MD Michael Fiegl
email: michael.fiegl@i-med.ac.at

Florian Falkner

Michael Steurer

Niklas Zojer

Georg Hopfinger

Ferdinand Haslbauer

Guntram Winder

Daniela Voskova

Johannes Andel

Alois Lang

Yvona Brychtova

Jiri Mayer

Richard Greil

Günther Gastl

The Austrian

Alemtuzumab Retreatment in CLL 1 Successful Alemtuzumab Retreatment in Progressive B-Cell Chronic Lymphocytic Leukemia: A Multicenter Survey in 30 Patients

Keywords: alemtuzumab, retreatment, chronic lymphocytic leukemia, CLL Funding: Bayer Schering

Although retreatment with alemtuzumab in relapsing B-cell chronic lymphocytic leukemia (CLL) may be beneficial, there has thus far been no thorough analysis available on this topic. Data were collected from 30 chemotherapy-pretreated patients with progressive CLL who had received alemtuzumab twice in consecutive, distinct therapy lines. The median dose of alemtuzumab retreatment was 402 mg (range, 43 -1090 mg). Retreatment with alemtuzumab induced an overall response rate of 47%. From the start of alemtuzumab retreatment, median progression-free survival (PFS) and overall survival (OS) were 6.3 months and 20.0 months, respectively. Response rates, PFS and OS upon alemtuzumab retreatment were correlated with response to initial alemtuzumab treatment, the time interval between the initial course of alemtuzumab and start of retreatment, and the hemoglobin concentration prior to retreatment. Reported toxicities from 24 cases included infections (50%), febrile reactions upon alemtuzumab administration (38%), exanthema (21%) and grade 4 neutropenia (13%) and thrombocytopenia (17%). We conclude that alemtuzumab retreatment represents an effective and tolerable therapeutic option for selected patients with CLL.

Introduction

Chronic lymphocytic leukemia (CLL) can be treated with various therapies in a sequential manner. The selection of standard therapies depends on patient characteristics and disease features. For younger (<60) and physically fit patients, a combination of fludarabine, cyclophosphamid and rituximab (FCR regimen) has proven highly effective as first-and second line treatment option [START_REF] Hallek | First-Line Treatment with Fludarabine (F), Cyclophosphamide (C), and Rituximab (R) (FCR) Improves Overall Survival (OS) in Previously Untreated Patients (pts) with Advanced Chronic Lymphocytic Leukemia (CLL): Results of a Randomized Phase III Trial On Behalf of An International Group of Investigators and the German CLL Study Group[END_REF][START_REF] Robak | Rituximab plus fludarabine and cyclophosphamide prolongs progression-free survival compared with fludarabine and cyclophosphamide alone in previously treated chronic lymphocytic leukemia[END_REF], whereas for elderly and frail patients, chlorambucil is still a good choice [START_REF] Eichhorst | Treatment of elderly patients with chronic lymphocytic leukemia[END_REF]. For CLL patients with 17p deletion, alemtuzumab-based regimens are recommended as first line therapy [START_REF] Hillmen | Alemtuzumab compared with chlorambucil as first-line therapy for chronic lymphocytic leukemia[END_REF][START_REF] Eichhorst | ESMO Guidelines Working Group. Chronic lymphocytic leukemia: ESMO minimum clinical recommendations for diagnosis, treatment and follow-up[END_REF]. In addition, treatment with alemtuzumab is an established option for salvage therapy, particularly for patients who have failed alkylating agents and fludarabine.

Alemtuzumab (MabCampath , Genzyme) is a humanized monoclonal antibody that targets the CD52 cell surface glycoprotein, which is abundantly expressed on normal and malignant B-and T-lymphocytes. The therapeutic benefit of alemtuzumab has been demonstrated in phase II studies with pretreated CLL patients [START_REF] Osterborg | Phase II multicenter study of human CD52 antibody in previously treated chronic lymphocytic leukemia. European Study Group of CAMPATH-1H Treatment in Chronic Lymphocytic Leukemia[END_REF][START_REF] Rai | Alemtuzumab in previously treated chronic lymphocytic leukemia patients who also had received fludarabine[END_REF][START_REF] Keating | Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study[END_REF][START_REF] Moreton | Eradication of minimal residual disease in B-cell chronic lymphocytic leukemia after alemtuzumab therapy is associated with prolonged survival[END_REF][START_REF] Stilgenbauer | Subcutaneous alemtuzumab in fludarabine-refractory chronic lymphocytic leukemia: clinical results and prognostic marker analyses from the CLL2H study of the German Chronic Lymphocytic Leukemia Study Group[END_REF][START_REF] Cortelezzi | Low-dose subcutaneous alemtuzumab in refractory chronic lymphocytic leukaemia (CLL): results of a prospective, single-arm multicentre study[END_REF], in a phase II study with chemo-naïve CLL patients [START_REF] Lundin | Phase II trial of subcutaneous anti-CD52 monoclonal antibody alemtuzumab (Campath-1H) as first-line treatment for patients with B-cell chronic lymphocytic leukemia (B-CLL)[END_REF], and was confirmed in a phase III trial (CAM307) with chemo-naïve CLL patients [START_REF] Hillmen | Alemtuzumab compared with chlorambucil as first-line therapy for chronic lymphocytic leukemia[END_REF]. Routine use of alemtuzumab in clinical practice is feasible and relatively safe, as shown by the retrospective reports on pretreated CLL patients from our Austrian-Czech CLL Alemtuzumab Registry [START_REF] Fiegl | Austrian Collaborative Study Group on Alemtuzumab in Chronic Lymphocytic Leukemia (2006) Routine clinical use of alemtuzumab in patients with heavily pretreated B-cell chronic lymphocytic leukemia: a nation-wide retrospective study in Austria[END_REF][START_REF] Fiegl | Alemtuzumab in chronic lymphocytic leukaemia, other lymphoproliferative disease and autoimmune disorders[END_REF][START_REF] Fiegl | Clinical outcome of pretreated B-cell chronic lymphocytic leukemia following alemtuzumab therapy: a retrospective study on various cytogenetic risk categories[END_REF].

Currently, there is little data on how to treat CLL patients with disease progression after alemtuzumab treatment. In our previous Alemtuzumab Registry report, we noted that a remarkable number of patients with progressive disease having received a first course of alemtuzumab were fit enough to receive a second course of alemtuzumab.

Intriguingly, long lasting responses were observed among patients retreated with alemtuzumab. To date, there are no clinical trials published on the efficacy and safety of alemtuzumab retreatment. Only single case reports [START_REF] Rai | Alemtuzumab in previously treated chronic lymphocytic leukemia patients who also had received fludarabine[END_REF][START_REF] Rieger | Efficacy and tolerability of alemtuzumab (CAMPATH-1H) in the salvage treatment of B-cell chronic lymphocytic leukemia--change of regimen needed?[END_REF][START_REF] Eyged | Fifth succesfully retreatment with Alemtuzumab in patient with chronic lymphocytic leukemia[END_REF] and series of six [START_REF] Hui | Alemtuzumab in clinical practice: a British Columbia experience[END_REF] and seven CLL patients [START_REF] Thieblemont | Feasibility of Alemtuzumab retreatment in heavily pretreated refractory B-CLL/SLL[END_REF] have been published indicating clinical benefits from alemtuzumab retreatment.

In this survey, we retrospectively investigated the efficacy and toxicity of alemtuzumab retreatment in 30 patients with CLL. All cases had received a second course of alemtuzumab as monotherapy due to progressive disease (PD) after a treatment-free interval following the first alemtuzumab treatment. Patients' characteristics and clinical features predicting clinical benefit of alemtuzumab retreatment were analysed.

Patients and Methods

Patients

This retrospective survey was conducted retrieving clinical data from a large data base of 196 pretreated patients with CLL who received alemtuzumab as monotherapy. The kind and sequence of prior therapy regimens is detailed in Table 2. Thirty patients selected for this analysis had received a second course of alemtuzumab as the next therapy (-alemtuzumab retreatment‖). The indication to reinduce alemtuzumab was considered by the treating physician; arguments for choosing alemtuzumab rechallenge might have been lasting response upon alemtuzumab, good tolerability and massive pre-treatments with chemotherapy (Table 2). The data set included patient characteristics, pretreatment modalities, cumulative doses of alemtuzumab, response, reported toxicities and disease course.

Data were collected from 16 clinical centers in Austria, one hospital in South Tyrol (Italy), and a center in Brno (Czech Republic). In this survey, alemtuzumab retreatment was initiated between May 15, 2002 andDecember 27, 2007. Final follow-up data available for this analysis were obtained on May 25, 2010. The median observation time for this patient cohort was 20.9 months. Approval of the survey was obtained from the Ethics Committee at the Medical University of Innsbruck.

Treatment

Usually, the alemtuzumab dose was escalated during the first week of therapy by intravenous (IV) administration of 3 mg on day 1, 10 mg on day 2, and 30 mg on day 3. Subsequent doses of 30 mg were administered ordinarily three times per week for a projected 12 weeks, either intravenously (IV) (n = 5) or subcutaneously (SC) (n = alemtuzumab varied widely and were left to the discretion of clinicians from participating centers. Anti-infective prophylaxis, with valacyclovir and trimethoprimsulfamethoxazole, was given in accordance with accepted guidelines [START_REF] Osterborg | Management guidelines for the use of alemtuzumab in chronic lymphocytic leukemia[END_REF].

Response Evaluation

Patients who received at least one dose of alemtuzumab for retreatment were considered evaluable for safety and efficacy. Response or disease progression was evaluated according to the National Cancer Institute Working Group (NCI-WG) criteria [START_REF] Cheson | National Cancer Institutesponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment[END_REF] immediately after the last alemtuzumab administration, 2 months after the last antibody administration, and subsequently, in case of a delayed response. For documentation of CR, PR, or stable disease (SD), the clinical benefit had to persist for at least 2 months. PFS was defined as time from first dose of alemtuzumab retreatment to disease progression or death, whichever occurred first. OS was measured from initiation of alemtuzumab retreatment.

