ALBERT

Description: The two-dimensional scattering of water waves over a finite region of arbitrarily varying topography linking two semi-infinite regions of constant depth is considered. Unlike many approaches to this problem, the formulation employed is exact in the context of linear theory, utilizing simple combinations of Green's functions appropriate to water of constant depth and the Cauchy-Riemann equations to derive a system of coupled integral equations for components of the fluid velocity at certain locations. Two cases arise, depending on whether the deepest point of the topography does or does not lie below the lower of the semi-infinite horizontal bed sections. In each, the reflected and transmitted wave amplitudes are related to the incoming wave amplitudes by a scattering matrix which is defined in terms of inner products involving the solution of the corresponding integral equation system. This solution is approximated by using the variational method in conjunction with a judicious choice of trial function which correctly models the fluid behaviour at the free surface and near the joins of the varying topography with the constant-depth sections, which may not be smooth. The numerical results are remarkably accurate, with just a two-term trial function giving three decimal places of accuracy in the reflection and transmission coefficients in most cases, whilst increasing the number of terms in the trial function results in rapid convergence. The method is applied to a range of examples.

Description: The behaviour of water waves over periodic beds is considered in a two-dimensional context and using linear theory. Three cases are investigated: the scattering of waves by a finite section of periodic topography; the Bloch problem for infinite periodic topography; and sloshing motions over periodic topography confined between vertical boundaries. Connections are established between these problems. A transfer matrix method incorporating evanescent modes is developed for the scattering problem, which reduces the computation to that required for a single period, without compromising full linear theory. The problem of the existence of Bloch waves can also be posed on a single period, leading to a close relationship between it and the scattering problem. Sloshing motions over periodic beds, which may be regarded as special cases of the Bloch problem, are also found to have a significant connection with wave scattering. Integral equations methods allied to the Galerkin approximation are used to resolve the three problems numerically. In particular, the full linear solution for Bragg resonance is presented, allowing the accuracy of existing approximations to this phenomenon to be assessed. The selection of results given illustrates the main features of the work.

Description: An investigation is carried out into the effect on wave propagation of an ice sheet of varying thickness floating on water of varying depth, in three dimensions. By deriving a variational principle equivalent to the governing equations of linear theory and invoking the mild-slope approximation in respect of the ice thickness and water depth variations, a simplified form of the problem is obtained from which the vertical coordinate is absent. Two situations are considered: the scattering of flexural-gravity waves by variations in the thickness of an infinite ice sheet and by depth variations; and the scattering of free-surface gravity waves by an ice sheet of finite extent and varying thickness, again incorporating arbitrary topography. Numerical methods are devised for the two-dimensional versions of these problems and a selection of results is presented. The variational approach that is developed can be used to implement more sophisticated approximations and is capable of producing the solution of full linear problems by taking a large enough basis in the Rayleigh-Ritz method. It is also applicable to other situations that involve wave scattering by a floating elastic sheet. © 2004 Cambridge University Press.

Description: The scattering and trapping of water waves by three-dimensional submerged topography, infinite and periodic in one horizontal coordinate and of finite extent in the other, is considered under the assumptions of linearized theory. The mild-slope approximation is used to reduce the governing boundary value problem to one involving a form of the Helmholtz equation in which the coefficient depends on the topography and is therefore spatially varying. Two problems are considered: The scattering by the topography of parallel-crested obliquely incident waves and the propagation of trapping modes along the periodic topography. Both problems are formulated in terms of 'domain' integral equations which are solved numerically. Trapped waves are found to exist over any periodic topography which is 'sufficiently' elevated above the unperturbed bed level. In particular, every periodic topography wholly elevated above that level supports trapped waves. Fundamental differences are shown to exist between these trapped waves and the analogous Rayleigh-Bloch waves which exist on periodic gratings in acoustic theory. Results computed for the scattering problem show that, remarkably, there exist zeros of transmission at discrete wavenumbers for any periodic bed elevation and for all incident wave angles. One implication of this property is that total reflection of an incident wave of a particular frequency will occur in a channel with a single symmetric elevation on the bed. The zeros of transmission in the scattering problem are shown to be related to the presence of a 'nearly trapped' mode in the corresponding homogeneous problem. The scattering of waves by multiple rows of periodic topography is also considered and it is shown how Bragg resonance - well-established in scattering of waves by two-dimensional ripple beds - occurs in modes other than the input mode.

