Piia Valonen

Water diffusion in a rat glioma during ganciclovir-thymidine kinase gene therapy-induced programmed cell death in vivo: correlation with cell density.

To study the characteristics of diffusion magnetic resonance imaging (MRI) contrast in a rat brain BT4C glioma during progression of ganciclovir (GCV)‐thymidine kinase gene therapy‐induced programmed cell death (PCD) in vivo.

Early gene therapy-induced apoptotic response in BT4C gliomas by magnetic resonance relaxation contrast T1 in the rotating frame.

The design and evaluation of therapeutic gene transfection protocols and vectors are under extensive development. Magnetic resonance imaging (MRI) techniques can aid considerably in the development of experimental treatment approaches, as well as in determining treatment response by observing gross tissue morphology. However, through a unique set of contrast parameters, namely T1, T2, and diffusion, more information about tissue status can be obtained while delineating and classifying tumor characteristics in more detail. We show here that T1 relaxation in the rotating frame, T1ρ, provides unique in vivo MRI contrast. Ganciclovir treatment of HSV-tk+BT4C gliomas, which effectively eradicates these tumors, resulted in significantly prolonged T1ρ relaxation times in MRI already after 3 days of treatment, whereas conventional contrast parameters were elevated after 6–8 days of therapy. Interestingly, the prolonged T1ρ values were observed while an increase in tumor volume was still taking place. The regions of elevated T1ρ relaxation coincided with high apoptotic activity as determined by histology, suggesting that T1ρ MRI contrast could be used as a novel early indicator of cytotoxic cell damage in gliomas.

Quantitative evaluation of spontaneously and surgically repaired rabbit articular cartilage using intra-articular ultrasound method in situ.

During the last decade, a major effort has been devoted to developing surgical methods for repairing localized articular cartilage lesions. Despite some promising results no ultimate breakthrough in surgical cartilage repair has been achieved. Improvements in repair techniques would benefit from more sensitive and quantitative methods for long-term follow-up of cartilage healing. In this study, the potential of a new ultrasound technique for detecting the compositional and structural changes in articular cartilage after surgery, using recombinant human type II collagen gel and spontaneous repair was, investigated. Rabbit knee joints containing intact (n = 13) and surgically (n = 8) or spontaneously (n = 5) repaired tissue were imaged in situ at 6 months after the operation using a clinical intravascular high-frequency (40 MHz) ultrasound device. Based on the ultrasound raw data, ultrasound reflection coefficient (R), integrated ultrasound reflection coefficient (IRC), apparent integrated backscattering coefficient (AIB) and ultrasound roughness index (URI) were determined for each sample. URI was significantly higher in both repair groups than in intact cartilage (p < 0.05). The reflection parameters (R and IRC) were significantly lower in surgically repaired cartilage (p < 0.05) than in intact cartilage. Furthermore, AIB was significantly higher in surgically repaired cartilage than in intact tissue (p < 0.05). To conclude, the integrity of the rabbit articular cartilage repair could be quantitatively evaluated with the nondestructive ultrasound approach. In addition, clinically valuable qualitative information on the changes in cartilage integration, structure and composition could be extracted from the ultrasound images. In the present study, the structure and properties of repaired tissue were inferior to native tissue at 6 months after the operation. The applied ultrasound device and probes are FDA approved and, thus, applicable for the quantitative in vivo evaluation of human articular cartilage.

Water spin dynamics during apoptotic cell death in glioma gene therapy probed by T1ρ and T2ρ

Longitudinal and transverse relaxations in the rotating frame, with characteristic time constants T1ρ and T2ρ, respectively, have potential to provide unique MRI contrast in vivo. On‐resonance spin‐lock T1ρ with different spin‐lock field strengths and adiabatic T2ρ with different radiofrequency‐modulation functions were measured in BT4C gliomas treated with Herpes Simplex Virus thymidine kinase (HVS‐tk) gene therapy causing apoptotic cell death. These NMR tools were able to discriminate different treatment responses in tumor tissue from day 4 onward. An equilibrium two‐site exchange model was used to calculate intrinsic parameters describing changes in water dynamics. Observed changes included increased correlation time of water associated with macromolecules and a decreased fractional population of this pool. These results are consistent with destructive intracellular processes associated with cell death and the increase of extracellular space during the treatment. Furthermore, association between longer exchange correlation time and decreased pH during apoptosis is discussed. In this study, we demonstrated that T1ρ and T2ρ MR imaging are useful tools to quantify early changes in water dynamics reflecting treatment response during gene therapy. Magn Reson Med 59:1311–1319, 2008.

