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.

OBJECTIVES:To evaluate weight loss maintenance after 4 years of nonpharmacological, nonsurgical obesity treatment, including a very low calorie diet (VLCD), diet and behavioural support. Furthermore, to assess weight development amongst completers and noncompleters beyond the active 4-year treatment period.DESIGN:Clinical trial.SETTING:Two Swedish county hospitals.SUBJECTS:A total of 113 patients were randomized to a 2-year treatment programme with or without an initial VLCD period. The 87 patients who completed the 2-year programme were offered the chance to continue a support programme for another 2 years. A total of 55 patients completed the entire 4-year programme.INTERVENTIONS:All the patients took part in a comprehensive support programme, including a hypocaloric diet and behavioural support, either as single treatment (non-VLCD group) or following the VLCD period (VLCD group).RESULTS:Significant 4-year weight losses were found in both groups, 7.6 +/- 12.2 kg (VLCD group) and 6.3 +/- 8.5 kg (non-VLCD group), (P < 0.01, n.s. between groups). The completers (n = 55) had maintained a weight loss of 3.3 +/- 10.7 kg (P < 0.05) 8 years after randomization. After 6 years, the noncompleters (n = 58) had gained 3.2 +/- 9.7 kg compared with baseline (P < 0.05). The difference in weight change between completers and non-completers was highly significant (P < 0.01).CONCLUSIONS:Highly significant weight losses can be maintained after a 4-year comprehensive treatment programme, including a hypocaloric diet and behavioural support. An initial VLCD period did not significantly affect the long-term weight loss. The posttreatment long-term weight loss was larger amongst completers than amongst patients who did not complete the treatment.

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 FcεR (common FcRγ-chain)- and FcγRII-deficient mice demonstrated that CTA1-DD exerted unaltered enhancing effects, indicating that FcγR-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.

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 round 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, father, it binds directly to B cells of all isotypes, including naive IgD(+) cells, No binding was observed to macrophages or dendritic cells. Immunizations in Fc is an element of R (common FcR gamma-chain)- and Fc gamma RII-deficient mice demonstrated that CTA1-DD exerted unaltered enhancing effects, indicating that Fc gamma R-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 Bel-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.

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.

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 21–60 y, BMI ≥30.0 kg/m 2 . 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.

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.