Abstract

Comparison of PET and DW/PW-MRI in Acute Ischemic Stroke

Wolf-Dieter Heiss

Background and Purpose: The concept of penumbra is based on animal experiments in which potentially reversible functional disturbances can be observed when blood flow decreases beyond a critical threshold. With reperfusion within a limited time window, these functional disturbances are reversible without leading to morphological damage and irreversible neurological deficits. In the last 20 years, surrogate markers of penumbra and irreversible tissue damage were studied, especially in regard to patients who could benefit from treatment in acute ischemic stroke. Positron emission tomography (PET) has been the gold standard but was replaced by diffusion- (DWI) and perfusion-weighted imaging (PWI) because of its wider distribution and the less complex logistics involved. There are some limitations with conventional DWI/PWI: increased diffusion signals can be reversible, and the determination of the threshold of critical perfusion by PWI is still a matter of ongoing debate.

Methods: Comparative studies with PET and MRI were performed in 3 groups of patients: (1) in 12 acute stroke patients, results from DWI (median, 6.5 hours after symptom onset) and ¹¹C-flumazenil (FMZ) PET (median, 85 minutes between DWI and PET) were compared with infarct extension 24 to 48 hours later on T2-weighted MRI. (2) in 11 acute stroke patients, results from PWI (median, 8 hours after symptom onset) were compared with cerebral blood flow measurements obtained with [¹⁵O]H2O PET (interval, 60 minutes between PWI and PET). (3) In 13 patients with acute (n = 6) or chronic stroke (n = 7), results from PWI/DWI were compared with PET of cerebral blood flow and oxygen consumption to detect mismatch or increased oxygen extraction fraction as surrogate markers of penumbra.

Results: From regions with increased DWI intensity, decreased apparent diffusion coefficient
(ADC) and decreased FMZ binding probability curves were computed for eventual infarction, and 95% prediction limits were determined. These limits predicted 83.5% (FMZ), 84.7 (DWI), and 70.9% (ADC) of the final infarct volume. However, the false-positive predictions were much higher for the DWI variables (5.1 and 3.6 cm³ for DWI and ADC versus a median of 0 for FMZ). (2) The comparison of volumes generated by different time to peak (TTP) thresholds (PWI) and hypoperfusion <20 ml/100g per minute (PET) indicates that a TTP delay of 4 to 6 seconds yields a fair estimate of hypoperfusion. (3) The PWI/DWI mismatch with TTP >4 seconds did not reliably correspond to the penumbra as assessed by PET (oxygen extraction fraction >150%). Only 8 of 13 patients with a mismatch had areas of penumbra. In these cases, the penumbra volume was overestimated by MRI.

Conclusion: DWI correlates with FMZ results and, with a few exceptions, yields a good estimate of acute tissue damage and final infarct volume. PWI measures seem to be less reliable; the TTP prolongation of >4 seconds assessed only 83% of the volume of hypoperfusion <20 ml/100g per minute. The mismatch volume imprecisely depicts increased oxygen extraction fraction, and, despite its clinical role for selection of patients for eventual therapy, it does not seem to be a reliable correlate of penumbra.

Identification and Functional Characterization of Distinct Dendritic Cell Subpopulations in Murine Skin

Keisuke Nagao, Florent Ginhoux, Wolfgang W. Leitner, Clare L. Bennett, Björn E. Clausen, Miriam Merad, and Mark C. Udey

Normal murine skin contains at least three subpopulations of dendritic cells (DC) that can be distinguished based on differential expression of the C-type lectin langerin and the adhesion molecule EpCAM. EpCAM was easily detected on DC surfaces while langerin was not. Langerhans cells (LC) in epidermis, dermis and lymph nodes all expressed abundant EpCAM and langerin. Dermal DC did not express EpCAM, but a minor subpopulation was langerin-positive. LC migrated from skin explants in the absence of exogenous cytokine, while emigration of langerin-positive dermal DC was CCL21-dependent. EpCAM-negative, langerin-negative dermal DC did not migrate ex vivo. Studies of mice that had been conditionally depleted of langerin-expressing cells and bone marrow chimeras revealed that EpCAM-negative langerin-positive DC repopulated the dermis well before Ep-CAM-positive langerin-positive LC appeared in epidermis, that EpCAM-postive and EpCAM-negative DC were found exclusively in the epidermis and dermis respectively, and that DC populations in each compartment could proliferate. EpCAM-positive langerin-positive LC were not present in the epidermis, dermis or lymph nodes of TGFB1^-/- Rag2^-/- mice, whereas EpCAM-negative langerin-positive dermal DC were frequent. Differential expression of EpCAM and langerin by DC defines three subpopulations in murine skin that are phenotypically distinct and that may represent different lineages. Carefully timed epicutaneous immunizations of mice that had been conditionally depleted of langerin-expressing cells via gene gun indicated that LC are required for optimal production of Th2-dependent IgG1 antibodies, and they may blunt Th1-dependent IgG2a/c antibody production. These results indicate that distinct skin DC subpopulations have specialized functions as well.

