Long-term outcomes after gene therapy for adenosine deaminase severe combined immune deficiency.

Patients lacking functional adenosine deaminase activity have severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy [GT]). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a phase 2 clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND (myeloproliferative sarcoma virus, negative control region deleted, dl587rev primer binding site)-ADA gammaretroviral vector (gRV) and infused following busulfan reduced-intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8 to 11 years. Nine of 10 patients have sufficient immune reconstitution to protect against serious infections and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of 9 evaluable patients with the highest gene marking and B-cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID but also highlight risks of genotoxicity with gRVs. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

Gene therapy for adenosine deaminase-deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans.

We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34(+) cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m(2)). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.

A Cross-Sectional Analysis of Dietary Intake and Nutritional Status of Patients on Haemodialysis Maintenance Therapy in a Country of Sub-Saharan Africa.

Malnutrition is common among dialysis patients, but there is insufficient literature on the problem from resource-poor settings of the sub-Saharan region. We conducted a cross-sectional investigation of dietary intake and nutritional status of haemodialysis (HD) patients to inform the current status of this population group in the region. HD patients aged ≥18 years, with dialysis vintage of ≥3 months, at one nephrology unit in Tanzania were assessed for their habitual diet and nutrient intake. Anthropometric measures and biochemistry tests were also performed. The diet was predominantly starchy food based, accompanied by a limited selection of vegetables. Fruits and animal protein were also minimally consumed (1 portion/day each). Fruit consumption was higher in females than males (median (25th, 75th) = 2 (1, 2.3) versus 0.5 (0, 1.7) portions, p = 0.008). More than 70% of participants had suboptimal measures for protein and energy intake, dietary iron, serum albumin, muscle mass, and hand grip strength (HGS). Inadequacies in protein and energy intake and dialysis clearance (URR) increased with the increase in body weight/BMI and other specific components (MAMC and FMI). Consumption of red meats correlated significantly and positively with serum creatinine (r = 0.46, p = 0.01), potassium (r = 0.39, p = 0.03), and HGS (r = 0.43, p = 0.02) and was approaching significance for a correlation with serum iron (r = 0.32, p = 0.07). C-RP correlated negatively with albumin concentration (r = -0.32, p = 0.02), and participants with C-RP within acceptable ranges had significantly higher levels of haemoglobin (p = 0.03, effect size = -0.28). URR correlated negatively with haemoglobin concentration (r = -0.36, p = 0.02). Patients will benefit from improved nutritional services that deliver individually tailored and culturally practical dietary advice to enable them to make informed food choices whilst optimizing disease management.

Clinical efficacy of gene-modified stem cells in adenosine deaminase-deficient immunodeficiency.

BACKGROUND: Autologous hematopoietic stem cell transplantation (HSCT) of gene-modified cells is an alternative to enzyme replacement therapy (ERT) and allogeneic HSCT that has shown clinical benefit for adenosine deaminase-deficient (ADA-deficient) SCID when combined with reduced intensity conditioning (RIC) and ERT cessation. Clinical safety and therapeutic efficacy were evaluated in a phase II study. METHODS: Ten subjects with confirmed ADA-deficient SCID and no available matched sibling or family donor were enrolled between 2009 and 2012 and received transplantation with autologous hematopoietic CD34+ cells that were modified with the human ADA cDNA (MND-ADA) γ-retroviral vector after conditioning with busulfan (90 mg/m2) and ERT cessation. Subjects were followed from 33 to 84 months at the time of data analysis. Safety of the procedure was assessed by recording the number of adverse events. Efficacy was assessed by measuring engraftment of gene-modified hematopoietic stem/progenitor cells, ADA gene expression, and immune reconstitution. RESULTS: With the exception of the oldest subject (15 years old at enrollment), all subjects remained off ERT with normalized peripheral blood mononuclear cell (PBMC) ADA activity, improved lymphocyte numbers, and normal proliferative responses to mitogens. Three of nine subjects were able to discontinue intravenous immunoglobulin replacement therapy. The MND-ADA vector was persistently detected in PBMCs (vector copy number [VCN] = 0.1-2.6) and granulocytes (VCN = 0.01-0.3) through the most recent visits at the time of this writing. No patient has developed a leukoproliferative disorder or other vector-related clinical complication since transplant. CONCLUSION: These results demonstrate clinical therapeutic efficacy from gene therapy for ADA-deficient SCID, with an excellent clinical safety profile. TRIAL REGISTRATION: ClinicalTrials.gov NCT00794508. FUNDING: Food and Drug Administration Office of Orphan Product Development award, RO1 FD003005; NHLBI awards, PO1 HL73104 and Z01 HG000122; UCLA Clinical and Translational Science Institute awards, UL1RR033176 and UL1TR000124.

Changes in the human plasma and urinary metabolome associated with acute dietary exposure to sucrose and the identification of potential biomarkers of sucrose intake.