Description: Introduction: FL is generally responsive to conventional-dose chemotherapy but long term disease-free survival (DFS) is uncommon. High-dose chemo-radiotherapy followed by ASCT has the potential to induce remission in this disease but the long-term benefit of this modality remains to be determined. Methods: Between 1990 and 2003, we transplanted 52 pts originally diagnosed with low-grade FL (31 grade 1, 21 grade 2). Twenty-five (48%) had biopsy-proven large cell transformation (FL grade 3 or diffuse large cell lymphoma) before ASCT. The median number of prior therapies was 2 (range: 1 to 7). Prior to ASCT, 45 pts (87%) were responsive to salvage therapy with 20 pts (38%) in CR. Five pts (10%) had chemo-resistant disease at the time of ASCT. High-dose regimens included BCNU-cyclophosphamide-etoposide (31%), melphalan/TBI (27%), and cyclophosphamide/TBI (25%). Thirty-eight pts (73%) received peripheral stem cells (PSCT) and 14 pts (27%) received autologous bone marrow (BM) with 4-hydroxyperoxycyclophosphamide (4-hc) purging in 9 cases (17%). The median age was 49 yrs (range: 29–65). Results: There was 1 treatment-related death during the first 100 days. After ASCT, 36 pts (69%) achieved a CR, 2 (4%) had a PR, and 7 (13%) had stable disease. Among those in CR, 20 (56%) had a CR pre-ASCT, 14 (41%) had a lesser response, and 1 (3%) was chemo-resistant. Median follow-up (f/u) of survivors was 5.3 yrs (range: 1.7 months to 12.4 yrs). The median overall survival (OS) has not yet been reached. The median event-free survival (EFS) is 3.4 yrs (range: 1.7 months to 12.4 yrs). Among complete responders, more than 50% are disease free at last follow-up (range 1.7 months to 12.1 yrs). Variables favorably affecting EFS and OS are age 〈 60 yrs (p = 0.007, 0.015 respectively), achievement of a CR after ASCT (p = 0.002, 0.001), absence of transformation (p = 0.038, 0.017), BM vs. PSCT (p = 0.042, 0.086), and 4-hc BM purging (p = 0.044, 0.059). Number of prior regimens, response prior to ASCT, type of preparative regimen, and addition of TBI, were not significantly associated with EFS, DFS, or OS. In multivariable analysis, achievement of CR after ASCT and age 〈 60 yrs are the only significant predictors of EFS and OS. Adjusted for age, 53% of pts with a CR after ASCT are alive and event-free at last f/u (range: 2.4 months to 12.4 yrs) (Figure 1). In contrast, the median EFS among pts without a CR is 0.5 yrs (range: 1.7 months to 5.3 yrs). Conclusion: ASCT is a reasonable therapeutic approach to FL, resulting in long term EFS for some pts, even with relapsed, refractory and/or transformed disease. In our experience, significant predictors of EFS and OS after ASCT are complete response and age

Description: CD4+CD25+ T-regulatory (Treg) cells have been shown to critically regulate self- and allograft tolerance in several model systems. Studies of human Treg cells have been restricted by the small number present in peripheral blood and their naturally hypoproliferative state. To better characterize Treg suppressor cell function, we determined methods for the isolation and expansion of these cells. Stringent magnetic microbead-based purification was required for potent suppressor cell line generation. Culture stimulation with cell-sized Dynabeads coated with anti-CD3 and anti-CD28 monoclonal antibodies, CD4+ feeder cells, and interleukin 2, provided for marked expansion in cell number (100-fold), with retention and enhancement of suppressor function. The potent Treg cell lines suppressed proliferation in dendritic cell-driven allo-mixed lymphocyte reaction (MLR) cultures by more than 90%. The Treg-derived suppressor cells functioned early in allo-MLR because expression of activation antigens and accumulation of cytokines was nearly completely prevented. Importantly, cultured Treg cells also suppressed activated and matured dendritic cell-driven responses. These results demonstrate that short-term suppressor cell lines can be generated, and they can express a very potent suppressive activity. This approach will enable more detailed biologic studies of Treg cells and facilitate the evaluation of cultured Treg cells as a novel form of immunosuppressive therapy. (Blood. 2004;104:453-461)