Statistical Considerations

Comparison of response rates between patient subgroups (based on cytogenetic categories and other baseline characteristics prior to alemtuzumab) was carried out using Pearson's chi-square test. Event-related data (PFS and OS) were estimated using the Kaplan-Meier method, and comparison of outcomes between categorical subgroups was made using the log-rank test. Univariate analysis was performed to determine correlations between baseline characteristics and PFS or OS. The significance level was set at P < 0.05, in two-sided tests.

Results

Patient Characteristics

Out of 196 patients followed in our registry, who had been treated first-line with chemotherapy and later with alemtuzumab in second or higher line, 30 patients (15%) received retreatment with single-agent alemtuzumab and were therefore included in this study. Importantly, between the two lines of alemtuzumab therapy, no other CLL treatment was administered. Patient demographics, baseline characteristics and therapy modalities at the start of alemtuzumab retreatment are detailed in Table 1. For all individual cases, the cumulative alemtuzumab doses, treatment responses and time intervals between both alemtuzumab series are detailed in Table 2. Briefly, 22 patients were male (73%), and their median age was 66 years (range 50 -81 years). The majority of the cases (63%) were refractory towards fludarabine, and presented with stage Rai 4 (53%). The median number of previous treatment lines (including the initial alemtuzumab course) was 4 (range, 2 -12). The median interval between the last dose of alemtuzumab course I and start of alemtuzumab course II was 10.9 months (range, 0.9 -39.6 months). For the 30 patients, the median dose of alemtuzumab course I was 552 mg (range 123 -1093 mg) and in course II, 402 mg (range, 43 -1090 mg). Various clinical parameters with potential impact on the results of alemtuzumab retreatment with respect to response, clinical benefit, PFS and OS were tested for their predictive value (Table 1). Results of this data analysis are presented in Table 3.

Response upon alemtuzumab retreatment

Individual responses upon alemtuzumab treatment I and II are summarized in Table 2. Briefly, 14 patients (47%) achieved a PR by alemtuzumab retreatment. Six patients (20%) manifested SD and 9 patients (30%) PD. One patient was not evualuable for response since he died during alemtuzumab retreatment for unknown reasons.

Notably, 11 out of 13 patients (85%) who responded to the first alemtuzumab course responded again to the second course. The remaining two patients showed longlasting SD (with PR at the blood level but persistent splenomegaly). Of note, 3 out of 11 patients (27%) with SD upon alemtuzumab course I showed PR upon alemtuzumab course II. Six out of six patients (100%) who were retreated with alemtuzumab although having progressed during or shortly after the first alemtuzumab course again experienced PD (Tables 2 and3). Response to alemtuzumab retreatment was correlated with the following characteristics at start of retreatment: Rai stage, presence or absence of bulky lymphadenopathy, peripheral blood leukocyte count, hemoglobin level, response to the initial alemtuzumab course, and the time interval between the alemtuzumab courses (Table 3). In this case series, response to alemtuzumab retreatment was not dependent on gender, age, platelet count or ß 2 -microglobulin level, and most remarkably not on cytogenetic risk category, number of prior therapies, and refractoriness toward fludarabine. Regarding cases categorized by cytogenetic risk (n = 20), response to alemtuzumab retreatment was achieved in 1/2 (50%), 5/7 (71%), 0/3 (0%), 1/3 (33%) and 2/5 (40%) of patients with 13q deletion, normal interphase karyotype, trisomy 12, 11q deletion, and 17p deletion, respectively. Progression-free survival after alemtuzumab retreatment Disease progression of CLL was documented at the time when the first signs of PD were recognized. During the follow-up period of this survey, all patients with alemtuzumab retreatment progressed, with the exception of one who was censored due to early death (see above). The median PFS after start of alemtuzumab retreatment was 6.3 months (range, 0.9 months to 26.9 months) (Figure 1). Short PFS was related to the following clinical characterstics at start of alemtuzumab retreatment (Table 3): higher age; presence of anemia (Figure 2A) and bulky lymphadenopathy; lack of response upon initial alemtuzumab therapy, and a short time interval between the finish of the initial alemtuzumab therapy and start of alemtuzumab retreatment (Figure 2B). As expected, PFS was significantly associated with the cumulative alemtuzumab dose applied and the clinical response achieved (Table 3).

Next, we investigated whether PFS, after starting alemtuzumab course I and II, differed when calculated for the cohort of these 30 patients. The median PFS after alemtuzumab course I was 8.9 months and after course II still 6.3 months. (p = 0.150) (Figure 1). In the subset of responders (n = 13) to the initial alemtuzumab course (Table 2), median PFS was 9.4 months after alemtuzumab course I and 11.9 months after course II (P = 0.312). However, also non-responders to alemtuzumab course I (n = 17) benefited with a median PFS of 7.5 months, although this patient group showed a PFS of only of 3.3 months upon alemtuzumab retreatment (P = 0.002).

Overall survival after alemtuzumab retreatment

Median OS after initiation of alemtuzumab retreatment was 20.0 months for the total cohort of 30 CLL patients. Poor OS was associated with following clinical parameters present at start of alemtuzumab retreatment: higher age, presence of bulky lymphadenopathy, presence of anemia, lack of response upon initial alemtuzumab therapy; and short time interval between the alemtuzumab courses (Table 3). On the contrary, an improved OS was seen in responders to alemtuzumab retreatment.

Disease Progression

Patients with disease progression during or shortly after alemtuzumab therapy presented primarily with increasing peripheral blood lymphocyte counts (n=16; 53%), progressive lymphadenopathy (n=7; 23%), increasing splenomegaly (n=2; 7%), and other clinical or laboratory features (rising LDH, cachexia, or other non specified signs) (n= 5; 17%).

Toxicity

CLL treatment following alemtuzumab retreatment

Twenty-two of the thirty cases received one or more further systemic therapies due to progressive disease after alemtuzumab retreatment (Table 2). The following therapies were chosen: a third course of alemtuzumab in 8 patients, of whom 6 had a PR to the second alemtuzumab therapy; rituximab-containing chemo-immuno therapy (n = 8); rituximab monotherapy (n = 2); chemotherapy (n = 3); and dexamethasone plus rituximab (n = 1). In the 8 patients receiving a third alemtuzumab therapy, a PR was achieved in one and SD in two cases. Alemtuzumab administration was cessated in one case due to septicemia (Table 2). One patient received combination therapy with cyclophosphamide, fludarabine, alemtuzumab and rituximab and achieved an ongoing CR.

Discussion

In our previous retrospective reports [START_REF] Fiegl | Austrian Collaborative Study Group on Alemtuzumab in Chronic Lymphocytic Leukemia (2006) Routine clinical use of alemtuzumab in patients with heavily pretreated B-cell chronic lymphocytic leukemia: a nation-wide retrospective study in Austria[END_REF][START_REF] Fiegl | Alemtuzumab in chronic lymphocytic leukaemia, other lymphoproliferative disease and autoimmune disorders[END_REF][START_REF] Fiegl | Clinical outcome of pretreated B-cell chronic lymphocytic leukemia following alemtuzumab therapy: a retrospective study on various cytogenetic risk categories[END_REF], we demonstrated that routine use of alemtuzumab in CLL patients was beneficial and safe, in line with prospective clinical studies [START_REF] Osterborg | Phase II multicenter study of human CD52 antibody in previously treated chronic lymphocytic leukemia. European Study Group of CAMPATH-1H Treatment in Chronic Lymphocytic Leukemia[END_REF][START_REF] Rai | Alemtuzumab in previously treated chronic lymphocytic leukemia patients who also had received fludarabine[END_REF][START_REF] Keating | Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study[END_REF][START_REF] Moreton | Eradication of minimal residual disease in B-cell chronic lymphocytic leukemia after alemtuzumab therapy is associated with prolonged survival[END_REF][START_REF] Stilgenbauer | Subcutaneous alemtuzumab in fludarabine-refractory chronic lymphocytic leukemia: clinical results and prognostic marker analyses from the CLL2H study of the German Chronic Lymphocytic Leukemia Study Group[END_REF][START_REF] Cortelezzi | Low-dose subcutaneous alemtuzumab in refractory chronic lymphocytic leukaemia (CLL): results of a prospective, single-arm multicentre study[END_REF][START_REF] Lundin | Phase II trial of subcutaneous anti-CD52 monoclonal antibody alemtuzumab (Campath-1H) as first-line treatment for patients with B-cell chronic lymphocytic leukemia (B-CLL)[END_REF]. Interestingly, it became evident that a considerable portion of patients received alemtuzumab retratment subsequently. A prospective clinical study addressing the role of alemtuzumab retreatment in CLL is unlikely to be performed in the foreseeable future. Thus, we aimed at investigating the utility of alemtuzumab retreatment in retrospect and at identifying factors that can be used for adequate patient selection.

Who were the patients subjected to alemtuzumab rechallenge? Most likely those in whom a benefit could be expected, e.g. due to response upon prior alemtuzumab, or because other therapy options were not available, e.g. due to massive pretreatment (Table 2). For the physicians using alemtuzumab retreatment, another pro-argument might have been good tolerability during first course of alemtuzumab; indeed, in our series the rate of grade IV hematoxicity was definitely low in both first and second course of alemtuzumab, when compared to the literature [START_REF] Osterborg | Phase II multicenter study of human CD52 antibody in previously treated chronic lymphocytic leukemia. European Study Group of CAMPATH-1H Treatment in Chronic Lymphocytic Leukemia[END_REF][START_REF] Moreton | Eradication of minimal residual disease in B-cell chronic lymphocytic leukemia after alemtuzumab therapy is associated with prolonged survival[END_REF][START_REF] Stilgenbauer | Subcutaneous alemtuzumab in fludarabine-refractory chronic lymphocytic leukemia: clinical results and prognostic marker analyses from the CLL2H study of the German Chronic Lymphocytic Leukemia Study Group[END_REF] and to the whole series of our CLL/alemtuzumab registry (with grade IV neutropenia and thrombocytopenia of 33% and 19%, respectively [START_REF] Fiegl | Alemtuzumab in chronic lymphocytic leukaemia, other lymphoproliferative disease and autoimmune disorders[END_REF]). Similarly, Hui et al. observed low hematological toxicity in the 6 patients who received a second course of alemtuzumab [START_REF] Hui | Alemtuzumab in clinical practice: a British Columbia experience[END_REF].

