Comparison of filgrastim, pegfilgrastim, and lipegfilgrastim added to chemotherapy for mobilization of CD34+ cells in non-Hodgkin lymphoma patients.

BACKGROUND: Data are limited on the long-acting granulocyte-colony stimulating factors (G-CSFs) pegfilgrastim (PEG) and lipegfilgrastim (LIPEG) compared with filgrastim (FIL) regarding the mobilization efficiency of CD34+ cells, graft cellular composition, and engraftment. STUDY DESIGN AND METHODS: In this prospective nonrandomized study, 36 patients with non-Hodgkin lymphoma received FIL, 67 received PEG, and 16 patients received LIPEG as a cytokine after chemotherapy. We analyzed the mobilization and collection of CD34+ cells, cellular composition of blood grafts, and hematologic recovery after auto-SCT according to the type of G-CSF used. RESULTS: Patients in the LIPEG group had fewer apheresis sessions (1 vs. 2, p = 0.021 for FIL and p = 0.111 for PEG) as well as higher median blood CD34+ cell counts at the start of the first apheresis (LIPEG 74 × 106 /L vs. FIL 31 × 106 /L, p = 0.084 or PEG 27 × 106 /L, p = 0.021) and CD34+ yields of the first apheresis (FIL 5.1 × 106 /kg vs. FIL 2.3 × 106 /kg, p = 0.105 or PEG 1.8 × 106 /kg, p = 0.012). Also, the costs associated with G-CSF mobilization and apheresis were lower in the LIPEG group. The graft composition was comparable except for the higher infused CD34+ cell counts in the LIPEG group. The engraftment kinetics were significantly slower in the FIL group. CONCLUSION: LIPEG appears to be more efficient compared with PEG after chemotherapy to mobilize CD34+ cells for auto-SCT demonstrated as fewer sessions of aphereses needed as well as 2.8-fold CD34+ cell yields on the first apheresis day. Early hematologic recovery was more rapid in the LIPEG group. Thus further studies on LIPEG in the mobilization setting are warranted.

Preemptive plerixafor injection added to pegfilgrastim after chemotherapy in non-Hodgkin lymphoma patients mobilizing poorly.

Filgrastim is usually combined with chemotherapy to mobilize hematopoietic progenitor cells in non-Hodgkin lymphoma (NHL) patients. Limited information is available on the efficacy of a preemptive plerixafor (PLER) injection in poor mobilizers after chemotherapy and pegfilgrastim. In this prospective study, 72 patients with NHL received chemotherapy plus pegfilgrastim, and 25 hard-to-mobilize patients received also PLER. The usefulness and efficacy of our previously developed algorithm for PLER use in pegfilgrastim-containing mobilization regimen were evaluated as well as the graft cellular composition, hematological recovery, and outcome after autologous stem cell transplantation (auto-SCT) according to the PLER use. A median 3.4-fold increase in blood CD34+ cell counts was achieved after the first PLER dose. The minimum collection target was achieved in the first mobilization attempt in 66/72 patients (92%) and 68 patients (94%) proceeded to auto-SCT. An algorithm for PLER use was fulfilled in 76% of the poor mobilizers. Absolute numbers of T-lymphocytes and NK cells were significantly higher in the PLER group, whereas the number of CD34+ cells collected was significantly lower. Early neutrophil engraftment was slower in the PLER group, otherwise hematological recovery was comparable within 12 months from auto-SCT. No difference was observed in survival according to the PLER use. Chemotherapy plus pegfilgrastim combined with preemptive PLER injection is an effective and convenient approach to minimize collection failures in NHL patients intended for auto-SCT. A significant effect of PLER on the graft cellular composition was observed, but no difference in outcome after auto-SCT was detected.

Intermittent versus on-demand use of a very low calorie diet: a randomized 2-year clinical trial.