Conditioning Dendritic Cells to Imprint Gut-Tropic Effector/Memory Lymphocytes

J. Rodrigo Mora

We and others have previously shown that T cells are instructed to express gut-homing receptors (integrin α4β7 and chemokine receptor CCR9) when they are activated with antigen-presenting dendritic cells (DC) from gut-associated lymphoid tissues (GALT-DC), such as Peyer's patches or mesenteric lymph nodes. The ability of GALT-DC to imprint gut-tropism on T cells is explained by their selective capacity to produce retinoic acid (RA), a vitamin A metabolite. More recently, we have shown that GALT-DC and RA also induce gut-homing capacity on activated B cells. These mechanisms are important in vivo, because mice deficient in the RA precursor, vitamin A, lacked IgA antibody-secreting cells (IgA-ASC) in their small intestine. Interestingly, RA also synergized with IL-6 or IL-5 to induce IgA-ASC. Thus, GALT-DC and RA shape gut mucosal immunity by linking lymphocyte migration and function (IgA secretion).
An important unresolved question is how GALT-DC are themselves “imprinted” to secrete RA (and hence to induce gut-tropic lymphocytes). Likely sources of conditioning signals for DC in the gut are the intestinal epithelial cells (IEC). IEC form intimate contacts with DC in the intestinal mucosa and similar to GALT-DC they can metabolize vitamin A into RA. Since RA is known to modulate some functional properties of DC, such as antigen presentation, we hypothesized that IEC-derived RA instructs intestinal DC to imprint gut-homing lymphocytes. In agreement with our hypothesis, preliminary experiments show that spleen-DC pretreated with RA acquire the capacity to induce α4β7, CCR9 and gut-tropism on T cells upon activation. These findings suggest that RA may play a pivotal role imprinting GALT-DC to induce gut-homing lymphocytes. Our next step will be to address the in vivo role of RA and IEC in GALT-DC programming.

Multidisciplinary Management of Malignant Gliomas

Victor A. Levin

This talk will cover the incidence and frequency of brain tumor histologies in the US and current expectations for low-grade astrocytoma and oligodendroglioma tumors (WHO grade 2), mid-grade anaplastic astrocytoma, oligodendroglioma, and oligoastrocytoma tumors (WHO grade 3), and high-grade glioblastoma tumors (WHO grade 4).
For astrocytoma and oligodendroglioma, >90% resection yields 70% survival at 5-yr, post-surgery irradiation between 45-54 Gy yields 63% 5-yr survival, and adjuvant PCV chemotherapy about 80% 6-yr survival. For anaplastic gliomas, median survival is >51 months with irradiation and adjuvant chemotherapy. For glioblastoma median survival is 9 months for RT only, increases to about 13 months with adjuvant chemotherapy, and better adjuvant chemotherapies achieve ≥ 25% survival at 2 years.
Also discussed will be radiation necrosis in patients surviving > 1 year. In addition to neurocognitive slowing and MRI changes in white matter, some patients progress to radiation necrosis and more severe neurological deficits. Under investigation for the treatment of radiation necrosis is IV bevacizumab, an antibody directed against VEGF-A. Preliminary results show found patients had reduction in their T2 FLAIR, Gd-contrast, and dexamethasone dependence. We will discuss why progression-free survival (PFS) is a good surrogate for response to treatment and why new therapies should seek to improve on PFS at 6 months of 38% for anaplastic astrocytomas and 24% for glioblastoma. Also discussed will be therapies directed at angiogenesis, cell surface receptors, internal signaling pathways, and chromatin integrity. Future progress in the treatment of gliomas requires better drugs and the integration of molecular markers into clinical trials.