SCOPE: The intake of sucrose is of public health concern but limited information is available on the metabolic effects of short-term exposure. Our aim was to use metabolomics to investigate the metabolic impact of acute sucrose exposure. METHODS AND RESULTS: We performed a randomized, parallel, single-dose feeding study on healthy females (n = 90, aged 29.9 ± 4.7 years, BMI 23.3 ± 2.5 kg/m(2) ) consuming either 0, 50, or 100 g sucrose in 500 mL water. Blood and urine samples were taken before and 24 h post sucrose intake. Urine and plasma samples underwent detailed metabolite profiling analysis using established protocols. Flow-injection electrospray MS fingerprinting analysis showed that 3 h after intake was the most informative time point in urine and plasma and out of 120 explanatory signals, highlighted 16 major metabolite signals in urine and 25 metabolite signals in plasma that were discriminatory and correlated with sucrose intake over time. The main confirmed metabolites positively correlated with intake were sucrose, fructose, and erythronic acid, while those negatively correlating with intake included fatty acids and derivatives, acyl-carnitines, and ketone bodies. GC-TOF-MS profiling analysis confirmed the fingerprinting data. CONCLUSION: Acute exposure to sucrose identified a number of metabolites correlated with sucrose intake and several compounds attributed to metabolic fasting.

Barriers to domestic violence help seeking: implications for intervention.

Data were obtained from the Chicago Women's Health Risk Study, in which 491 abused women were interviewed in public health centers and a hospital. Responses of a subgroup of these women who did not seek help through the identified interventions are examined. Common themes emerge across the help-seeking interventions studied: individual thresholds for the seriousness of the violence, a perceived requirement to end the relationship, and certain specific barriers. The responses provide a glimpse into abused women's reasons for not seeking particular interventions. Implications for public health, mental health, domestic violence counseling agencies, and the police are discussed.

Comparative measurements of total body water in healthy volunteers by online breath deuterium measurement and other near-subject methods.

BACKGROUND: We developed a new near-subject approach, using flowing afterglow-mass spectrometry (FA-MS) and deuterium dilution, which enables the immediate measurement of total body water (TBW) from single exhalations. OBJECTIVES: The objectives were to show the efficacy of the new FA-MS method in measuring TBW in healthy subjects and to compare these measurements with values derived from multifrequency bioelectrical impedance analysis, skinfold-thickness (SFT) measurements, and both recent and historical published regression equations. DESIGN: After baseline measurement of breath deuterium abundance, 24 healthy subjects ingested 0.3 g D(2)O/kg body wt. A second breath sample was taken after 3 h to measure the increase in deuterium, from which TBW was calculated. Bioelectrical impedance analysis was carried out with a multifrequency analyzer, and SFT was measured by a single trained observer. Methods were compared with the use of Pearson's correlation coefficient and Bland-Altman analyses. RESULTS: TBW measures obtained by all methods were highly correlated (r = 0.95-0.98, P < 0.001), especially those between FA-MS, SFT measurement, and recent regression equations. The mean values obtained were within 2% of those published for age-matched control subjects and varied by 1-6% when all methods were compared. Systematic bias was greatest when FA-MS was compared with bioelectrical impedance analysis, which tended to underestimate TBW in smaller, female subjects. No bias related to subject size was observed in a comparison of FA-MS with SFT measurement or with more recent regression equations. CONCLUSIONS: FA-MS is a simple and effective new approach to TBW measurement in healthy subjects. The difficulty of using population-derived equations to estimate TBW in individual subjects is emphasized.

Update on gene therapy of inherited immune deficiencies.

Gene therapy has been under development as a way to correct inborn errors for many years. Recently, patients with two forms of inherited severe combined immunodeficiency (SCID), adenosine deaminase and X-linked, treated by three different clinical investigative teams, have shown significant immune reconstitution leading to protective immunity. These advances irrefutably prove the concept that hematopoietic progenitor cell gene therapy can ameliorate these diseases. However, due to proviral insertional oncogenesis, two individuals in one of the X-SCID studies developed T-cell leukemia more than two years after the gene transfer. Depending upon the results of long-term follow-up, the successes together with the side effects highlight the relative merits of this therapeutic approach.

No influence of high- and low-carbohydrate diet on the oral glucose tolerance test in pregnancy.