Thus far, the topic of alemtuzumab retreatment has not been systematically investigated. Egyed et al. presented a patient with CLL with 11q deletion who meanwhile had received five courses of alemtuzumab within a five-year interval and had responded well to each course [START_REF] Eyged | Fifth succesfully retreatment with Alemtuzumab in patient with chronic lymphocytic leukemia[END_REF]. Thieblement et al. presented a case series from a total of 17 patients with CLL relapsing after alemtuzumab, seven of whom received alemtuzumab retreatment. The median interval between the two alemtuzumab courses was 10 months. Within the first month of retreatment, normalization of blood lymphocytosis was observed in all cases, and four patients had a response of lymphadenopathies and splenomegaly subsequently. Median time to progression was 5.8 months [START_REF] Thieblemont | Feasibility of Alemtuzumab retreatment in heavily pretreated refractory B-CLL/SLL[END_REF]. Hui et al. documented 6 patients who received alemtuzumab retreatement. Among those, two achieved PR and three SD [START_REF] Hui | Alemtuzumab in clinical practice: a British Columbia experience[END_REF].

For T-PLL, alemtuzumab is considered a standard therapy option for first and following therapy lines. In T-PLL, alemtuzumab retreatment dosing and response have been studied retrospectively [START_REF] Dearden | High remission rate in T-cell prolymphocytic leukemia with CAMPATH-1[END_REF]. The authors found that 13 of 39 patients with T-PLL treated initially with alemtuzumab received alemtuzumab retreatment, and the median dose of retreatment was 263 mg (range, 40 mg -1325 mg). Notably, six patients responded (46%) to the second alemtuzumab course, and response to retreatment was seen only in patients who had achieved a complete response upon first alemtuzumab treatment.

In our study, a quite favourable response rate of 47% upon alemtuzumab retreatment was observed. Most important, it was possible to identify factors predicting responsiveness to alemtuzumab retreatment (Table 3). Clinically relevant is the finding that presence of anemia at start of alemtuzumab retreatment is clearly indicating inferior outcome, corresponding to our observation that anemia is an independent risk factor of inferior outcome at first course of alemtuzumab treatment correspond closely with favourable PFS and OS after start of alemtuzumab retreatment (Table 3). This data compares well with results achieved with retreatment of CLL using purine analogs [START_REF] Leporrier | Role of fludarabine as monotherapy in the treatment of chronic lymphocytic leukemia[END_REF][START_REF] Juliusson | Retreatment of chronic lymphocytic leukemia with 2-chlorodeoxyadenosine (CdA) at relapse following CdA-induced remission: no acquired resistance[END_REF]; of indolent lymphoma using rituximab [START_REF] Lemieux | Second treatment with rituximab in B-cell non-Hodgkin's lymphoma: efficacy and toxicity on 41 patients treated at CHU-Lyon Sud[END_REF]; of myeloma using bortezomib [START_REF] Sood | Retreatment with bortezomib alone or in combination for patients with multiple myeloma following an initial response to bortezomib[END_REF][START_REF] Petrucci | Efficacy and Safety of Retreatment with Bortezomib in Patients with Multiple Myeloma: Interim Results From RETRIEVE, a Prospective International Phase 2 Study[END_REF], and of solid tumors such as ovarian [START_REF] Nguyen | Prognostic factors associated with response in platinum retreatment of platinum-resistant ovarian cancer[END_REF] and lung cancer [START_REF] Smit | Randomized phase II and pharmacogenetic study of pemetrexed compared with pemetrexed plus carboplatin in pretreated patients with advanced non-small-cell lung cancer[END_REF] using platinum-based chemotherapy. There is strong evidence from retreatments with various agents in different maligancies that the response upon initial treatment and the interval between initial treatment and retreatment were predictive for both clinical benefit and disease outcome [START_REF] Leporrier | Role of fludarabine as monotherapy in the treatment of chronic lymphocytic leukemia[END_REF][START_REF] Juliusson | Retreatment of chronic lymphocytic leukemia with 2-chlorodeoxyadenosine (CdA) at relapse following CdA-induced remission: no acquired resistance[END_REF][START_REF] Lemieux | Second treatment with rituximab in B-cell non-Hodgkin's lymphoma: efficacy and toxicity on 41 patients treated at CHU-Lyon Sud[END_REF][START_REF] Sood | Retreatment with bortezomib alone or in combination for patients with multiple myeloma following an initial response to bortezomib[END_REF][START_REF] Petrucci | Efficacy and Safety of Retreatment with Bortezomib in Patients with Multiple Myeloma: Interim Results From RETRIEVE, a Prospective International Phase 2 Study[END_REF][START_REF] Nguyen | Prognostic factors associated with response in platinum retreatment of platinum-resistant ovarian cancer[END_REF][START_REF] Smit | Randomized phase II and pharmacogenetic study of pemetrexed compared with pemetrexed plus carboplatin in pretreated patients with advanced non-small-cell lung cancer[END_REF].

In conclusion, our results from this retrospective survey corroborate and expand preliminary data indicating that for carefully selected cases with relapsing or progressive CLL, alemtuzumab retreatment provides a valid therapeutic option.

Notably, recent guidelines on the routine use of alemtuzumab in CLL established by an international panel of experts underscore the option of alemtuzumab retreatment [START_REF] Osterborg | Management guidelines for the use of alemtuzumab in chronic lymphocytic leukemia[END_REF], based on recently presented, preliminary data from our study [30]. 15 25 Median interval between alemtuzumab courses I and II in months 10.9 (0.9 to 39.6).

*according to the hierarchical model by Döhner et al. [START_REF] Döhner | Genomic aberrations and survival in chronic lymphocytic leukemia[END_REF]. Data for subgroup analyses were not always available for all 30 patients. †Pearsons qui-square test ‡ Log-rank test. (A) According to absence or presence of anemia prior to alemtuzumab retreatment, PFS was 9.5 months and 3.1 months in patients with hemoglobin ≥11 g/dL or less, respectively (P <0.001). (B) Plotted according to the length of the treatment-free interval between end of initial alemtuzumab therapy and alemtuzumab retreatment, the median PFS was 9.9 months and 4.1 months in patients with an interval ≥10.9 months or shorter, respectively (P = 0.011).

  Data on toxicities upon alemtuzumab retreatment were available from 24 cases. Two patients experienced no side effects. Frequently documented toxicities were, in falling order: infections (n = 12 [50%]; including fever of unknown origin [n=7], sinusitis [n=2], pneumonia [n=1], endocarditis [n=1] and enteritis [n=1]); febrile reaction with shaking chills during or shortly after alemtuzumab administration (n = 9 [38%]), and exanthema (n = 5 [21%]). Thrombocytopenia and neutropenia of grade IV were observed in 4 (17%) and 3 cases (13%), respectively. Of note, severe thrombocytopenia and neutropenia was comparably low also during first course of alemtuzumab in these 24 patients (thrombocytopenia grade IV, n = 3 [13%]; neutropenia grade IV, n = 2 [8%]). One patient died shortly after start of alemtuzumab retreatment, and the cause remains unknown.

Figure 1 .Figure 2 .

 12 Figure 1. Kaplan-Meier plots illustrating progression-free survival (PFS) in 30patients with chemotherapy pretreated CLL who received alemtuzumab monotherapy. PFS for the initial alemtuzumab treatment series and alemtuzumab retreatment was 8.9 months and 6.3 months, respectively (P = n.s.).

  Fig. 2A

Table 1 .

 1 Patient demographics and baseline disease characteristics at alemtuzumab retreatment (n = 30)

	Characteristics

Table 2 .

 2 Individual patients' characteristics, alemtuzumab dosing, treatment-free intervals, and responses in CLL patients treated with alemtuzumab (n = 30)

	Pat. Age * /Sex	Year diagn.	No. prior ther.	† Kind of prior therapies	Dose 1 st course A (mg)

Table 3 .

 3 Univariate analysis for association of baseline characteristics and treatmentrelated variables with overall response rate, progression-free and overall survival upon alemtuzumab retreatment National Cancer Institute Working Group; NE, not evaluable; n.r., not recorded; OS, overall survival; PD, progressive disease; PR, partial response; SD, stable disease.

