-CD14 (monocyte phenotype) and polyclonal rabbit anti-human interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumour necrosis factor alpha (TNF-alpha), or control rabbit IgG. Binding of rabbit antibodies was detected using goat anti-rabbit IgG fluorescein isothiocyanate (FITC). FITC fluorescence was increased in CD14 PE positive cells with the three anti-cytokine antibodies following LPS stimulation, compared with controls. There was a reproducible dose related response in monocyte IL-1 beta and TNF-alpha expression following LPS stimulation, with early peaks in TNF-alpha (2 h), compared with IL-1 beta (4 h), and IL-1 alpha (12 h). Specificity of this cytokine detection system was confirmed by inhibition studies using the corresponding recombinant human cytokines, by an absence of staining in CD14 negative or unpermeabilised MNCs, and by the characteristic cytoplasmic localisation of the different cytokines visualised with UV immunochemistry. Hence, the methods described here provide a reproducible, semiquantitative and specific assay for the detection of cell associated monokines. The technique may be applicable to the analysis of a variety of different cytokines in other phenotypically defined cell populations.     

The role of major and minor histocompatibility antigens in active enhancement of rat kidney allograft survival by blood transfusion

Rats given a blood transfusion do not reject a subsequent kidney allograft from the same donor strain. This effect is strain-specific so that pretransplant blood transfusion from a LEW (RT1l) rat will protect a LEW kidney in a DA (RT1a) recipient but not a PVG/c (RT1c) kidney. The same is true in other combinations. Using DA or PVG.RT1a congenic recipients we have shown that sharing of all or part of the MHC or of minor alloantigens by the blood transfusion and kidney transplant donors is sufficient to prolong allograft survival. Activation of suppression against minor alloantigens appears to require two signals: one is the minor alloantigen and the other may be a major histocompatibility complex alloantigen.     

Cyclic AMP inhibits macrophage suppressor function and enhances lymphocyte proliferation.

The effects of increasing the level of cyclic AMP (cAMP) activity on lymphocyte proliferation in the rat mixed lymphocyte reaction (MLR) were investigated. Dibutyryl cAMP (dbcAMP) and prostaglandin E2 (PGE2) produced a dose-dependent reduction in proliferation in the lymph node (LN) MLR, but produced a substantial increase in proliferation in the spleen MLR at the lower concentrations used (10(-5)-10(-4) M dbcAMP; 10(-7)-10(-6) M PGE2). Enhancement of proliferation was dependent on the presence of macrophages and was probably due to inhibition of macrophage activation. This was based on the following findings: (1) spleen MLR proliferation was lower than that in the LN MLR; (2) depletion of spleen macrophages increased proliferation in the spleen MLR and addition of these macrophages to the LN MLR reduced proliferation; (3) macrophage depletion from the spleen MLR abolished the proliferation-enhancing effect of dbcAMP. In conclusion, cAMP enhances lymphocyte proliferation in this system, apparently as a consequence of suppressing the inhibitory influence of macrophages.     

Environmental education

The need for a new profession devoted to environmental matters is asserted. The qualities of such a profession are sketched, and it is argued that new initiatives in environmental education are needed in the form of graduate, professional programs with primary emphasis on practice. An example 2-year program is presented. A fundamental requirement is scientific competence; undergraduate preparation in the sciences or engineering is mandatory. The graduate curriculum itself is built on three primary cores: environmental science and engineering, business and management, and public policy. Additionally, an environmental round table is proposed as a focal point for academic, industrial, governmental, and public discussion on environmental matters. The round table would provide oversight for the professional educational program and an affiliated research institute.