Immune reconstitution following hematopoietic stem-cell transplantation

BACKGROUND: Reconstitution of the immune system following allogeneic stem-cell transplantation is a complex process that requires successful engraftment of the hematopoietic stem cell, as well as adequate thymic function. As the majority of patients have reduced thymic function due to age, hormonal changes, as well as the damage caused by conditioning and GvHD, immune recovery is often delayed and incomplete. Following graft infusion there is rapid proliferation of natural killer (NK) cells that appear to proceed directly from the hematopoietic stem cell. B-cell function is dependent on specific maturation development in the BM micro-environment, as well as CD4 help. The CD8 population expands rapidly due to proliferation of many memory cells that react against Class I Ags, as well as viral molecules. Expansion of T-helper cells originates mainly from the memory pool that is present in the bone marrow graft. Naive cells require competent thymus hence the CD4 cell counts may be subnormal with clinical immunodeficiency. Controversy remains as to the capacity of the thymus to recover and thus extra thymic proliferation of T cells have been postulated. However these cells appear to have a limited capacity to expand and a fixed repertoire. DISCUSSION: Donor lymphocyte infusions may contribute a competent CD4 population that can cause GvHD, but have limitations in the capacity to respond to new antigens. Future research needs to be concentrated on improving the capacity of the thymus to reconstitute a functional naive population.     

In vivo imaging of islet transplantation

Type 1 diabetes mellitus is characterized by the selective destruction of insulin-producing beta cells, which leads to a deficiency in insulin secretion and, as a result, to hyperglycemia. At present, transplantation of pancreatic islets is an emerging and promising clinical modality, which can render individuals with type 1 diabetes insulin independent without increasing the incidence of hypoglycemic events. To monitor transplantation efficiency and graft survival, reliable noninvasive imaging methods are needed. If such methods were introduced into the clinic, essential information could be obtained repeatedly and noninvasively. Here we report on the in vivo detection of transplanted human pancreatic islets using magnetic resonance imaging (MRI) that allowed noninvasive monitoring of islet grafts in diabetic mice in real time. We anticipate that the information obtained in this study would ultimately result in the ability to detect and monitor islet engraftment in humans, which would greatly aid the clinical management of this disease.     

Autologous stem-cell transplantation in refractory autoimmune diseases after in vivo immunoablation and ex vivo depletion of mononuclear cells

Autoimmune diseases that are resistant to conventional treatment cause severe morbidity and even mortality. In the present study we demonstrate that complete remissions can be achieved in refractory polychondritis and systemic lupus erythematosus (SLE), even at advanced stage, with the use of autologous stem-cell transplantation (SCT). Remissions persisted after reconstitution of the immune system. In the treatment of advanced systemic sclerosis (SSc), stable disease may be achieved with autologous SCT.     

In vivo hyperoxic preconditioning prevents myocardial infarction by expressing bcl-2

Preconditioning with oxidative stress has been demonstrated in vitro to stimulate the cellular adaptation to subsequent severe oxidative stress. However, it is uncertain whether this preconditioning works in vivo. In the present study, we examined in vivo the beneficial effect of oxidative preconditioning. After rats were pretreated with whole-body hyperoxygenation (100% O(2) at 3 atmosphere for 20 mins, four cycles with 20-min intermission), isolated hearts were subjected to 45-min ischemia followed by 90-min reperfusion. This hyperoxic preconditioning significantly reduced infarct size, cytochrome-c release, DNA fragmentation, and terminal deoxynucleotidyl transferase-mediated dUTD nick-end labeling-positive cell frequency in the left ventricle, biphasically with an early (30-min) and a delayed (48-hr) effect after the hyperoxygenation. Mechanistically, the NF-kappaB activity and Bcl-2 expression were enhanced in the hearts, and a NF-kappaB inhibitor, pyrrolidine dithiocarbamate, abolished the Bcl-2 induction as well as the infarct-limiting effect. An antioxidant, N-acetylcysteine, and protein kinase C (PKC) inhibitors chelerythrine and G? 6983 also blocked the preconditioning effects. These results indicate that hyperoxia induces myocardial tolerance against ischemia-reperfusion injury in association with Bcl-2 induction by NF-kappaB activation through reactive oxygen species and PKC-dependent signaling pathway.     

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as ε-N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of ε-N-methyllysines (1.40, 1.66, and 5.62 mol% for ε-N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of ε-N-methyllysines in histone H1.     

In vivo electrotransfer of interleukin-10 cDNA prevents endothelial upregulation of activated NF-kappaB and adhesion molecules following an atherogenic diet

OBJECTIVES: Interleukin (IL)-10 has anti-atherogenic properties. However, the molecular mechanisms involved in IL-10 protection against atherosclerosis in vivo remain poorly understood. In this study, we examined the effect of IL-10 cDNA in vivo electrotransfer on diet-induced, endothelial activation. METHODS: C57BL/6J mice were fed an atherogenic diet for 10 days. Expression of VCAM-1 and ICAM-1 was examined in the aortic sinus, a region predisposed to atherogenesis in mice, using immunohistochemistry. NF-kappaB activation was examined using a monoclonal antibody that selectively reacts with the activated form of the p65 subunit. RESULTS: We detected a low basal expression of activated NF-kappaB, VCAM-1 and ICAM-1 in the endothelium of the aortic sinus. Endothelial expression of activated NF-kappaB, VCAM-1 and ICAM-1 was markedly increased after 10 days on the atherogenic diet (p < 0.001). In vivo electrotransfer of a murine IL-10-encoding plasmid completely prevented diet-induced endothelial upregulation of activated NF-kappaB, VCAM-1 and ICAM-1 (p < 0.01). CONCLUSION: In vivo electrotransfer of IL-10 cDNA prevents diet-induced endothelial activation. These results suggest that the protective effects of IL-10 may already occur in the very early stages of atherogenesis.     