	Characteristics	No.	ORR	P value	†	PFS, mo.	P value ‡	OS, mo.	P value ‡
		pat	*	n (%)			(median)		(median)
	All patients	30		14 (47)	-		6.3	-	20.0	-
							(Fig. 1)
	CLL stage
	Rai 1-2	13		9 (69)	0.030		9.5	0.153	46.0	0.494
	Rai 3-4	17		5 (29)			4.6		18.5
	Hemoglobin
	≥11 g/dL	18		14 (78)	<0.001		9.5	<0.001	49.5	0.001
	<11 g/dL	10		0 (0)			3.1		9.4
							(Fig. 2A)
	Leuko
	<50.000 G/L	14		10 (71)	0.040		4.6	0.267	16.7	0.222
	>50.000 G/L	15		5 (33)			7.4		46.0
	Lymph nodes
	<5 cm	16		10 (63)	0.023		9.3	0.004	37.7	0.015
	5 cm	11		2 (18)			3.2		8.4
	Response to initial
	alemtuzumab therapy
	CR/PR	13		11 (79)	<0.001		11.9	<0.001	57.0	<0.001
	Rest	17		3 (21)			3.3		15.5
	Interval between alem-
	tuzumab courses I and II
	<10.9 months	15		11 (27)	0.028		4.1	0.011	11.3	0.002
	≥10.9 months	15		10 (67)			9.9		49.9
							(Fig. 2B)
	Cumulative dose
	<400 mg	14		5 (36)	0.256		3.2	0.013	11.3	0.352
	≥400 mg	14		8 (57)			7.5		25.5
	Response to alemtuzu-
	mab retreatment
	PR	14			-		9.5	<0.001	49.8	<0.001
	SD	6					4.6		17.8
	PD	9					2.7		7.4
	NE	1					0.9		0.9

Abbreviations: CR, complete response; NCI-WG,


M.D Thomas Wisniewski
email: thomas.wisniewski@nyumc.org

Ph.D Einar M Sigurdsson

Murine models of Alzheimer's disease, and their use in developing immunotherapies

Keywords: Transgenic mice, amyloid β, congophilic angiopathy, tau, immunization, neurofibrillary tangles, immunomodulation, Alzheimer's disease

Alzheimer's disease (AD) is one of a category of neurodegenerative diseases characterized by a conformational change of a normal protein into a pathological conformer with a high β-sheet content that renders it resistant to degradation and neurotoxic. In AD the normal soluble amyloid β (sAβ) peptide is converted into oligomeric/fibrillar Aβ. The oligomeric forms of Aβ are

Introduction:

Treatments for AD available currently provide largely symptomatic relief with only minor effects on the course of the disease; hence, there is an urgent need for better therapeutic

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M A N U S C R I P T interventions. Besides immunomodulation, numerous other approaches are being studied, which include anti-Aβ aggregation agents, secretase inhibitors/modulators blocking Aβ production, tau aggregation blockers, agents targeting mitochondria, stem cell therapies and various neuroprotective strategies [1; 2]. Currently it appears that the greatest hope for an intervention that will significantly impact disease progression in the near future comes from immunization approaches [START_REF] Brody | Active and passive immunotherapy for neurodegenerative diseass[END_REF][START_REF] Wisniewski | Amyloid-β immunization for Alzheimer's disease[END_REF][START_REF] Wisniewski | Vaccination as a therapeutic approach for Alzheimer's disease[END_REF]. In AD Tg mouse models Aβ directed immunization has been hugely successful using a wide variety of methods. Despite this, significant unanswered questions remain for the current and future human trials as to what is the best design of a vaccine, what is the best target and when should therapy start? A key issue which needs to be addressed is how to target the early initiating events in AD and not just the tombstone lesions which are the end result of a long chain of pathological processes.

Pathogenesis of Familial and Sporadic Alzheimer's disease:

The diagnostic neuropathological lesions of AD are the accumulation of Aβ as neuritic plaques and congophilic angiopathy, as well as aggregation of abnormally phosphorylated tau in the form of neurofibrillary tangles (NFTs). Missense mutations in APP or in the presenilin (PS)

genes PS 1 and 2 can cause early onset, familial forms of AD (FAD) affecting <4% of AD patients. The most common form of AD is sporadic and late-onset. The dominant theory for the causation of AD has been the amyloid cascade hypothesis [6; 7]. This theory currently suggests that accumulation of Aβ peptides particularly in a highly toxic oligomeric form is the primary pathogenic driver, that downstream leads to tau hyperphosphorylation, NFT formation and ultimately to synaptic and neuronal loss. Extensive evidence supports this hypothesis in FAD patients and in models of FAD: 1) Inherited forms of AD linked with mutations in the APP gene

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or in the PS1 or 2 genes are associated with changes in APP processing that favor over production of sAβ or production of more aggregation prone forms of sAβ such as Aβ1-42 [START_REF] Hardy | A hundred years of Alzheimer's disease research[END_REF]. 2) Down's syndrome, where there is an extra copy of the APP gene due to trisomy 21, is associated with AD related pathology at a very early age [START_REF] Lemere | Sequence of deposition of heterogeneous amyloid β-peptides and APO E in Down syndrome: implications for initial events in amyloid plaque formation[END_REF]. 3) In transgenic and other models of coexpressed amyloid β and tau, amyloid β oligomer formation precedes and accentuates tau related pathology, consistent with the hypothesis that NFT formation is downstream from Aβ aggregation [START_REF] Gotz | Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Abeta 42 fibrils[END_REF][START_REF] Oddo | Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction[END_REF][START_REF] King | Tau-dependent microtubule disassembly initiated by prefibrillar beta-amyloid[END_REF][START_REF] Sigurdsson | Local and distant histopathological effects of unilateral amyloid-beta 25-35 injections into the amygdala of young F344 rats[END_REF][START_REF] Sigurdsson | Bilateral injections of amyloid-beta 25-35 into the amygdala of young Fischer rats: behavioral, neurochemical, and time dependent histopathological effects[END_REF][START_REF] Frautschy | Effects of injected Alzheimer beta-amyloid cores in rat brain[END_REF] 4) In transgenic mouse models of mutant APP over-expression (where there is no tau pathology) therapeutic prevention and/or removal of Aβ is associated with cognitive benefits in experimental mice [START_REF] Schenk | Immunization with amyloid-β attenuates Alzheimer disease-like pathology in the PDAPP mice[END_REF][START_REF] Sigurdsson | Immunization with a non-toxic/non-fibrillar amyloid-β homologous peptide reduces Alzheimer's disease associated pathology in transgenic mice[END_REF][START_REF] Morgan | Aβ peptide vaccination prevents memory loss in an animal model of Alzheimer's disease[END_REF][START_REF] Pearson | Aβ peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease[END_REF][START_REF] Sigurdsson | An attenuated immune response is sufficient to enhance cognition in an Alzheimer's disease mouse model immunized with amyloid-β derivatives[END_REF][START_REF] Asuni | Aβ derivative vaccination in alum adjuvant prevents amyloid deposition and does not cause brain microhemorrhages in Alzheimer's model mice[END_REF] Importantly, in transgenic mouse models of both mutant APP and tau over-expression (with both amyloid and tau related pathology) prevention of Aβ pathology leads to both amelioration of cognitive deficits and attenuates early but not late tau pathology [START_REF] Oddo | Temporal profile of amyloid-beta (Abeta) oligomerization in an in vivo model of Alzheimer disease. A link between Abeta and tau pathology[END_REF][START_REF] Blurton-Jones | Pathways by which Abeta facilitates tau pathology[END_REF][START_REF] Mckee | Ibuprofen reduces Abeta, hyperphosphorylated tau and memory deficits in Alzheimer mice[END_REF]. In addition, it has been shown that reducing the level of endogenous mouse tau can prevent behavioral deficits in APP Tg mice without affecting Aβ levels [START_REF] Roberson | Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model[END_REF] and that exogenous Aβ extracted from AD Tg mice can accelerate plaque deposition in predisposed young Tg mice [START_REF] Eisele | Induction of cerebral betaamyloidosis: intracerebral versus systemic Abeta inoculation[END_REF]. However, evidence proving that Aβ is central in the common late-onset sporadic form of AD is more limited: 1) A correlation has been shown between biochemically extracted Aβ peptide species from sporadic AD brains with cognitive decline [START_REF] Naslund | Correlation between elevated levels of amyloid beta-peptide in the brain and cognitive decline[END_REF]. A comparison of several neuropathological features with cognitive states in AD revealed that the strongest predictor of cognitive dysfunction was Aβ load in the entorhinal cortex [START_REF] Cummings | Beta-amyloid deposition and other measures of neuropathology predict cognitive status in Alzheimer's disease[END_REF]. 2) Isolated Aβ peptide dimers/oligomers from sporadic AD brains have been documented to impair synaptic structure and function [START_REF] Shankar | Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory[END_REF]. 3) Aβ extracted from sporadic AD patients has been shown to induce amyloid deposits when injected into transgenic mice [START_REF] Meyer-Luehmann | Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host[END_REF]. A significant problem for the amyloid cascade hypothesis comes from the autopsy data from the initial human active vaccination trial

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targeting full-length Aβ. Post-mortem analysis was available from nine subjects in the active immunization arm [START_REF] Holmes | Long term effects of Aβ42 immunization in Alzheimer's disease: immune response, plaque removal and clinical function[END_REF]. All these individuals showed partial or near complete plaque removal and reduced Aβ load compared to age-matched non-immunized controls. Despite this, there were no differences between placebo and active immunization groups in terms of long-term survival outcome, time to severe dementia and in outcome measures such as ADAS-Cog, MMSE or DAD. This may be related to immunization having begun too late in the disease process;

alternatively, one can use this data to suggest that the amyloid cascade hypothesis is an oversimplification. A number of investigators have suggested alternative theories, whereby accumulation of Aβ and tau hyperphosphorylation are dual pathways both downstream from a common upstream pathogenic deficit (which remains to be identified) [START_REF] Small | Linking Abeta and tau in late-onset Alzheimer's disease: a dual pathway hypothesis[END_REF][START_REF] Castellani | Alzheimer disease pathology as a host response[END_REF][START_REF] Shioi | FAD mutants unable to increase neurotoxic Abeta 42 suggest that mutation effects on neurodegeneration may be independent of effects on Abeta[END_REF]. In such a scenario it is essential for immunotherapy to address both of these pathologies to be highly effective. In this review we discuss the various approaches which have been tried to target parenchymal amyloid deposition, vascular amyloid, oligomeric Aβ and tau related pathology.