OBJECTIVES: To compare two different very low calorie diet (VLCD)-based weight maintenance strategies. DESIGN AND SETTING: A randomized 2-year clinical trial performed at the Department of Body Composition and Metabolism, Sahlgrenska University Hospital, Sweden. SUBJECTS: A total of 334 patients, body mass index (BMI) >30 kg m-2, aged 18-60 years. INTERVENTIONS: All the patients started with 16 VLCD weeks. Subjects in the intermittent group were then scheduled to use VLCD for 2 weeks every third month, whilst patients in the on-demand group were instructed to use VLCD whenever their body weight passed an individualized cut-off level. Irrespective of the treatment group, all the subjects were recommended a hypocaloric diet during VLCD-free periods. MAIN OUTCOME MEASURES: Changes in body weight, body composition, anthropometric variables and cardiovascular risk factors. RESULTS: Completers in both groups maintained highly significant weight losses after 2 years: 7.0 +/- 11.0 kg (6.2 +/- 9.5%) in the intermittent group and 9.1 +/- 9.7 kg (7.7 +/- 8.1%) in the on-demand group (P < 0.001, ns between groups). Male completers in the on-demand group lost significantly more weight than men in the intermittent group, 14.5 +/- 11.0 kg vs. 4.0 +/- 10.5 kg, respectively (P < 0.01). Most cardiovascular risk factors improved during the first year, whilst anthropometric measures, insulin, HDL- and LDL-cholesterol were also significantly improved after 2 years of treatment. CONCLUSION: Clinically significant weight reductions were achieved after 2 years of VLCD-based treatment. The structure of VLCD treatment during the maintenance phase did not affect weight loss in the total study population, whilst male subjects might benefit from the VLCD on-demand strategy.

The ADP-ribosylating CTA1-DD adjuvant enhances T cell-dependent and independent responses by direct action on B cells involving anti-apoptotic Bcl-2- and germinal center-promoting effects.

We recently developed a novel immunomodulating gene fusion protein, CTA1-DD, that combines the ADP-ribosylating ability of cholera toxin (CT) with a dimer of an Ig-binding fragment, D, of Staphylococcus aureus protein A. The CTA1-DD adjuvant was found to be nontoxic and greatly augmented T cell-dependent responses to soluble protein Ags after systemic as well as mucosal immunizations. Here we show that CTA1-DD does not appear to form immune complexes or bind to soluble Ig following injections, but, rather, it binds directly to B cells of all isotypes, including naive IgD+ cells. No binding was observed to macrophages or dendritic cells. Immunizations in FcepsilonR (common FcRgamma-chain)- and FcgammaRII-deficient mice demonstrated that CTA1-DD exerted unaltered enhancing effects, indicating that FcgammaR-expressing cells are not required for the adjuvant function. Whereas CT failed to augment Ab responses to high m.w. dextran B512 in athymic mice, CTA1-DD was highly efficient, demonstrating that T cell-independent responses were also enhanced by this adjuvant. In normal mice both CT and CTA1-DD, but not the enzymatically inactive CTA1-R7K-DD mutant, were efficient enhancers of T cell-dependent as well as T cell-independent responses, and both promoted germinal center formation following immunizations. Although CT augmented apoptosis in Ag receptor-activated B cells, CTA1-DD strongly counteracted apoptosis by inducing Bcl-2 in a dose-dependent manner, a mechanism that was independent of the CD19 coreceptor. However, in the presence of CD40 stimulation, apoptosis was low and unaffected by CT, suggesting that the adjuvant effect of CT is dependent on the presence of activated CD40 ligand-expressing T cells.

Hydrophobicity engineering of cholera toxin A1 subunit in the strong adjuvant fusion protein CTA1-DD.