Evaluation of cancer virotherapy with attenuated replicative Semliki forest virus in different rodent tumor models.

Semliki Forest virus (SFV) is one of the latest candidates for a virotherapeutic agent against cancer, and recent studies have demonstrated its efficacy in tumor models. In the present study, we examined the antitumor efficacy of an avirulent SFV strain A7(74) and its derivative, a replication‐competent SFV vector VA7‐EGFP, in a partially immunodeficient mouse tumor model (subcutaneous A549 human lung adenocarcinoma in NMRI nu/nu mouse) and in an immunocompetent rat tumor model (intracranial BT4C glioma in BDIX rat). When subcutaneous mouse tumors were injected 3 times with VA7‐EGFP, intratumorally treated animals showed almost complete inhibition of tumor growth, while systemically treated mice displayed only delayed tumor growth (intravenous injection) or no response at all (intraperitoneal injection). This was at least partially due to a strong type I interferon (IFN) response in the tumors. The animals did not display any signs of abnormal behavior or encephalitis, even though SFV‐positive foci were detected in the brain after the initial blood viremia. Intracranial rat tumors were injected directly with SFV A7(74) virus and monitored with magnetic resonance imaging. Tumor growth was significantly reduced (p < 0.05) with one virus injection, but the tumor size continued to increase after a lag period and none of the treated animals survived. Three virus injections or T‐cell suppression with dexamethasone did not significantly improve treatment efficacy. It appeared that the local virotherapy induced extensive production of neutralizing anti‐SFV antibodies that most likely contributed to the insufficient treatment efficacy. In conclusion, we show here that SFV A7(74) is a potential oncolytic agent for cancer virotherapy, but major immunological hurdles may need to be overcome before the virus can be clinically tested.

Blood graft composition after plerixafor injection in patients with NHL

Plerixafor is used to mobilize CD34+ hematopoietic stem cells from bone marrow to circulation. Limited data are available in regard to graft cellular content collected after plerixafor.

Protein disulfide isomerase in ALS mouse glia links protein misfolding with NADPH oxidase-catalyzed superoxide production

Protein disulfide isomerase (PDI) is an oxidoreductase assisting oxidative protein folding in the endoplasmic reticulum of all types of cells, including neurons and glia. In neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), up-regulation of PDI is an important part of unfolded protein response (UPR) that is thought to represent an adaption reaction and thereby protect the neurons. Importantly, studies on animal models of familial ALS with mutant Cu/Zn superoxide dismutase 1 (SOD1) have shown that the mutant SOD1 in astrocytes or microglia strongly regulates the progression of the disease. Here, we found an early up-regulation of PDI in microglia of transgenic (tg) mutant SOD1 mice, indicating that in addition to neurons, UPR takes place in glial cells in ALS. The observation was supported by the finding that also the expression of a UPR marker GADD34 (growth arrest and DNA damage-inducible protein) was induced in the spinal cord glia of tg mutant SOD1 mice. Because mutant SOD1 can cause sustained activation of NADPH oxidase (NOX), we investigated the role of PDI in UPR-induced NOX activation in microglia. In BV-2 microglia, UPR resulted in NOX activation with increased production of superoxide and increased release of tumor necrosis factor-α. The phenomenon was recapitulated in primary rat microglia, murine macrophages and human monocytes. Importantly, pharmacological inhibition of PDI or its down-regulation by short interfering RNAs prevented NOX activation in microglia and subsequent production of superoxide. Thus, results strongly demonstrate that UPR, caused by protein misfolding, may lead to PDI-dependent NOX activation and contribute to neurotoxicity in neurodegenerative diseases including ALS.