Overview of Transgenic Mouse Models:

A) Models of Amyloid Plaque Deposition

The majority of successful models to generate Aβ deposits have used transgenes for human APP with missense mutations found in familial form of Alzheimer's disease (FAD) (see Table 1). The first transgenic model (PDAPP) was published by Games et al 1995 [35], used the Indiana mutation V717F, under the control of the PDGF promoter. This model shows a robust increase of Aβ deposition between 6 and 9 months, with the expression of APP being ~18 fold greater than endogenous levels. Minimal CAA can be found in these mice from about 18 months.

In old mice, the size of amyloid fibrils and the association of plaques to dystrophic neurites were

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shown to be analogous to the pathology detected in patients with AD [START_REF] Masliah | Neurofibrillary pathology in transgenic mice overexpressing V717F beta-amyloid precursor protein[END_REF]. The next AD transgenic model to be developed used the Swedish APP mutation (APP 695, K670N/M671L), under the control of the PrP mouse promoter [START_REF] Hsiao | Correlative memory deficits, Aβ elevation and amyloid plaques in transgenic mice[END_REF]. This model is referred to as Tg2576 and is the most widely studied Tg model of amyloid deposition. The brain Aβ levels start increasing at 6 months and Aβ parenchymal deposits start developing between 9 and 12 months [37; 38].

Congophilic angiopathy can be abundant in these mice at advanced ages (>18 months) [START_REF] Fryer | Apolipoprotein E markedly facilitates age-dependent cerebral amyloid angiopathy and spontaneous hemorrhage in amyloid precursor protein Transgenic mice[END_REF]. The over-expression of the mutant APP is about 5 fold. This model was extensively used to examine the role of inflammation, including astrogliosis [START_REF] Irizarry | APP Sw transgenic mice develop age-related Aβ deposits and neuropil abnormalities, but no neuronal loss in CA1[END_REF], microgliosis [START_REF] Frautschy | Microglial response to amyloid plaques in APPsw transgenic mice[END_REF], cytokine production [START_REF] Tan | Microglial activation resulting from CD40-CD40L interaction after betaamyloid stimulation[END_REF],

and oxidative stress [43; 44]. Several behavioral tests have shown age dependent impairment with cognitive tasks [18; 20; 37; 45-47]. Further models such as the APP23 were generated using the APP751 isoform, expressed under control of the murine thy-1 promoter with 7 fold overexpression [START_REF] Sturchler-Pierrat | Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology[END_REF]. Plaques are evident at 6 months of age in the hippocampus and cortex with older animals having plaques also in the thalamus and olfactory nucleus. Another line, APP22, expresses the Swedish (APP 695, mutated K670N/M671L) and London mutation (APP V717I) under control of thy-1 promoter. These Tg mice develop amyloid deposits at 8 months with two fold over-expression of APP. The transgenic CRND8 mouse line was generated under the control of the PrP promoter using the Swedish and the Indiana mutations (V717F) [START_REF] Chishti | Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695[END_REF]. Amyloid β deposits start quite early (from the age of 3 months) since the two mutations involve both the β and γ secretase APP cleavage sites. In these mice the pathology starts in the subiculum and the frontal cortex, followed by spread to the rest of the cortex and hippocampus and then the thalamus, striatum and cerebral vasculature. The mThy-1 APP 751 mouse [START_REF] Rockenstein | Early formation of mature amyloid-beta protein deposits in a mutant APP transgenic model depends on levels of Abeta(1-42)[END_REF] is analogues in terms of spread of the pathology to the CRND8 line. A comparison of transgenic mice overexpressing mutated (V717I and Swedish mutation) and wild -type human APP showed that

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cognitive deficits could be seen as early as 3 months, well before amyloid deposition suggesting that Aβ peptide soluble aggregates could be related to these phenotypic traits [START_REF] Moechars | Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain[END_REF]. Other single Tg lines were generated by expression of either the Flemish (APPA692G) or the Dutch mutation (APPE693Q) [START_REF] Kumar-Singh | Behavioral disturbances without amyloid deposits in mice overexpressing human amyloid precursor protein with Flemish (A692G) or Dutch (E693Q) mutation[END_REF]. These FAD mutations are associated with extensive CAA, but in the Tg mice they led to behavioral abnormalities in the absence of amyloid deposition.

The next step in creating Tg models of Aβ parenchymal deposition was to cross APP mutation lines with Tg mice expressing FAD linked presenilin (PS) 1 mutations. Over 160 mutations in PS-1, located on chromosome 14q, and 11 mutations on PS-2, located on chromosome 1, have been linked to FAD [START_REF] Brouwers | Molecular genetics of Alzheimer's disease: an update[END_REF]. PS is part of the γ-secretase complex and FAD linked mutations are associated with an increase in Aβ42 production [START_REF] Borchelt | Familial Alzheimer's disease-linked presenilin 1 variants elevate Aβ1-42/1-40 ratio in vitro and in vivo[END_REF][START_REF] Citron | Mutant presenilins of Alzheimer's disease increase production of 42residue amyloid β-protein in both transfected cells and transgenic mice[END_REF][START_REF] Wolfe | gamma-Secretase in biology and medicine[END_REF]. Crossing PS1 mutation mice with APP Tg models dramatically increases amyloid deposition, thought to be due to increased Aβ42 production [START_REF] Duff | Increased amyloid-β42(43) in brains of mice expressing mutant presenilin 1[END_REF][START_REF] Borchelt | Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins[END_REF][START_REF] Holcomb | Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes[END_REF][START_REF] Radde | Abeta42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology[END_REF]. In these various Tg models the ratio of Aβ 40 to 42, as well as the expression of "chaperone" proteins affects the type and distribution of amyloid deposits [START_REF] Mcgowan | A decade of modeling Alzheimer's disease in transgenic mice[END_REF][START_REF] Wilhelmus | Heat shock proteins and amateur chaperones in amyloid-Beta accumulation and clearance in Alzheimer's disease[END_REF][START_REF] Wisniewski | Preventing Aβ fibrillization and deposition: β-sheet breakers and pathological chaperone inhibitors[END_REF]. The critical role of Aβ42 in "seeding" amyloid deposition was illustrated in Tg mice that express Aβ1-40 or Aβ1-42 in the absence of human APP over-expression by the fusion of Aβ42 and Aβ40 to the C-terminal end of the BRI protein [START_REF] Mcgowan | Abeta42 is essential for parenchymal and vascular amyloid deposition in mice[END_REF]. Mutations in BRI are associated with the cerebral amyloidoses of British and Danish dementias [65; 66]. The Aβ1-42 expressing mice developed extensive cored plaques, diffuse plaques and CAA (at older ages), in contrast to the Aβ1-40 mice which did not develop amyloid deposits at any age [START_REF] Mcgowan | Abeta42 is essential for parenchymal and vascular amyloid deposition in mice[END_REF]. Although Aβ42 appears to be essential for seeding, Aβ40 can influence the amount of CAA, perhaps related to the fact that the major biochemical component of CAA is Aβ40 [START_REF] Prelli | Differences between vascular and plaque core amyloid in Alzheimer's disease[END_REF].

The expression of several Aβ binding proteins can also influence Aβ deposition and clearance. In particular the expression of apolipoprotein E (apoE) isoforms is a significant factor [63; 68]. Many studies have shown that the inheritance of the apoE4 allele is the single most important genetic risk factor for late-onset AD identified so far [63; 68]. The role of apoE in AD is complex with it being involved in both the aggregation state of Aβ and its clearance [62; 63; 68; 69]; however, one suggested role for apoE has been as a "pathological chaperone" that can induce a β-sheet conformation in Aβ [START_REF] Wisniewski | Apolipoprotein E: a pathological chaperone protein in patients with cerebral and systemic amyloid[END_REF][START_REF] Wisniewski | Apolipoprotein E: binding to soluble Alzheimer's beta-amyloid[END_REF][START_REF] Wisniewski | Alzheimer's disease and soluble Aβ[END_REF][START_REF] Sadowski | Blocking the apolipoproteinE/Amyloid β interaction reduces the parenchymal and vascular amyloid-β deposition and prevents memory deficit in AD transgenic mice[END_REF]. The critical role of apoE in Aβ deposition was shown when PDAPP and Tg2576 mice were crossed to murine apoE knock-out (KO) mice resulting in a complete lack of true amyloid plaques [74; 75]. The effects of different human apoE isoforms on Aβ deposition is somewhat more complex. In PDAPP mice expressing human apoE there was a marked delay in amyloid deposition compared to murine apoE expressing mice or apoE KO mice; however, the apoE4 expressing mice had increased levels of Aβ deposits compared to apoE3 mice [39; 76; 77]. These crosses have also been performed using mice that express human apoE isoforms under the control of endogenous mouse regulatory elements to examine this issue under more physiological conditions. When these knock-in apoE3 and apoE4 mice were crossed to Tg2576 mice or more recently in PDAPP mice, the apoE4 mice had significantly more parenchymal amyloid deposition and more CAA then the apoE3 mice [78; 79]. In humans the significance of apoE expression has been well documented by autopsy studies and by neuroimaging, showing that apoE4 expressing individuals have a higher amyloid burden and glucose utilization abnormalities even in presymptomatic stages of AD [START_REF] Reiman | Preclinical evidence of Alzheimer's disease in persons homozygous for the ε4 allele for apolipoprotein E[END_REF][START_REF] Reiman | Functional brain abnormalities in young adults at genetic risk for lateonset Alzheimer's dementia[END_REF][START_REF] Reiman | Fibrillar amyloid-beta burden in cognitively normal people at 3 levels of genetic risk for Alzheimer's disease[END_REF][START_REF] Kok | Apolipoprotein E-dependent accumulation of Alzheimer disease-related lesions begins in middle age[END_REF]. The expression of different apoE isoforms is an important consideration for immunomodulation, as current human data suggest that apoE4 carriers are more likely to experience inflammatory/hemorrhagic complications from immunomodulation and in some clinical trials apoE4 carriers are being excluded or are limited to lower doses of anti-Aβ antibodies [4; 5; 84].

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The effect of immunomodulation in different human apoE isotype expressing AD Tg models is a subject of on-going investigation.

