Martina Vendrame

Infusion of Human Umbilical Cord Blood Cells in a Rat Model of Stroke Dose-Dependently Rescues Behavioral Deficits and Reduces Infarct Volume

Background and Purpose— Intravenously delivered human umbilical cord blood cells (HUCBC) have been previously shown to improve functional recovery of stroked rats. To extend these findings, we examined the behavioral recovery and stroke infarct volume in the presence of increasing doses of HUCBC after permanent middle cerebral artery occlusion (MCAO). Methods— Rats were subjected to MCAO and allowed to recover for 24 hours before intravenous infusion of 104 up to 3 to 5×107 HUCBC. Behavioral tests (spontaneous activity, step test, elevated body swing test) were performed 1 week before MCAO and at 2 and 4 weeks after HUCBC infusion. On completion of behavioral testing, animals were euthanized and brain infarct volumes quantified. HUCBC were identified by immunofluorescence for human nuclei and by polymerase chain reaction (PCR) using primers specific for human glycerol 3-phosphate dehydrogenase. Results— At 4 weeks after infusion, there was a significant recovery in behavioral performance when 106 or more HUCBC were delivered (p=0.001 to p=0.05). Infarct volume measurements revealed an inverse relationship between HUCBC dose and damage volume, which reached significance at the higher HUCBC doses (107 cells, p<0.01; 3 to 5×107 cells, p<0.05). Moreover, HUCBC were localized by immunohistochemistry and PCR analysis only in the injured brain hemisphere and spleen. Conclusions— These results extend previous observations of HUCBC infusion in the MCAO rat stroke model by demonstrating a dose relationship between HUCBC, behavioral improvement, and neuronal sparing.

Cord blood rescues stroke-induced changes in splenocyte phenotype and function.

The neuroprotective mechanism of human umbilical cord blood cells (HUCBC) in the rat middle cerebral artery occlusion (MCAO) stroke model remains uncertain. Given the inflammatory sequelae that occur following stroke, we investigated whether HUCBC protection could be derived from the modulation of this immuno-inflammatory event, suggested by the attraction of the HUCBC to the spleen. We found that, following MCAO, rat spleen size was reduced concomitantly with their CD8+ T-cell counts. Interestingly, MCAO-induced spleen size reduction correlated with the extent of ischemic damage, however, HUCBC treatment rescued the spleen weight, splenic CD8+ T-cell counts, as well as the amount of brain injury. Additionally, splenocyte proliferation assays demonstrated that HUCBC treatment opposed MCAO-associated T-cell proliferation by increasing the production of IL-10 while decreasing IFN-gamma. Taken together, these results suggest a novel immunomodulatory mechanism by which HUCBC mediate protection in the rat MCAO model of stroke.

Neuronal expression of CD22: novel mechanism for inhibiting microglial proinflammatory cytokine production.

Although considered an immunologically privileged site, the central nervous system (CNS) can display significant inflammatory responses, which may play a pathogenic role in a number of neurological diseases. Microglia appear to be particularly important for initiating and sustaining CNS inflammation. These cells exist in a quiescent form in the normal CNS, but acquire macrophage‐like properties (including active phagocytosis, upregulation of proteins necessary for antigen presentation, and production of proinflammatory cytokines) after stimulation with inflammatory substances such as lipopolysaccharide (LPS). Recent studies have focused on elucidating the role of neurons in the regulation of microglial inflammatory responses. In the present study, we demonstrate, using neuron‐microglial cocultures, that neurons are capable of inhibiting LPS‐induced tumor necrosis factor‐α (TNF‐α) production by microglia. This inhibition appears to be dependent on secretion of substances at axon terminals, as treatment with the presynaptic calcium channel blocker ω‐conotoxin abolishes this inhibitory effect. Moreover, we show that conditioned medium from neuronal cultures similarly inhibits microglial TNF‐α production, which provides additional evidence that neurons secrete inhibitory substances. We previously demonstrated that the transmembrane protein‐tyrosine phosphatase CD45 plays an important role in negatively regulating microglial activation. The recent characterization of CD22 as an endogenous ligand of this receptor led us to investigate whether neurons express this protein. Indeed, we were able to demonstrate CD22 mRNA and protein expression in cultured neurons and mouse brain, using reverse transcriptase‐polymerase chain reaction and antibody‐based techniques. Furthermore, we show that neurons secrete CD22, which functions as an inhibitor of microglial proinflammatory cytokine production.

Mobilized peripheral blood cells administered intravenously produce functional recovery in stroke.