Treg Suppressive Mechanism by Apoptosis*

Pushpa Pandiyan and Michael Lenardo

CD4^＋CD25^＋Foxp3^＋ regulatory T (Treg) cells are believed to play an important role in suppressing autoimmunity and maintaining peripheral tolerance. The mechanism of Treg suppression still remains to be completely elucidated. Here we show that Tregs induce apoptosis of effector CD4^＋ T cells in vitro and in a mouse inflammatory bowel disease (IBD) model. Effector death required Bcl-2 interacting mediator of cell death (Bim) and Akt1/Protein Kinase-B induced dephosphorylation of the bcl-2 antagonist of cell death (BAD) protein. Treg cells, most likely consume IL-2 and other cytokines from effector cells causing cytokine deprivation death because exogenous addition of cytokines restored the effector survival. We also show that Treg cells express receptors for gamma chain cytokines and are dependent on an exogenous supply of these cytokines for survival. This result was validated in vivo by the accumulation of Treg cells in Bim^-/- and Bcl-2 tg mice which have arrested cytokine deprivation apoptosis. We also found that CD25 and Foxp3 expression were down-regulated in the absence of these cytokines. Our study reveals that the survival of Treg cells is strictly dependent on cytokines and cytokine producing cells because they do not produce cytokines. Thus, cytokine deprivation induced apoptosis regulates Treg homeostasis in the periphery and is a prominent event in Treg-inhibition of T cell responses.

Innate Immune Responses to Bacteria in the Lungs

Joseph P. Mizgerd

Acute lower respiratory tract infections are a public health concern. The outcome of these infections is determined by innate immune responses (such as neutrophil recruitment and activation), necessary for host defense but also contributing to lung injury. Innate immune responses in the lungs require the coordinated expression of diverse mediators including adhesion molecules, chemokines, colony stimulating factors, and cytokines that are not expressed or are expressed at low levels in uninfected lungs, but are locally expressed at high levels during infection. Roles for so many diverse factors suggests programs of gene expression which coordinate regulation. NF-κB transcription factors are critical to the gene expression program directing innate immunity in the lungs. NF-κB RelA is required for the induction of innate immunity genes mediating host defense, while NF-κB p50 counteracts this gene induction and is essential to preventing lung injury. The transcription factor STAT3 is also essential to fighting lung infections, mediating both antibacterial host defense and prevention of lung injury. These transcription factors have cell-specific roles during infection. An improved knowledge of the molecular mechanisms directing innate immunity in the lungs will provide new directions for preventing and curing acute lower respiratory tract infections.

Immune Regulation by Dendritic Cells and
Regulatory T Cells

Gosse J. Adema

Dendritic cells (DCs) and regulatory T cells (Treg) play a central role in our immune system. DC are professional antigen-presenting cells that attract, activate and instruct T- and B-lymphocytes, while Treg actively suppress leukocytes to prevent autoimmune responses harmful to the host. Key questions are how these pivotal cells are regulated in health and disease and how their unique immune-modulating capacity can be exploited to the benefit of patients. Others and we have explored the expression and function of Toll-like Receptors (TLRs) and of C-type lectins (CLRs) on DC and Treg. Recent data from our lab demonstrate that TLRs are not only important in innate immune function but also affect Treg. The TLR2 ligand Pam3Cys temporarily abrogates Treg mediated suppression and induces their proliferation by directly acting on the Treg themselves. These data establish a direct link between TLRs, Treg and the control of immune responses and fit a model in which effector T cell activity is favored over Treg suppression upon pathogen encounter. Aside TLR ligands, pathogens contain complex carbohydrate structures that are recognized by CLRs. Next to their role in pathogen-recognition leading to phagocytosis and antigen presentation, CLRs are increasingly recognized as signaling receptors. DCIR and dectin-1 are two CLRs containing opposing signaling motives. We now report the existence of DCIR/TLR cross-talk to down modulate cytokine production but not co-stimulatory molecule expression by DCs. These data provide further support to the concept of CLR/TLR cross-talk as a means to modulate immune responses.