One of the limitations of the existing Tg amyloid models is the relative lack of neuronal loss, contrasting to what is found in AD [41; 85; 86]. Three Tg lines with some neuronal loss are the APP23 mice [48; 87], a line with both the Swedish and London FAD APP mutations, along with two PS1 mutations (M233T/L235P) [START_REF] Casas | Massive CA1/2 neuronal loss with intraneuronal and N-terminal truncated Abeta42 accumulation in a novel Alzheimer transgenic model[END_REF], and a model that expresses five familial AD mutations, three of those within the APP gene (Swedish, Florida and London) and two within the PS1 gene (M146L and L286V) ("Vassar" or 5xFAD Tg mice) [START_REF] Oakley | Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation[END_REF]. In the APP23 mice there is limited neuronal loss in old animals in the direct vicinity of thioflavin positive amyloid plaques in the CA1 sector of the hippocampus. In the model described by Casas et al. [START_REF] Casas | Massive CA1/2 neuronal loss with intraneuronal and N-terminal truncated Abeta42 accumulation in a novel Alzheimer transgenic model[END_REF] there is ~50% loss in the CA1/2 sectors of the hippocampus correlating with the accumulation of intraneuronal Aβ rather then extracellular plaques. In the Vassar model, large pyramidal neurons in cortical layer 5 and subiculum are lost. Synaptic markers decrease as well in these models with age.

Many other Tg lines that do not develop extensive neuronal loss have shown age-associated synaptic degeneration [60; 85; 86; 90; 91]. In the widely used Tg2576 mice synaptophysin immunoreactivity loss occurs in 21-25 month old mice with associated electrophysiological changes suggesting synaptic dysfunction [START_REF] Spires | Dendritic spine abnormalities in amyloid precursor protein transgenic mice demonstrated by gene transfer and intravital multiphoton microscopy 7[END_REF]. In addition, elegant 3D multi-photon microscopy studies in Tg2576 and APP/PS1 Tg mice have shown neurite displacement, dendritic spine loss, thinning of dendrites and dendritic breakage adjacent to amyloid plaques suggesting that dense cored amyloid plaques do contribute at least partially to the cognitive abnormalities found in these mice [92; 93]. However, it is likely that small soluble aggregates of Aβ are a more important cause of neuronal dysfunction since cognitive deficits in these mouse lines occur well before the appearance of widespread cored amyloid plaques. This hypothesis is supported by a neurites adjacent to cored amyloid deposits [35; 37; 59].In particular the S199/S202 site recognized by the monoclonal Ab AT8 is found in many mice, but not the more AD specific phosphorylation sites such as those at residues S396, S404 or S422. An additional important factor determining the degree of neuronal loss and tau pathology in Tg AD models may be the expression of other genes that play a significant role in disease that are downstream from Aβ accumulation. For example, APPSw crossed with mice with a KO of nitric oxide synthetase 2 (NOS2) have 30% hippocampal neuronal loss that correlates with behavioral abnormalities [START_REF] Colton | NO synthase 2 (NOS2) deletion promotes multiple pathologies in a mouse model of Alzheimer's disease[END_REF].

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It has been recently shown that active vaccination with Aβ1-42 with Freund's adjuvant can prevent the neuronal loss found in these mice [START_REF] Wilcock | Amyloid reduction by amyloid-beta vaccination also reduces mouse tau pathology and protects from neuron loss in two mouse models of Alzheimer's disease[END_REF].

B) Transgenic Models of Vascular Amyloid Deposition:

The vast majority of AD patients have some degree of congophilic angiopathy (CAA) at autopsy, with approximately 20% of AD patients having "severe" CAA [START_REF] Jellinger | Alzheimer disease and cerebrovascular pathology: an update[END_REF]. Furthermore CAA is present in about 33% of cognitively normal elderly, control populations [START_REF] Zhang-Nunes | The cerebral beta-amyloid angiopathies: hereditary and sporadic[END_REF].The population based Honolulu Asia Aging Study has shown a significant correlation between cognition and the presence of CAA [START_REF] Pfeifer | Cerebral amyloid angiopathy and cognitive function: the HAAS autopsy study[END_REF]. Many of the Tg models of parenchymal amyloid discussed above and Interestingly in human autopsy tissue it is the capillary CAA level which correlates best with the presence of other AD related pathology [107]. When the APPSwDI mice were crossed with NOS2 KO mice there was an increase in amyloid deposition in association with a 30% neuronal loss in the hippocampus [START_REF] Wilcock | Amyloid reduction by amyloid-beta vaccination also reduces mouse tau pathology and protects from neuron loss in two mouse models of Alzheimer's disease[END_REF]. The Tg2576 and the APPSwDI mice with and without NOS2 KO have been shown to have significant blood-brain barrier abnormalities which mirror what is found in AD autopsy tissue [108]. Importantly, the neuronal loss and behavioral abnormalities have been shown to be preventable in the APPSwDI/NOS2 KO mice by Aβ1-42 vaccination [START_REF] Wilcock | Amyloid reduction by amyloid-beta vaccination also reduces mouse tau pathology and protects from neuron loss in two mouse models of Alzheimer's disease[END_REF].

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C) Transgenic Models of Tau Pathology:

A number of tau mutations have been reported to be associated with frontal temporal A difficulty with active immunization aimed at just the removal or prevention of parenchymal amyloid deposition is the autopsy data from the human trial. Despite the apparent success in amyloid clearance indicated by the limited autopsy data, the clinical cognitive benefits were very modest when the active vaccination group was compared to the placebo group [158].

No difference between the antibody responders and the placebo group was found on the ADAS-Cog, Disability Assessment for Dementia, Clinical Dementia Rating scale, MMSE or on the

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Clinical Global Impression of Change. It was only on a nine-item composite NTB that antibody responders had a slight benefit compared to the placebo group. These data can be used to suggest that vaccination in this cohort was started too late, at a time point when irreversible neurodegeneration has already occurred; hence, tau related pathology was largely unaffected by vaccination and thus the cognitive benefits were small. Alternatively it can be suggested that the amyloid cascade hypothesis must be an over-simplification of the pathogenesis of sporadic AD.

The latter view is supported by the follow up study of the 80 patients in the initial phase I AN1782 trial, of whom 8 came to autopsy [START_REF] Holmes | Long term effects of Aβ42 immunization in Alzheimer's disease: immune response, plaque removal and clinical function[END_REF]. This study showed that despite evidence of very significant amyloid plaque removal in 6 out of the 8 autopsy subjects, which correlated with the anti-Aβ titer, in the overall group there was no evidence of improved survival or attenuation of dementia progression [START_REF] Holmes | Long term effects of Aβ42 immunization in Alzheimer's disease: immune response, plaque removal and clinical function[END_REF].

Passive Immunization:

The easiest way to fulfill the goal of providing monoclonal anti-Aβ antibodies without risk of uncontrolled Th-1 mediated autoimmunity is by passive transfer. AD Tg model mice treated this way had a significantly reduced Aβ level and demonstrated cognitive benefit [139; 140; 174-176]. Potential problems with passive immunization include the need for repeated injections in a chronic disease, high cost, proper selection of antigen targets, blood-brain barrier penetration, the risk of hemorrhages and the development of an immune response to the injected antibodies. Several passive immunization trials are underway with the most advanced being the Phase III Bapineuzumab trial begun in Dec 2007 [START_REF] Wisniewski | Amyloid-β immunization for Alzheimer's disease[END_REF]. The Phase II trial using this anti-Aβ monoclonal antibody was a randomized, double-blind, placebo controlled trial testing 3 doses in received active agent and 28 placebos. Although the study did not attain statistical significance on the primary efficacy endpoint in the whole study population, in the sub-group of non-apoE4 carriers clinically significant benefits were documented using a number of scales including the Mini Mental State Examination (MMSE) and the Alzheimer's Disease Assessment Scale Battery, over the 18 month trial period. In addition among non-apoE4 carriers, evaluation of the MRI results showed less loss of brain volume in treated versus control patients. However, it was reported that some patients in the treatment group developed vasogenic edema, a significant adverse reaction. The Phase III trial is targeting to recruit 800 patients and run until December 2010.

A particular concern in association with passive immunization is cerebral microhemorrhage. The mechanism of this hemorrhage is thought to be related to Aβ deposition in the form of congophilic amyloid angiopathy (CAA) that causes degeneration of smooth muscle cells and weakening of the blood vessel wall. A number of reports have shown an increase in microhemorrhages in different AD mouse models following passive intra-peritoneal immunization with different monoclonal antibodies with high affinity for Aβ plaques and CAA [177][178][179]. Microhemorrhages following active immunization in animal models have also been reported in three studies [96; 180; 181]. In particular in the APPSwDI/NOS2 KO mouse model with the most extensive vascular amyloid, vaccination with Aβ1-42, while reducing the amyloid burden in association with behavioral benefits, led to a marked increase in microhemorrhages [START_REF] Wilcock | Amyloid reduction by amyloid-beta vaccination also reduces mouse tau pathology and protects from neuron loss in two mouse models of Alzheimer's disease[END_REF]. While this increase in microhemorrhages does not appear to be symptomatic in the mouse models this would be much less likely in humans. Strategically placed microhemorrhages in 30, reduced Aβ burden and improved cognition without increase in microhemorrhages [START_REF] Asuni | Aβ derivative vaccination in alum adjuvant prevents amyloid deposition and does not cause brain microhemorrhages in Alzheimer's model mice[END_REF].