In vivo cell biology: following the zebrafish trend

A deeper understanding of the mechanisms of cell behavior is essential if we want to comprehend how an organism develops and functions. Changes in cellular processes, including the orientation of cell divisions, cell shape, polarity, differentiation and migration, account for tissue rearrangements during development and homeostasis. The in vivo relevance of in vitro findings is being constantly debated and the need for in vivo systems becoming more pressing. The zebrafish (Danio rerio) might become the vertebrate system of choice for a wide spectrum of biological questions that need to be investigated in vivo at cellular and subcellular resolutions. Here, we discuss some recent studies in which the zebrafish was used to gain insight into cell-biological mechanisms. Although this model system has been predominantly appreciated for its amenability to forward genetics, current advances in imaging technology and an increasing number of transgenic lines are bringing it closer to its full potential.     

In vivo study on medullary H(+)-sensitive neurons

Using the micro pressure ejection technique, we examined responses of medullary neurons with nonphasic discharges (164 units) to direct application of acidified mock cerebrospinal fluid (CSF, pH 6.85-7.05) in decerebrated spontaneously breathing cats. We found 16 H(+)-sensitive cells; they were excited promptly on application of approximately 500 pl of acidified mock CSF in the vicinity of the neuron under investigation, whereas they were unaffected by microejection of the control mock CSF (pH 7.25-7.60). Of the 16 H(+)-sensitive cells, 10 units were further found to be excited by transcapillary stimulation of the central chemoreceptors by using a method of intravertebral arterial injection of CO2-saturated saline. The discharges increased in a similar time course to that of ventilatory augmentation. Distributions of these 10 specific H(+)-sensitive cells were found in the vicinity of nucleus tractus solitarii as well as deep in the ventrolateral medulla. The present results suggest a possibility that pH-dependent central chemoreceptors, if any, would be located in two distinct medullary regions described in this study.     

In vivo specific binding of [3H]1-nicotine in the mouse brain

[3H] 1-Nicotine was used as a receptor ligand in the intact mouse. It was injected i.v., and radioactivity in brain regions was assayed. Nonspecific binding was estimated by pretreatment with unlabelled 1-nicotine. Radioactivity entered the brain rapidly, was heterogeneously distributed, and declined after 5 min. Estimated specific binding was highest in the medial and posterior cortex, midbrain, thalamus/hypothalamus and medulla/pons; intermediate in the cerebellum, caudate/putamen, frontal and frontoparietal cortex; and lowest in the hippocampus and olfactory bulb. Autoradiography showed similar patterns. Coinjection of unlabelled 1-nicotine reduced specific binding so that it approached estimated nonspecific binding. Nicotinic agonists reduced radioactivity in the thalamus/hypothalamus, but nicotinic antagonists were less active. Non-nicotinic drugs did not reduce brain radioactivity. The results suggest that radiolabelled nicotine may be used for in vivo receptor studies despite problems in estimating nonspecific binding.     

1.9 A structure of the therapeutic antibody CAMPATH-1H fab in complex with a synthetic peptide antigen.

CAMPATH-1 antibodies have a long and successful history in the treatment of leukaemia, autoimmune disease and transplant rejection. The first antibody to undergo "humanisation", CAMPATH-1H, permits treatment with limited patient anti-globulin response. It recognises the CD52 antigen which is a small glycosylphosphatidylinositol(GPI)-anchored protein expressed on lymphocytes and mediates cell depletion. We present the 1.9 A structure of the CAMPATH-1H Fab complexed [corrected] with an analogue of the antigenic determinant of CD52. Analysis of the CAMPATH-1H binding site reveals that in contrast to most antibodies CDR L3 plays a dominant role in antigen binding. Furthermore CDR H3, which is essential for effective antigen recognition in most antibodies, participates in only two main-chain interactions in CAMPATH-1H. The CAMPATH-1H binding site is highly basic; ionic interaction with the enthanolamine phosphate of the CD52 GPI anchor has long been hypothesised to be important in antigen binding. The structure reveals a number of important specific ionic interactions, including Lys53H but not Lys52bH as had previously been suggested. Prolonged treatment with CAMPATH-1H can lead to patient anti-idiotype responses which may be exacerbated by the unusually high number of basic residues in the antibody. This suggests that a strategy where redundant basic residues are replaced with neutral counterparts may be effective in further reducing the immunogenicity of this versatile and widely used antibody.     

Glycosylation and biological activity of CAMPATH-1H expressed in different cell lines and grown under different culture conditions

CAMPATH-1H (where CAMPATH is a trade mark of Well-come groupcompanies), a humanized IgG antibody used in the therapy oflymphoma, leukaemia and rheumatoid arthritis, has been expressedin Chinese hamster ovary, Y0 myeloma and NS0 myeloma cell lines.These engineered cell lines were grown under different cultureconditions, and the antibody isolated and purified. N-Linkedoligosaccharides, on the CH₂ heavy chain region of the antibody,were isolated and analysed by hydrazlnolysis, high-performanceanion-exchange chromatography with pulsed amperometric detection,laser-desorption mass spectrometry and sequential exoglycosidasetreatment Both the glycosylation pattern and the biologicalactivity of CAMPATH-1H, as measured by antibody-dependent cell-mediatedcytotoxicity, were markedly affected by the cell line used toexpress the antibody. It is concluded that glycosylation ofthe antibody may be important in the clinical outcome of therapy. ADCC antibody CAMPATH glycosylation oligosac-charide