Filgratism (granulocyte colony stimulating factor, G-CSF)-mobilized peripheral blood progenitor cells (PBPCs) have replaced bone marrow (BM) as a preferred source of autologous stem cells, in light of the faster hematologic recovery and lesser supportive care requirement exhibited by PBPC transplants. Other hematopoietic stem cells, like the human umbilical cord blood-derived stem cells (hUCBs), and nonhematopoietic stem cells have been shown to improve motor function in rodent models of injury and degenerative disease. In the present study we transplanted either G-CSF-mobilized PBPCs or hUCBs in rats 24 h after permanent middle cerebral artery occlusion (MCAO), and assessed their behavioral abnormalities in spontaneous activity and spontaneous motor asymmetry. In both transplanted groups of rats we observed a significant reduction of the stroke-induced hyperactivity compared with nontransplanted, stroked animals. In addition, transplantation of G-CSF PBPC and hUCB cells prevented the development of extensive motor asymmetry. Our findings raise the possibility that PBPCs could provide a novel transplantation therapy to treat stroke.

Reduced Th1 and enhanced Th2 immunity after immunization with Alzheimer's β-amyloid1–42

Abstract It has been demonstrated that immunization of transgenic mouse models of Alzheimers disease (AD) with amyloid-β 1–42 peptide (Aβ 1–42 ) results in prevention of Aβ plaque formation and amelioration of established plaques in the brain. As the response of the T lymphocyte helper (Th) arm of the immune response had not yet been investigated after Aβ immunization, we i.p. immunized C57BL/6 mice with Aβ 1–42 , Aβ 1–40 , or phosphate-buffered saline (PBS), and examined markers of Th1 and Th2 immune responses in spleen and in splenocytes from these mice. Spleens from Aβ 1–42 -immunized mice demonstrated decreased interleukin-12 receptor beta chain expression compared to mice immunized with Aβ 1–40 or PBS. Consistently, following stimulation with concanavalin A or anti-CD3 antibody, primary splenocytes from Aβ 1–42 -immunized mice demonstrated elevated secretion of interleukin-4 and interleukin-10, and decreased levels of interferon-γ. To validate this Th1→Th2 shift in a transgenic mouse model of AD, we immunized Tg APP sw mice (line 2576) with Aβ 1–42 and found decreased Th1 (interleukin-2 and interferon-γ) and elevated Th2 (interleukin-4 and interleukin-10) cytokines in their stimulated primary splenocytes. Interferon-γ was markedly reduced and interleukin-10 was increased in blood plasma from these mice, effects that were associated with dramatically mitigated Aβ deposition after Aβ 1–42 immunization. Taken together, these results show enhanced Th2 and down-regulated Th1 immunity following immune challenge with Aβ 1–42 .

Galantamine and nicotine have a synergistic effect on inhibition of microglial activation induced by HIV-1 gp120

Chronic brain inflammation is the common final pathway in the majority of neurodegenerative diseases and central to this phenomenon is the immunological activation of brain mononuclear phagocyte cells, called microglia. This inflammatory mechanism is a central component of HIV-associated dementia (HAD). In the healthy state, there are endogenous signals from neurons and astrocytes, which limit excessive central nervous system (CNS) inflammation. However, the signals controlling this process have not been fully elucidated. Studies on the peripheral nervous system suggest that a cholinergic anti-inflammatory pathway regulates systemic inflammatory response by way of acetylcholine acting at the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) found on blood-borne macrophages. Recent data from our laboratory indicates that cultured microglial cells also express this same receptor and that microglial anti-inflammatory properties are mediated through it and the p44/42 mitogen-activated protein kinase (MAPK) system. Here we report for the first time the creation of an in vitro model of HAD composed of cultured microglial cells synergistically activated by the addition of IFN-gamma and the HIV-1 coat glycoprotein, gp120. Furthermore, this activation, as measured by TNF-alpha and nitric oxide (NO) release, is synergistically attenuated through the alpha7 nAChR and p44/42 MAPK system by pretreatment with nicotine, and the cholinesterase inhibitor, galantamine. Our findings suggest a novel therapeutic combination to treat or prevent the onset of HAD through this modulation of the microglia inflammatory mechanism.

CD45 isoform RB as a molecular target to oppose lipopolysaccharide-induced microglial activation in mice.

CD45 is a membrane-bound protein tyrosine phosphatase expressed on all hemopoietic cells with multiple splice variants, including RA, RB, RC and RO. Our previous studies have shown that cross-linking of CD45 with an anti-CD45 antibody markedly inhibits LPS-induced microglia activation. In order to determine which of the CD45 isoforms may be responsible for these effects, we have investigated the expression of CD45 isoforms on cultured microglial cells using flow cytometric analysis. Data reveal that CD45RB is the predominant isoform expressed in murine primary cultured microglial cells. Furthermore, incubation of these cultured cells with anti-CD45RB antibody results in a reduction of microglial activation induced by LPS as evidenced by TNF-alpha production. As a validation of these findings in vivo, brain homogenates from anti-CD45RB antibody (MG23G2)-injected animals that had been treated with LPS demonstrate a significant decrease in TNF-alpha levels compared to control mice treated with LPS plus vehicle. Taken together, these findings suggest that therapeutic agents that specifically stimulate the microglial CD45RB signaling pathway may be effective in suppressing microglial activation associated with several neurodegenerative disorders.