In transgenic mouse models, Aβ antibodies can in theory both prevent the deposition of vascular amyloidand remove it, with the former scenario contributing to vascular repair, whereas the latter may potentially promote bleeding. However, clearance of parenchymal Aβ complicates the picture as several mouse studies suggest it may lead to enhanced vascular amyloidosis, thereby canceling any direct effect of the antibodies on Aβ deposits in vessels. On the other hand, the autopsies from the AN1792 trial indicated no clearance of vascular amyloid and in one of these cases numerous cortical bleeds were found, which are typically rare in AD patients, [150]. This is an important issue since CAA is present in virtually all AD cases, with approximately 20% of AD patients having "severe" CAA [START_REF] Jellinger | Alzheimer disease and cerebrovascular pathology: an update[END_REF]. The need for vascular repair and The repeats (3R, 4R) correspond to the microtubule binding region of protein tau. Stabilization of microtubules by tau is essential for the maintenance of neuronal cell morphology and for transport within the neuron . In addition, tau has other roles such as interactions with kinesin -1 and the complex dynactin/dynein [190; 191]. Tau also plays a crucial role in neuronal cell architecture by interacting with the plasma membrane as well as cytoskeleton proteins such as actin, spectrin and neurofilament proteins. Several mutations have been detected in the tau gene in FTDP-17 and other tauopathies, however none have been linked to AD [109]. Most of these mutations affect the binding of tau to microtubules or enhance the aggregation of tau into fibrils.

Other intronic mutations that affect the splicing of Exon 10 induce an increase of isoforms with 4 repeats. In AD, tau is hyperphosphorylated at many phosphorylation sites with 9 phosphates per molecule in comparison to normal brain tau that has 2 to 3 phosphorylated residues [192]. Other studies suggested that changes in tau splice forms are related to neurodegeneration. In some animal models expressing mutated tau there is an increase of 4R versus 3R tau [193]. The functional significance of a shift in the 3Rtau/4Rtau ratio remains unclear, but four -repeat tau binds microtubules with a higher affinity than three-repeat tau [194].

Normal tau and PHF tau differ in molecular weight and banding pattern. Normal tau has 6 bands between 45 and 68 kDa, while PHF-tau has 4 bands between 60 and 74 kDa [195;196].

The diversity of tau isoforms is related to various post-translational modifications such as phosphorylation, glycosylation, glycation, ubiquitination, and nitration [197]. Tau has multiple phosphorylation sites that have been characterized using phospho-tau dependant antibodies. 71 out of the 85 potential phosphorylated sites have been shown to be phosphorylated in physiological or pathological conditions [198; 199]. More than 20 protein kinases have been added potential advantage that it could be effective against both Aβ and tau related pathologies.

Studies to address whether stimulation of the innate immune system will be effective to inhibit tau related pathology are underway. Targeting only oligomeric Aβ or tau would avoid potential interference with these physiological functions. A novel immunotherapeutic approach is to target the shared abnormal β-sheet conformation of amyloid proteins using conformationally specific antibodies or active immunization that favors such a conformational response [223; 224; 231; 232]. Such an approach has the advantage that both Aβ and tau related pathologies would be addressed concurrently.

Immunization

Conclusions:

Many studies are underway in AD Tg mouse models aiming to improve the efficacy and safety profile of immunomodulation in patients. Approaches which concurrently address the three AD related pathologies: neuritic plaques, CAA and NFTs will have the greatest chance of success. Abnormal protein conformation is thought to be not only the underlying pathogenesis of AD but also of a long list of neurodegenerative conditions, such as prion disease, Parkinson's disease and Huntington's chorea [233], with immunomodulation having the potential to be a disease altering therapeutic approach for all these disorders [START_REF] Wisniewski | Vaccination as a therapeutic approach for Alzheimer's disease[END_REF]. For example it has been shown that prion directed mucosal vaccination can prevent infection from an exogenous source [234; 235]. The development of safe and effective immunomodulation methods in Tg models that direct the immune system to clear highly toxic abnormal oligomeric conformers that underlie the pathogenesis of multiple neurodegenerative diseases has the greatest potential to halt the progression of a wide spectrum of human neurodegenerative conditions.

  dementia with parkinsonism linked to chromosome 17, but none have been shown to produce AD pathology[109; 110]. A number of transgenic mice models that express human tau with FTDP-17 mutations have been produced[111]. Some of these mice display neurofibrillaryA C C E P T E DM A N U S C R I P T tangles, neuronal death and behavioral deficits[112][113][114][115][116][117][118][119][120][121][122][123][124] In most of these models there is presumably a disruption of axonal transport due to the mutated tau expression and aggregation that induces synaptic and neuronal loss. In addition one Tg mouse model that expresses a mutated (N279K) tau shows behavioral deficits without formation of NFTs or neuronal loss[125]. . The distribution of tau pathology in many of these tauopathy models is distinct from AD, with pre-tangles and tangles often prominent in regions such as the brain stem and spinal cord , with lesser involvement of hippocampal and cortical regions[111].An additional tau mutation model is the triple transgenic line (3xTg-AD) which expresses the PS1M146V, APPSwe and TauP301L transgenes. This model develops extracellular amyloid deposits from 6 months of age, and tau related pathology starting at 10 to12 months of age[11; 126], first in the hippocampus and then progresses to the cortex[126]. Deficits in long term synaptic plasticity correlate with accumulation of intraneuronal Aβ[START_REF] Oddo | Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction[END_REF]. Recently, it has been reported that spatial and contextual learning and memory was affected in the 3x Tg-AD mice in an age depend manner, and the accumulation of intraneuronal Aβ correlates with cognitive deficits [127]. Variability in the pathology of this model has been reported [128]. Some lines have delayed pathology, with plaque deposition starting at 15 months [128]. In order to generate a more ideal model for AD, other researchers have used a single wild-type human tau to generate a transgenic model; however, most of these models did not develop NFTs, with the exception of two models: One expressing ON3R wild-type tau with a few NFTs in aged animals [116] and another with abundant NFTs that expresses all 6 human tau isoforms on a knockout background for murine tau [129; 130], that develops age-related cognitive deficits [131]. The relative absence of tau pathology in mice that express wild-type human tau is likely due to the endogenous tau inhibiting the formation of NFT-like pathology.A C C E P T E D M A N U S C R I P T Recently, a new model has been generated by crossing htau mice (with expression of 6 tau isoforms) with mice expressing the M146L PS1 mutation [57]. This model maintained on a mouse tau knockout background shows earlier and more advanced tau pathology than the htau model, and develops more severe cognitive deficits as well [132; 133] Supporting data for AD immunotherapy initially showed that anti-Aβ antibodies could inhibit Aβ peptide fibrillization/oligomerizaton and prevent cell culture based neurotoxicity [134; 135]. This lead to vaccination of AD Tg mice with Aβ1-42 or Aβ homologous peptides coinjected with Freund's adjuvant which demonstrated striking reduction in Aβ deposition and as a consequence elimination of behavioral impairments [16-21]. This has also been done with Aβ encoding DNA vaccines and Aβ peptide fragment (EFRH) phage vaccines [136-138]. Similar effects on Aβ load and behavior have been demonstrated in AD Tg mice by peripheral injections of anti-Aβ monoclonal antibodies indicating that the therapeutic effect of the vaccine can be based primarily on eliciting a humoral response [139; 140]. In the initial preclinical studies no toxicity was evident in the treated mice; however, some investigators suggested that use of nonfibrillogenic, non-toxic Aβ homologous peptides along with approaches that stimulate primarily humoral, Th-2 immunity, in contrast to a primary Th-1 cell mediated response might reduce potential toxicity [141-143]. The dramatic biological effect of vaccination in preclinical testing encouraged Elan/Wyeth in April 2000 to launch a randomized, multiple-dose, dose-escalation, double-blind Phase I clinical trial with a vaccine designated as AN1792, which contained preaggregated Aβ1-42 and QS21 as an adjuvant. This type of vaccine design was aimed to induce a A C C E P T E D M A N U S C R I P T strong cell-mediated immune response, since QS21 is known to be a strong inducer of Th-1 lymphocytes [144]. The initial trial was conducted in the UK and involved 80 patients with mild to moderate AD [145]. This trial was designed to assess the antigenicity and the toxicity of multiple dose immunization with the full length Aβ1-42 peptide with the QS21. 53% of patients developed an anti-Aβ humoral response. During the later stages of the phase I trial, the emulsifier polysorbate 80 was added causing a more pronounced proinflammatory Th1 response [146]. In the subsequent phase IIa trial, begun in October 2001, 372 patients were enrolled with 300 receiving the aggregated Aβ1-42 (AN1792) with QS21 in the polysorbate 80 formulation. This trial was prematurely terminated in January 2002 when 6% of vaccinated patients manifested symptoms of acute meningoencephalitis (18 out of 298 subjects) [144; 147; 148]. Autopsies performed on a limited number of trial patients suggested that striking Aβ clearance of parenchymal plaques had occurred, similar to what had been reported in the animal studies, confirming the validity of this approach for amyloid clearance in humans[148-153]. In these cases extensive areas of cerebral cortex were devoid of plaques, with residual plaques having a "moth-eaten" appearance or persisting as "naked" dense cores. This amyloid clearance in most cases was in association with microglia that showed Aβ immunoreactivity, suggesting phagocytosis. Additional striking features were the persistence of amyloid in cerebral vessels in most patients, as well as limited effects on tau immunoreactive NFTs and neuropil threads in regions of cerebral cortex where plaque clearing had apparently occurred, compared to regions without clearing [151-154]. A recent of five patients who had participated in the phase IIa Elan trial showed a reduction in dystrophic neurites , as well as a reduction in PHF-1 immunolabeled NFTs, but no reduction in Alz50 or thioflavin-2 positive tangles [155]. Furthermore, more neuritic dystrophy was found associated with the dense cored plaques that remained in the A C C E P T E D M A N U S C R I P T vaccinated AD patients [155]. Hence, this initial vaccination approach has not sufficiently addressed either NFT related or vascular pathology. Some cases also showed a deleterious Tcell reaction surrounding some cerebral vessels, suggestive of an excessive Th-1 immune response. It appeared that the immune reaction triggered by AN1792 was a double-edge sword, where the benefits of a humoral response against Aβ were overshadowed in some individuals by a detrimental T cell mediated inflammatory response [148; 156]. The likely involvement of an excess cell mediated response in mediating toxicity was supported by analysis of peripheral blood mononuclear cells from trial patients, which were stimulated in vitro with the Aβ peptide, followed by quantification of cytokine secretion by enzyme-linked immunosorbent spot assay [146]. The cells of most responder trial patients mounted IL-2 and IFN-γ positive responses indicative of a Class II (CD4+) Th-1 type response [146]. Not all patients who received AN1792 responded with antibody production. The majority mounted a humoral response and showed a modest but statistically significant cognitive benefit demonstrated as an improvement on some cognitive testing scales compared to baseline and a slowed rate of disease progression compared to the patients who did not form antibodies [145; 157], although the effect on cognition was not clearly evident when subjects from several different sites were analyzed together [158].The follow-up data from the Zurich cohort, who are a subset of the Elan/Wyeth trial [157; 159], indicated that the vaccination approach may be beneficial for human AD patients. In agreement with the findings in the Zurich cohort, immune responders with high antibody titers in the multicenter cohort scored significantly better in composite scores of memory functions as compared to low-and non-responders or to the placebo group of patients [146]. Active vaccination approaches under development in Tg mouse models are aiming to avoid the excessive Th1 stimulation associated with the human trial. Concurrently the formulation of any active vaccine A C C E P T E D M A N U S C R I P T also has to overcome the problem of immunosenescence in the target patient population. One promising approach taken by several investigators is to alter the sequence of the Aβ peptide immunogen in order to remove or alter the major Th1 stimulator sites in the carboxyl terminus and the middle portion of Aβ, while focusing on the major Th2 stimulator site in the amino terminus [20; 21; 142; 160-162]. These Aβ homologous peptide immunogens can be combined with various co-stimulator epitopes. An example of this approach is a combination with a synthetic, non-natural Pan HLA DR-binding epitope PADRE [162] or linkage to viral-like particles (VLP) [163-165] to induce a primarily humoral immune response. These can be further combined with other immunostimulator carriers. For example the Aβ Th2 amino terminal epitope can be combined with PADRE and macrophage derived chemokine (MDC) in a DNA epitope vaccine to drive robust Th2 responses [166]. The choice of adjuvant is also an important consideration. The use of polysorbate 80 and a strong Th1 stimulate adjuvant (QS21), in the AN1792 trial is one likely contributing factor to the encephalitis in a minority of patients. Use of adjuvants such as alum which drive primarily a Th2 response is preferable [21; 167]. The route of immunization also plays an important role. Stimulating mucosal immunity by vaccinating nasally, via the gut or transcutaneously has been shown to be beneficial and may favor a Th2response[168][169][170][171][172][173]

