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The antibody-binding Fc γ receptors (FcγRs) trigger life-saving immune responses and many therapeutic monoclonal antibodies require FcγR engagement for full effect. One proven strategy to improve the efficacy of antibody therapies is to increase receptor binding affinity, in particular binding to FcγRIIIa/CD16a. Currently, affinities are measured using recombinantly-expressed soluble extracellular FcγR domains and CD16a-mediated antibody-dependent immune responses are characterized using cultured cells. It is notable that CD16a is highly processed with multiple N-glycosylation sites, and preventing individual N-glycan modifications affects affinity. Furthermore, multiple groups have demonstrated that CD16a N-glycan composition is variable and composition impacts antibody binding affinity. The level of N-glycosylation at each site is not known though computational prediction indicates low to moderate potential at each site based on primary sequence (40–70%). Here we quantify occupancy of the extracellular domains using complementary mass spectrometry-based methods. All five sites of the tighter-binding CD16a V158 allotype showed 65–100% N-glycan occupancy in proteomics-based experiments. These observations were confirmed using intact protein mass spectrometry that demonstrated the predominant species corresponded to CD16a V158 with five N-glycans, with a smaller contribution from CD16a with four N-glycans. Occupancy was likewise high for the membrane-bound receptor at all detected N-glycosylation sites using CD16a purified from cultured human natural killer cells. Occupancy of the N162 site, critical for antibody binding, appeared independent of N169 occupancy based on analysis of the T171A mutant protein. The weaker-binding CD16a F158 allotype showed higher occupancy of >93% at each site.

Background: Diagnosis of the HIV-associated lipodystrophy syndrome is based on clinical assessment, in lack of a consensus about case definition and reference methods. Three bedside methods were compared in their diagnostic value for lipodystrophy., Patients and Methods: Consecutive HIV-infected outpatients (n=278) were investigated, 128 of which also had data from 1997 available. Segmental bioelectrical impedance analysis (BIA) and waist, hip and thigh circumferences were performed. Changes in seven body regions were rated by physicians and patients using linear analogue scale assessment (LASA). Diagnostic cut-off values were searched by receiver operator characteristics., Results: Lipodystrophy was diagnosed in 85 patients (31%). BIA demonstrated higher fat-free mass in patients with lipodystrophy but not after controlling for body mass index and sex. Segmental BIA was not superior to whole body BIA in detecting lipodystrophy. Fat-free mass increased from 1997 to 1999 independent from lipodystrophy. Waist-hip and waist-thigh ratios were higher in patients with lipodystrophy. BIA, anthropometry and LASA did not provide sufficient diagnostic cut-off values for lipodystrophy. Agreement between methods, and between patient and physician rating, was poor., Conclusion: These methods do not fulfil the urgent need for quantitative diagnostic tools for lipodystrophy. BIA estimates of fat free mass may be biased by lipodystrophy, indicating a need for re-calibration in HIV infected populations., (Copyright 2001 Harcourt Publishers Ltd.)

Background: Highly active antiretroviral treatment (HAART) reduces the risk of wasting in HIV infection and may alter the prognostic weight of wasting. The phase angle from bioelectrical impedance analysis (BIA) can be interpreted as a surrogate marker for the catabolic reaction to chronic HIV infection and opportunistic disease., Objective: Our objective was to assess the prognostic ability of the phase angle in HIV-infected patients in the era of HAART., Design: Two cross-sectional observation studies were conducted in 1996 and 1997 at a German university outpatient HIV clinic. In the 1996 and 1997 cohorts, HAART was prescribed to 17 of 212 and 168 of 257 patients at baseline and to 179 of 212 and 234 of 257 patients during observation, respectively. Whole-body BIA was assessed at 50 KHz. Time to clinical progression and survival were calculated by using Cox proportional hazard models with time-dependent covariates. Median observation times were 1000 and 515 d for the 1996 and 1997 cohorts, respectively., Results: Higher phase angle was associated with a lower relative mortality risk, adjusted for viral load and CD4(+) cell count, of 0.49 (95% CI: 0.30, 0.81) per degree in 1996 and of 0.33 (95% CI: 0.18, 0.61) in 1997. The influence of phase angle on time to clinical progression, adjusted for viral load and CD4(+) cell count, was not significant in 1996 but the relative risk was 0.58 (0.36, 0.83) in 1997., Conclusion: Despite the favorable effects of HAART on the nutritional status of HIV-infected persons, low phase angle remains an independent adverse prognostic marker of clinical progression and survival.