Protein engineering of the cholera toxin A1 subunit (CTA1) fused to a dimer of the Ig-binding D-region of Staphylococcus aureus protein A (DD) was employed to investigate the effect of specific amino acid changes on solubility, stability, enzymatic activity and capacity to act as an adjuvant in vivo. A series of CTA1-DD analogues were selected by a rational modeling approach, in which surface-exposed hydrophobic amino acids of CTA1 were exchanged for hydrophilic counterparts modeled for best structural fit. Of six different mutants initially produced, two analogues, CTA1Phe132Ser-DD and CTA1Pro185Gln-DD, were demonstrated to have 50 and 70% increased solubility, respectively, at neutral pH. The double mutant CTA1Phe132Ser/Pro185Gln-DD was at least threefold more soluble, demonstrating an additive effect of the two mutations. Only the Phe132Ser analogue retained full biological activity and stability compared with the native CTA1-DD fusion protein. Two mutants, Pro185Gln and Phe31His mutations, exhibited unaltered ADP-ribosyltransferase activity in vitro, but demonstrated markedly reduced adjuvant function. Since the Pro185 and Phe31 amino acids are located in close vicinity on the distal side of the molecule relative to the enzymatically active cleft, it is conceivable that this region is involved in mediating a biological function, separate from the enzymatic activity but intrinsic to the adjuvant activity of CTA1.

Effects on body weight of strict or liberal adherence to an initial period of VLCD treatment. A randomised, one-year clinical trial of obese subjects.

OBJECTIVES: To examine the impact on early and late weight loss of three different, initial very low calorie diet (VLCD) approaches in a one-year obesity treatment program. DESIGN: Randomised clinical trial. SUBJECTS: 121 obese subjects, aged 21-60y, BMI > or = 30.0kg/m2. INTERVENTIONS: The VLCD-strict group was prescribed a strict outpatient VLCD for 16 weeks, followed by a 36-week hypocaloric diet. The VLCD-mw group received the same treatment, but were hospitalised in a metabolic ward for the initial week. The VLCD-plus group was allowed two small meals weekly, but received otherwise the same recommendations as the VLCD-strict group. RESULTS: After 16 weeks, there was no difference in weight loss between the treatment groups in the intent-to-treat population, while among completers, the weight loss was about 7 kg larger in the VLCD-strict group compared to the VLCD-plus group (P < 0.05). At one year, these groups differed by approximately 4 kg, both according to intention-to-treat and among completers (P < 0.05, both differences). These differences were more prominent among females. The weight reduction in the VLCD-mw group was generally not superior to the VLCD-strict group. CONCLUSIONS: In the short-term, strict VLCD only reduced weight better than a liberal VLCD approach among completers. However, after one year, a strict VLCD regimen seemed beneficial compared to a liberal VLCD for all patients. There was no extra weight loss if the VLCD period was initiated on a metabolic ward.

Adjuvanticity of the cholera toxin A1-based gene fusion protein, CTA1-DD, is critically dependent on the ADP-ribosyltransferase and Ig-binding activity.

The ADP-ribosylating enterotoxins, cholera toxin (CT) and Escherichia coli heat-labile toxin, are among the most powerful immunogens and adjuvants yet described. An innate problem, however, is their strong toxic effects, largely due to their promiscuous binding to all nucleated cells via their B subunits. Notwithstanding this, their exceptional immunomodulating ability is attracting increasing attention for use in systemic and mucosal vaccines. Whereas others have separated adjuvanticity from toxicity by disrupting the enzymatic activity of the A1 subunit by site-directed mutagenesis, we have constructed a nontoxic molecule that combines the full enzymatic activity of the A1 subunit with a B cell targeting moiety in a gene fusion protein, the CTA1-DD adjuvant. Despite its more selective binding properties, we found comparable adjuvant effects of the novel CTA1-DD adjuvant to that of CT. Here we unequivocally demonstrate, using a panel of mutant CTA1-DD molecules, that the immunomodulating ability of CTA1-DD is dependent on both an intact enzymatic activity and the Ig-binding ability of the DD dimer. Both agents, CT and CTA1-DD, ADP-ribosylate intact B cells. However, contrary to CT, no increase in intracellular cyclic AMP in the targeted cells was detected, suggesting that cyclic AMP may not be important for adjuvanticity. Most remarkably, CTA1-DD achieves similar immunomodulating effects to CT using a ganglioside-GM1 receptor-independent pathway for internalization.