Production of monocytic cells from bone marrow stem cells: therapeutic usage in Alzheimer’s disease

Accumulation of amyloid β (Aβ) is a major hallmark in Alzheimer’s disease (AD). Bone marrow derived monocytic cells (BMM) have been shown to reduce Aβ burden in mouse models of AD, alleviating the AD pathology. BMM have been shown to be more efficient phagocytes in AD than the endogenous brain microglia. Because BMM have a natural tendency to infiltrate into the injured area, they could be regarded as optimal candidates for cell‐based therapy in AD. In this study, we describe a method to obtain monocytic cells from BM‐derived haematopoietic stem cells (HSC). Mouse or human HSC were isolated and differentiated in the presence of macrophage colony stimulating factor (MCSF). The cells were characterized by assessing the expression profile of monocyte markers and cytokine response to inflammatory stimulus. The phagocytic capacity was determined with Aβ uptake assay in vitro and Aβ degradation assay of natively formed Aβ deposits ex vivo and in a transgenic APdE9 mouse model of AD in vivo. HSC were lentivirally transduced with enhanced green fluorescent protein (eGFP) to determine the effect of gene modification on the potential of HSC‐derived cells for therapeutic purposes. HSC‐derived monocytic cells (HSCM) displayed inflammatory responses comparable to microglia and peripheral monocytes. We also show that HSCM contributed to Aβ reduction and could be genetically modified without compromising their function. These monocytic cells could be obtained from human BM or mobilized peripheral blood HSC, indicating a potential therapeutic relevance for AD.

Aging aggravates ischemic stroke-induced brain damage in mice with chronic peripheral infection.

Ischemic stroke is confounded by conditions such as atherosclerosis, diabetes, and infection, all of which alter peripheral inflammatory processes with concomitant impact on stroke outcome. The majority of the stroke patients are elderly, but the impact of interactions between aging and inflammation on stroke remains unknown. We thus investigated the influence of age on the outcome of stroke in animals predisposed to systemic chronic infection. Th1‐polarized chronic systemic infection was induced in 18–22 month and 4‐month‐old C57BL/6j mice by administration of Trichuris muris (gut parasite). One month after infection, mice underwent permanent middle cerebral artery occlusion and infarct size, brain gliosis, and brain and plasma cytokine profiles were analyzed. Chronic infection increased the infarct size in aged but not in young mice at 24 h. Aged, ischemic mice showed altered plasma and brain cytokine responses, while the lesion size correlated with plasma prestroke levels of RANTES. Moreover, the old, infected mice exhibited significantly increased neutrophil recruitment and upregulation of both plasma interleukin‐17α and tumor necrosis factor‐α levels. Neither age nor infection status alone or in combination altered the ischemia‐induced brain microgliosis. Our results show that chronic peripheral infection in aged animals renders the brain more vulnerable to ischemic insults, possibly by increasing the invasion of neutrophils and altering the inflammation status in the blood and brain. Understanding the interactions between age and infections is crucial for developing a better therapeutic regimen for ischemic stroke and when modeling it as a disease of the elderly.

1H MR spectroscopic imaging of phospholipase‐mediated membrane lipid release in apoptotic rat glioma in vivo

The purpose of the current study was to determine regional spatiotemporal differences and to gain insight on the mechanisms responsible for lipid accumulation during apoptotic cell death using in vivo MR spectroscopic imaging in combination with histology and biochemical membrane lipid analyses. Rats bearing BT4C gliomas were treated with ganciclovir (GCV) for 14 days, and combined in vivo quantitative MR spectroscopic imaging (MRSI) of gliomas with histology and a biochemical analysis of major cell membrane constituents. By using 1H MRSI in vivo in combination with histology, we were able to demonstrate previously unattainable regional lipid concentration differences in tumors during GCV‐induced apoptosis, with 5‐μL tissue volume resolution. Our results also show that, during treatment, phospholipase A2 (PLA2) expression is significantly elevated by 37 ± 13% (P < 0.05) and tumor cell membranes loose a significant proportion of unsaturated fatty acyl moieties (56 ± 6 mmol/kg, P < 0.05). These changes are reflected in both histology and significant MR‐visible lipid accumulation, demonstrating that phospholipid hydrolysis in tissue undergoing apoptosis can be imaged with MRSI. Our work demonstrates the versatility of 1H MRSI in studying apoptosis in vivo, which is likely to pave way for the use of MRSI in both experimental and clinical anticancer trials. Magn Reson Med 59:1232–1238, 2008.