Description: IPI-504 is a novel inhibitor of Hsp90 based on the geldanamycin pharmacophore. When placed in rat, monkey, and human blood, IPI-504 rapidly converts to the known and well-studied compound 17-allylamino-17-demethoxy-geldanamycin (17-AAG). 17-AAG is the subject of multiple clinical trials for the treatment of hematologic and solid tumors. However, 17-AAG suffers from poor aqueous solubility necessitating the use of sub-optimal formulations to deliver this agent to patients. IPI-504 is over 1000-fold more soluble than 17-AAG in aqueous solution. In vitro, both 17-AAG and IPI-504 bind tightly to, and selectively inhibit Hsp90 derived from cancer cells. The cytotoxic effect of IPI-504, as well as its ability to stimulate the degradation of Hsp90 client proteins and increase the intracellular levels Hsp70, were monitored in two human multiple myeloma cells lines (RPMI-8226 and MM1.S). The effects of IPI-504 were compared to 17-AAG. We demonstrate that the actions of IPI-504 are bioequivalent to 17-AAG and that both compounds induce apoptosis in these cells and stimulate the degradation of HER2 and c-Raf. In addition, both agents stimulate Hsp70 protein levels. In all cases the EC50s are virtually the same for both molecules (~200–400 nM). Furthermore, IPI-504 inhibits the secretion of immunoglobulin light chain from the RPMI-8226 multiple myeloma cells (EC50 ~300 nM). Importantly, IPI-504 is active in tumor xenograft models of multiple myeloma. The data indicate that active metabolites of IPI-504 accumulate in these xenografts long after these metabolites are cleared from the plasma compartment, suggesting that they preferentially accumulate in tumor cells based on their increased affinity to Hsp90 derived from tumor cells. In conclusion, we have developed IPI-504 as a novel, potent inhibitor of Hsp90 with greatly increased solubility over 17-AAG, and that IPI-504 is an active anti-tumor agent in vitro and in vivo.

Description: Plasma hemoglobin (Hb) is a scavenger of nitric oxide (NO), which is a likely contributor to the pathogenesis and treatment of clinical abnormalities such as pulmonary hypertension and vasocclusive episodes in sickle cell anemia (SCA) and possibly in thalassemia syndromes, where pulmonary hypertension has been described most frequently in splenectomised patients with thalassemia intermedia (TI). Since plasma Hb consists of both free Hb and RBC-derived microvesicle Hb (Greenwalt. Vox Sang1991;61:14), the relationship between plasma Hb and circulating vesicles may be significant. We have measured plasma Hb and plasma vesicle levels in adults with SCA and thalassemia intermedia (TI). Patients with SCA were all untransfused and TI patients had no transfusions during the previous 3 months. Plasma Hb values in healthy adult controls (1.77±0.2, n=7) were significantly lower than in SCA (11.21±2.08mg/dl, n=15, p=0.003) or in splenectomised TI patients (48.46 ±3.66mg/dl, n=5, p=0.0038). Plasma Hb levels were significantly greater in TI as compared to levels in SCA (p=0.001). Vesicle numbers in SCA were 12.59±3.65 x103/ul (n=21, p〈 0.001, SCA v control) and in TI were 24.19 ±12.23 (n=7, p〈 0.001, TI v control). Thus both plasma Hb and circulating vesicle levels in SCA and TI were significantly greater than healthy controls. Plasma Hb was significantly greater in TI than in SCA and circulating vesicles markedly greater in TI than in SCA. Furthermore there was a significant correlation between plasma Hb and vesicle numbers in SCA, TI and normal controls (n=26, R2=0.59, p=0.0015). An analysis of splenectomised versus non-splenectomised TI patients revealed a further trend; both plasma Hb and vesicle numbers were significantly higher in splenectomised (n=5, n=7 respectively) than in non-splenectomised patients (n=4, n=4 respectively). Plasma Hb and vesicle numbers were respectively 48.5±3.6 and 24.19±12.2 in splenectomised patients compared to 17.18±5.58 (p=0.014) and 4.36±0.81 (p=0.008) in non-splenectomized TI patients. These findings show that raised plasma Hb levels are related to increments in vesicle numbers in TI and SCA. Splenectomy in TI, which is associated with increased risk of pulmonary hypertension and thrombosis, is associated with increased vesicle numbers and plasma Hb. We suggest that the scavenger characteristics of Hb containing vesicles for NO may differ from the scavenger characteristics of free Hb and thus require detailed study..