  patients have been shown to correlate with cognitive deficits [182; 183].Interestingly, gentler clearance of Aβ with immunogens eliciting moderate antibody response may prevent further A C C E P T E D M A N U S C R I P T cerebral bleeding. For example, vaccination of the Tg2576 model with an Aβ derivative, K6Aβ1-

  regeneration during Aβ immunotherapy represents another argument for early treatment as well as an argument favoring subtle clearance over a longer time period.Immunotherapy Targeting Tau Pathology:Neurofibrillary tangles (NFTs) are intraneuronal inclusion bodies that consist of an accumulation of paired helical filaments (PHFs), which biochemically are mainly composed of abnormally phosphorylated tau. NFTs are a major pathologic hallmark of AD. Recently there has been an increased focus on phosphorylated tau as an immunotherapeutic target[184][185][186][187]. In the CNS, human tau is expressed in 6 isoforms arising from alternative mRNA splicing from a single gene on chromosome 17q21, containing16 Exons[188; 189]. The size range of the six isoforms is between 352 and 441 amino acids, which differ by the absence or presence of 29 (Exon 2or four semi-homologous repeat of 31 or 32 amino acids, encoded by Exon 10.

  phosphorylation of tau proteins, with glycogen synthase kinase-3β (GSK-3β) and cyclin-independent kinase (cdk5) thought to play the most important role in phosphorylation under pathological condition[198][199][200][201].Recently, it has been shown that active immunization of homozygous Tg JNPL3 P301L mice[202] with a phospho-tau peptide (containing the phosphorylated PHF-1 epitope Ser 396, Ser 404) for three to six months could reduce tau-related pathology[203]. This particular phospho-epitope was chosen because of its immunogenicity and prominent involvement in tau pathology[187]. Histological and biochemical analyses showed a reduction of aggregated tau in the brain and improved performance on motor tasks[203]. Motor impairments are prominent in this model as tau pathology is particularly advanced in brain stem and spinal cord.This study clearly documented that it is possible to reduce tau related pathology with active immunization.Purified antibodies from high titer mice entered the brain and neurons following an intra-carotid injection and bound to pathological tau.At first examination it is difficult to understand how an antibody response to a protein which is accumulating intra-cellularly can have beneficial effects. However, such an outcome is supported by a study of immunization in a Parkinson's disease transgenic mouse model with αsynuclein showing a reduction of intracellular α-synuclein aggregates[204]. Another study has shown that antibodies against Aβ can be internalized in AD neuronal culture models of Aβ accumulation and clear intra-neuronal Aβ aggregates via the endosomal-lysosomal pathway[205]. Furthermore recent evidence has shown that extracellular tau aggregates can be internalized and promote the fibrillization of intracellular full length tau in a tissue culture model[206] and that injection of fibrillar tau brain extract into the brains of transgenic wild-type expressing mice can induce the formation of human tau into filaments, as well as the spread ofA C C E P T E D M A N U S C R I P Tpathology from the site of injection into neighboring brain regions[207]. This type of "infectivity" of abnormal protein conformation from outside the cell has also been demonstrated for polyglutamine aggregates[208] and is well characterized in prion disease[209; 210]. Aβ has also been shown to have such "infectious" properties in vivo, being able to induce an acceleration of both further Aβ and tau related pathology[13-15; 211; 212]. Hence, if the spread of PHF pathology in AD can occur via such a prion like mechanism, anti-phosphorylated tau antibodies would not need to enter cells in order to be effective.Immunotherapy by Stimulation of the Innate Immune System.An alternative, non-mutually exclusive approach to enhance vaccine design is to stimulate innate immunity and enable microglia/macrophages to clear amyloid and/or NFTs. Over 20 yrs ago, H.Wisniewski noted that while brain-resident macrophages were unable to phagocytose amyloid, brain-infiltrating macrophages are plaque competent [213]. A number of recent studies suggest that only a small percentage of plaques are associated with peripheral origin macrophages and that these are required for plaque clearance [214-216]. Vaccination approaches based on this knowledge are now being developed. Stimulation of peripheral macrophages to enter the CNS and phagocytose amyloid has been achieved by stimulation of the Toll-like receptor 9 using unmethylated cytosine-guanosine oligonuceotides (CpG) [217; 218], via blockade of the CD40/CD40L interaction [219] and by blockade of TGFβ-Smad2/3 innate signaling pathway [220]. Significantly amyloid clearance by CpG stimulation was effective against CAA and parenchymal amyloid without any associated increase in cerebral microhemorrhages [217]. These innate immunity stimulatory approaches can be used alone or in combination with adaptive immunity stimulation. Stimulating the innate immune system has the A C C E P T E D M A N U S C R I P T

  Targeting Aβ and Tau Oligomers: Abundant evidence both in vivo and in vitro suggests that the most toxic species of Aβ are oligomers or Aβ derived diffusible ligands (ADDLs) [221; 222] with a similar line of evidence suggesting that tau oligomers are the most toxic form of phosphorylated tau [120; 186].Active vaccination or use of monoclonal antibodies that specifically target Aβ oligomers, tau oligomers or preferably both would be an ideal way to block AD related toxicity. A small number of pre-clinical studies targeting Aβ oligomers suggest that this methodology is potentially powerful and in the need of further development[223][224][225][226][227]. However, various proteins/peptides exist to some extent in a β-sheet conformation , which raises concerns about possible side effects of this otherwise promising approach[228]. On the other hand, indiscriminate clearance of all forms of Aβ may not be ideal as emerging evidence suggests that monomeric Aβ peptides have normal physiological functions in the brain such as neuroprotection and modulatingLTP [229; 230], with normally phosphorylated tau also having a role[185].

  R I P T series of Tg lines produced byMucke et al. [94] expressing wild-type and mutant APP which do not have amyloid deposition but have age-associated synaptophysin immunoreactivity loss that correlates with brain soluble Aβ levels. It should be mentioned though that overexpression of APP may conceivably have synaptotoxic effects by itself or through other APP fragments other than Aβ. The lack of more marked synaptic and neuronal loss in APP and APP/PS1 mice, unless multiple mutations are expressed, is likely related to the fact that none of these Tg lines develop significant tau related pathology. Phosphorylated tau epitopes have been noted in dystrophic

  Table 1 also have some vascular amyloid deposition with a variable age of onset [100].However, the Tg model with the most extensive vascular amyloid in association with lower levels of parenchymal amyloid is the APPSwDI mouse which incorporates 3 APP mutations:

	Swedish, Dutch E693Q and Iowa D694N [101-103]. These Dutch and Iowa mutations are
	associated with hereditary cerebral hemorrhage with amyloidosis (HCHWA) [104; 105]. The
	parenchymal Aβ deposits in these mice largely does not stain with Congo red and represents
	non-fibrillar, diffuse amyloid similar to the neuropathology of HCHWA-Dutch [106]. A caveat

with the APPSwDI mice is that the vascular amyloid deposition is mainly in capillaries, in contrast to AD CAA which is primarily in arterioles with less capillary involvement.

This manuscript was supported by NIH grants AG20245,AG15408, AG020197, AG032611 and the Alzheimer's Association.

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