A novel concept in mucosal adjuvanticity: the CTA1-DD adjuvant is a B cell-targeted fusion protein that incorporates the enzymatically active cholera toxin A1 subunit.

A promising novel concept in mucosal adjuvant research is demonstrated here. The adjuvant and toxic effects of the cholera toxin (CT) have been successfully separated in a gene fusion protein, CTA1-DD. This protein consists of the ADP-ribosylating A1 subunit of CT linked to a synthetic analogue of protein A. The CTA1-DD protein was found to exert comparable adjuvant activity to that of CT after systemic as well as mucosal immunizations with soluble protein antigens, such as KLH or ovalbumin (OVA). However, contrary to CT it was completely non-toxic. The CTA1-DD approach to the construction of a potential vaccine adjuvant is unique and highly promising. Conceptually, the CTA1-DD fusion protein demonstrates that: (i) contrary to CT the CTA1-DD is a highly targeted adjuvant, directed to B cells and possibly other antigen-presenting cells; (ii) it is possible to introduce ADP-ribosyltransferase activity into cells via an alternative pathway to the GM1 receptor pathway used by CTB; (iii) the adjuvant effect of CTA1-DD, and possibly also of CT, depend on the enzymatic activity; and (iv) one possible mechanism, shared by CT, that may explain the adjuvant effect of CTA1-DD is its ability to induce expression of the costimulatory molecule CD86 on B cells.

Genetically engineered nontoxic vaccine adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit.

Cholera toxin (CT) is an exceptionally potent adjuvant but, unfortunately, also very toxic. Here we present a powerful new approach to separate toxicity from adjuvanticity by constructing a fusion protein that combines the enzymatically active cholera toxin A1 subunit (CTA1) with targeting to B cells. The CTA1 was genetically linked at its C-terminal end to two Ig-binding domains, DD, of staphylococcal protein A and produced in Escherichia coli. The highly purified, monomeric CTA1-DD fusion protein, with a molecular mass of 37 kDa, was found to exhibit strong ADP-ribosyltransferase activity and bound, via the DD moiety, to both Fc and Fab fragments and to all IgG subclasses--IgE, IgA, and IgM. After i.v. injection of the fusion protein, FACS analysis revealed binding of CTA1-DD to splenic IgM+ B cells, but not CD3+ T cells, indicating cell-specific targeting in vivo. Strikingly, we found that the adjuvant ability of CTA1-DD to enhance systemic IgG as well as mucosal IgA responses to the unrelated Ags, OVA, or keyhole limpet hemocyanin, administered i.v or intranasally, was comparable to that of intact CT. In addition, the enhancing effect on specific IgG1, IgG2a, and IgG2b responses mimicked that of CT and suggested involvement of both Th1 and Th2 CD4+ T cell activity. The CTA1-DD, as well as CT, up-regulated expression of the CD80 and CD86 molecules on the targeted B cells, indicating that enhanced T cell costimulation may be responsible for the adjuvant effect. Contrary to CT, however, CTA1-DD was completely nontoxic. Thus, the CTA1-DD adjuvant should find general applicability in systemic and mucosal vaccines, and the strategy used may also be explored for other regimens requiring targeted immunomodulation.

Andrena wilkella male bees discriminate between enantiomers of cephalic secretion components.

Diastereomers of the spiroacetal, 2,8-dimethyl-1,7-dioxaspiro [5.5]undecane, represent main components of the cephalic secretion from males of the solitary bee,Andrena wilkella. The major compound proved to be of high enantiomeric purity, showing (2S,6R,8S) configuration. Only the naturally occurring enantiomer attracted patrolling males in the field; its antipode was behaviorally inactive and in a racemic mixture did not inhibit response. The (E,Z) diastereomers were also found to be almost inactive. EAG studies gave the same result as the behavioral tests. The biological function of the spiroacetal is discussed in view of the evolution of the mating behavior inA. wilkella.