Description: High-dose melphalan followed by ASCT is a common component of the early treatment for patients with multiple myeloma. Daily subcutaneous injections of filgrastim (Neupogen) at 5 ug/kg/day until ANC 〉 500/ul are routinely administered at our center from day +4 following ASCT, in order to accelerate hematopoietic recovery and lessen neutropenic complications. Pegfilgrastim (Neulasta) as a single 6 mg fixed dose subcutaneous injection has been shown to have similar efficacy and ease of use when compared to filgrastim in the non-transplant setting, but little data is available in the transplant setting. We began using pegfilgrastim day +1 following ASCT for patients with multiple myeloma and performed a retrospective cohort study comparing those who received filgrastim (n=6) with those who received pegfilgrastim (n=11). Transplants occurred between July 2002 and January 2004 and included all patients transplanted for myeloma in that time period for whom sufficient data was available. All patients had at least 2 x 106 CD34+ cells/kg peripheral stem cells harvested after cytoxan and filgrastim mobilization. Main outcome measures were: days from stem cell infusion to WBC nadir, days to ANC〉500/ul, and days to ANC〉1000/ul. Subjects were excluded if CBCs were drawn less frequently than every four days. There were no significant differences between the filgrastim and pegfilgrastim groups with respect to the following demographic variables: age, gender, hemoglobin, creatinine, calcium, albumin and beta-2 microglobulin at diagnosis. The groups were also balanced with respect to SPEP, UPEP, presence of lytic lesions and number of prior lines of therapy. The median number of CD34+ cells infused was similar: 5.7 x 106 in the filgrastim group vs 4.8 x 106 in the pegfilgrastim group (p=0.28). After transplant, median number of days to WBC nadir in the filgrastim group (FG) was 7 (range 5–9) vs 6 (range 5–8) in the pegfilgrastim group (PG) (p=0.31). However, median number of days to ANC〉500/ul in the FG was 11.5 (range 11–17) vs 10 (range 9–12) for PG (p=0.02). Similarly, median number of days to ANC〉1000/ul was 12 (range 11–17) for FG vs 11 (range 10–13) for PG (p=0.03). Five of six patients in the FG had neutropenic fever after transplant, compared to five of eleven patients in the PG (p=0.30). Currently, no significant differences in infection or relapse rates between groups have been noted and there were no deaths in either group. In this retrospective cohort study, pegfilgrastim was safe and at least equivalent to filgrastim for accelerating hematopoiesis after ASCT for multiple myeloma. Furthermore, there was no significant difference in the incidence of neutropenic fever, infection and survival, suggesting a similar clinical utility.

Description: CD4+CD25+ regulatory T cells (Tr) are negative regulators of immune responses. Studies of human Tr are restricted by their small numbers in peripheral blood and their hypoproliferative state. A recently established method achieved in vitro expansion and generation of Tr cell lines (Godfrey et al; Blood 2004,104:453-61). This approach facilitates the evaluation of cultured Tr cells as a novel form of immunosuppressive therapy and provides a system for molecular analysis of Tr. Activation of Ras and MAP kinases is mandatory for IL-2 production, viability and cell cycle progression of T cells. In anergic T cells activation of these signaling events is impaired, whereas activation of Rap1 is retained. Subsequently, anergic cells have defective IL-2 production, impaired cell cycle progression, and increased susceptibility to apoptosis. In the current study, we sought to determine the signaling and biochemical properties of Tr. Human CD4+CD25+ (Tr) and control CD4+CD25− (Tc) cell lines were generated from human cord blood cells. We examined activation of Ras, Rap1 and MAP kinases as well as cell cycle progression and cell viability, in response to TCR/CD3-plus-CD28 mediated stimulation. Stimulation was done for 15 min, 2 and 16 hrs for assessment of signaling events or for 24, 48 and 72 hrs for assessment of cell cycle progression and viability. Although activation of Rap1 was not affected, activation of Ras was reduced in Tr as compared to Tc. Activation of JNK and Erk1/2 MAP kinases was also significantly impaired. Both Tr and Tc entered the cell cycle and expressed cyclin E and cyclin A at 24 and 48 hrs of culture. However, p27 was downregulated only in Tc and not in Tr and hyperphosphorylation of Rb, which is the hallmark of cell cycle progression, was detected only in the Tc and not in the Tr population. At 72 hrs of culture, expression of cyclin E and cyclin A was dramatically diminished in Tr whereas it remained unchanged in Tc. More strikingly, expression of p27 in Tr was increased to levels higher than background. Since Tr do not produce IL-2, we examined whether addition of exogenous IL-2 would downregulate p27 and rescue Tr from defective cell cycle progression, similarly to its effect on anergic cells. Addition of exogenous IL-2 resulted in decrease of p27, sustained increase of cyclin E and cyclin A and cell cycle progression. Besides inhibiting cell cycle progression, p27 also promotes apoptosis. Therefore, we examined whether Tr had a higher susceptibility to apoptosis. As determined by Annexin V staining, Tr had a high degree of apoptosis only at 72 hrs of culture, when p27 expression was highly upregulated. Exogenous IL-2 reversed both p27 upregulation and apoptosis. Addition of IL-2 to Tr, also resulted in sustained IL-2-receptor-mediated activation of Erk1/2 at levels equivalent to those of Tc. Thus Tr cells share many biochemical and molecular characteristics of anergy, including defective TCR/CD3-plus-CD28-mediated activation of Ras and MAP kinases, increased expression of p27, defective cell cycle progression and high susceptibility to apoptosis. Moreover, these results suggest that TCR/CD3-mediated and IL-2 receptor-mediated signals converge at the level of MAP kinases to determine the fate of Tr cells towards expansion or cell cycle arrest and subsequent apoptosis.