Objective: Bioelectrical impedance analysis (BIA) is widely used as bedside assessment of body composition. Body cell mass (BCM) and intracellular water (ICW) are clinically important body compartments. Estimates of ICW obtained from BIA by different calculation approaches were compared to a reference method in male HIV-infected patients., Patients: Representative subsample of clinically stable HIV-infected outpatients, consisting of 42 men with a body mass index of 22.4 +/- 3.8 kg/m(2)(range, 13-l31 kg/m(2))., Methods: Total body potassium was assessed in a whole body counter, and compared to 50 kHz monofrequency BIA and multifrequency bioelectrical impedance spectroscopy. Six different prediction equations for ICW from BIA data were applied. Methods were compared by the Bland-Altman method., Results: BIA-derived ICW estimates explained 58% to 73% of the observed variance in ICW (TBK), but limits of confidence were wide (-16.6 to +18.2% for the best method). BIA overestimated low ICW (TBK) and underestimated high ICW (TBK) when normalized for weight or height. Mono- and multifrequency BIA were not different in precision but population-specific equations tended to narrower confidence limits., Conclusion: BIA is an unreliable method to estimate ICW in this population, in contrast to the better established estimation of total body water and extracellular water. Potassium depletion in severe malnutrition may contribute to this finding but a major part of the residual between methods remains unexplained., (Copyright 2000 Harcourt Publishers Ltd.)

Background and Aims: Shifts from intracellular to extracellular water are features of a catabolic reaction to sepsis. Bedside assessment of fluid shifts was carried out in neutropenic patients at high risk of systemic infection., Methods: Multifrequency bioelectrical impedance analysis was performed in 41 patients with leukemia or high-malignant lymphoma and chemotherapy-induced neutropenia., Results: Hydration was stable during afebrile periods except for transient intra- and extracellular dehydration after chemotherapy. The risk of over-hydration and dehydration increased 3-fold during fever. Over-hydration was more severe when occurring during fever. Extracellular water was highly variable and tended to increase, and intracellular water was slowly depleted. During sepsis, these alterations were enhanced. Changes in hydration status did not predict subsequent progression to sepsis because it developed more slowly than other symptoms of infection., Conclusions: Extracellular over-hydration and intracellular dehydration are observed in febrile infection in neutropenia, similar to severe sepsis. If the technical limits of bioelectrical impedance are taken into account, this method may be useful for non-invasive monitoring of these features of metabolic stress., (Copyright 2000 Harcourt Publishers Ltd.)

Aims: To compare nutritional counseling with and without oral supplements in HIV-infected patients with recent weight loss., Design: Randomized non-blinded controlled trial, stratified for change in antiretroviral treatment at baseline., Patients: HIV-infected patients with recent weight loss (> 5% of total, and >3% in the last month)., Intervention: Nutritional counseling to increase dietary intake by 600 kcal/day over 8 weeks; in group A (n=24) by normal food, and in group B (n=26) by a range of fortified drink supplements with a calorific value of 0.6 to 1.5 kcal/ml., Methods: Body composition by bioelectrical impedance analysis, dietary intake by 24 h recall., Results: Fat free mass increased from baseline to week 8 (P<0.05) with no difference between groups A and B (P=0.97). Body cell mass and weight gain were not significant and equal between groups. Assessed at weeks 2 and 4, group B patients consumed 11 +/- 6 kcal/kg as supplements, and their total energy intake was 6 kcal/kg higher than in group A (P<0.01). Total energy intake was not different between groups at weeks 6 and 8., Discussion: Nutritional counseling and oral supplements are both feasible methods to restore food energy intake in malnourished HIV-infected patients. Although normal food intake is partially replaced, oral supplements may improve the adherence to a weight gain regimen., (Copyright 1999 Harcourt Publishers Ltd.)

Background: Triple antiretroviral treatment including protease inhibitors (PIs) delays the clinical progression of HIV infection and may thus reduce the risk of malnutrition. However, fat redistribution (lipodystrophy) was recognized recently as a metabolic side effect of PIs., Objective: The study aimed to assess the effect of triple antiretroviral treatment on body composition and on the prevalence of malnutrition., Design: Two cross-sectional studies, 1 in 1996 (t96; n = 247) and 1 in 1997 (t97; n = 266), were conducted in HIV-infected outpatients. Among patients who participated in both studies, 111 patients started a new antiretroviral treatment including a PI between t96 and t97 and were studied longitudinally. Total body water (TBW), intracellular water (ICW), extracellular water (ECW), and fat mass were estimated by monofrequency bioelectrical impedance analysis (BIA)., Results: Prevalence of malnutrition was reduced by 30-50% from t96 to t97, depending on the definition used. In the longitudinal study, TBW and the ratio between ICW and ECW increased and fat mass decreased (P < 0.001). BIA indicated a greater increase in ICW in 23 (21%) patients with clinically apparent fat redistribution than in patients without this syndrome, but estimates of fat mass changes were not significantly different., Conclusions: Triple antiretroviral treatment may protect HIV-infected patients against the development of malnutrition. Whole-body BIA data suggest an increase in appendicular body cell mass associated with improved antiretroviral treatment. However, the method is unreliable in detecting fat redistribution, and current prediction equations will need to be recalibrated for HIV-infected patients receiving highly active antiretroviral treatment.