OBJECTIVE: Our objective was to determine the influence of the carbohydrate content of the diet preceding the oral glucose tolerance test (OGTT) in pregnancy on the test results and to evaluate the necessity of the recommended preparatory high-carbohydrate diet. STUDY DESIGN: Thirty-four women from our outpatient clinic were enrolled in this prospective study. After giving informed consent, each women underwent a 90-min lesson (supervised by a dietary assistant) covering the carbohydrate, protein and fat content of different foods. Women were then randomized and in a crossover design started a diet with either a low or a high carbohydrate content. We were aiming at a carbohydrate intake of 40% in the low-carbohydrate week (LCH) and 50% in the high-carbohydrate week (HCH). Compliance was monitored by a detailed food diary which the women kept and which included the weight of the foods they consumed. RESULTS: The actual dietary intakes as calculated from the food diaries showed that the mean caloric intake was 1801 +/- 314 kcal in the LCH and 2118 +/- 312 kcal in the HCH week (<0.001). During the LCH diet, CH intake was 39 +/- 6.1% and 49 +/- 6.6% in the HCH week (P < 0.001). The carbohydrate intake per kilogram bodyweight was 30 +/- 5.3 kcal vs. 35 +/- 5.2 kcal (P < 0.001). The number of patients diagnosed with gestational diabetes was two in the LCH and three in the HCH week (not significant). The sum of the OGTT values (fasting, 1 h and 2 h) after the LCH was 18.9 +/- 2.1 mmol/l vs. 18.8 +/- 2.1 mmol/l after the HCH (P = 0.51). No differences could be found in both groups regarding the fasting, 1-h, or 2-h glucose values. Including patients with a CH difference of at least 5%, 10%, and 15% carbohydrate between the weeks, we still did not observe any differences in the OGTT sum. We also looked at a possible influence of the CH content of the diet on the day before the test and of the last meal before the OGTT results and observed there was none. CONCLUSION: This is the first study which has observed the influence of the previous day's meal on the test results. We conclude from our results that the carbohydrate percentage of the preparatory diet did not influence the results of an OGTT, even when we increased the difference in carbohydrate intake stepwise up to 15%. This might indicate that a preparatory diet before the OGTT is not necessary for women with normal nutritional behavior.

Relationship of demographic, dietary, and clinical factors to the hydration status of patients on peritoneal dialysis.

OBJECTIVES: To establish which clinical factors are associated with an increased proportion of extracellular fluid (ECF) in peritoneal dialysis (PD) patients. DESIGN: A single-center, cross-sectional analysis of 68 stable PD patients. METHOD: Bioelectrical impedance measurements (RJL, single frequency; RJL Systems, Clinton, Michigan, USA) of resistance and reactance were used to determine the proportion of ECF comprising total body water (TBW) in 68 stable PD patients attending for routine clearance and membrane studies. All patients underwent detailed dietetic, adequacy, and membrane function tests. Blood pressure and antihypertensive requirements were also documented. RESULTS: Significant gender differences in body composition were observed, such that women had lower absolute TBW and fat-free mass per kilogram body weight, but proportionately more ECF for a given TBW, mean ECF:TBW 0.5 +/- 0.03 versus 0.44 +/- 0.05, p < 0.005. In view of this, patients were split into two groups, defined as "over-" or "normally" hydrated, either by using the single discriminator (median ECF:TBW = 0.47) for the whole population, which resulted in groups distorted by gender, or by using different discriminators according to gender (women: 0.49, men 0.45). In both analyses, overhydrated patients were older, had significantly lower plasma albumin, less total fluid removal per kilogram body weight, and higher peritoneal solute transport. When split by a single discriminator, the overhydrated patients had lower sodium removal and significantly less intracellular fluid volume due to an excess of women in this group who also had less residual function and had been on dialysis longer. Using gender-specific discrimination, overhydrated patients were heavier due to expansion of the ECF volume: 20 +/- 4.1 L versus 16 +/- 3.3 L, p < 0.001. Stepwise multivariate analysis found age (p = 0.001), albumin (p = 0.009), and fluid losses per kilogram body weight (p = 0.025) to be independent predictors of gender-adjusted hydration status. Sodium intake did not vary according to hydration status. CONCLUSION: Gender influences the assessment of hydration status of PD patients when employing bioimpedance, such that women tend to have more ECF. Taking this into account, age, albumin, and achieved fluid removal appear to be independently associated with hydration status, whereas peritoneal solute transport is not. Advice on dietary sodium should take account of hydration status and achievable losses.

The disturbances of the zinc concentration in the blood in selected neoplasms.

A study was carried out on 92 patients (58 males and 34 females) aged 42-76 treated for malignant neoplasm of the gastrointestinal tract (54 patients with colorectal carcinoma, 38 with gastric carcinoma). In all patients, the zinc serum concentration was measured and the results obtained were referred to some epidemiological-clinical factors (sex, age, primary cause of cancer, the stage of clinical progression, and histological type). The results showed that the most pronounced hypozincemia occurred in male patients with mucous membrane carcinoma of the stomach.

A multi-centre, randomised trial to assess whether increased dietary fibre intake (using a fibre supplement or high-fibre foods) produces healthy bowel performance and reduces laxative requirement in free living patients on peritoneal dialysis.

BACKGROUND: Constipation is a significant problem for many patients on peritoneal dialysis (PD). Due in part to dietary restrictions it is a common cause of technique failure and poor dialysis efficacy. Both consequences have an economic cost as well as contributing to a poor patient experience. OBJECTIVE: This study aimed to investigate whether an appropriate daily bowel habit could be achieved through a higher fibre intake, minimal use of laxatives and with no adverse effect on potassium, phosphate and fluid balance. METHODS: One hundred and seven patients who had been on PD for at least three months were recruited from seven renal units. They were asked to record daily bowel habits (Bristol Stool Form Scale: BSFS) and laxative use for four weeks. From this group 41 suitable patients with regular laxative use were identified and invited to enter the Intervention stage, Stage 2. Patients were randomised into one of three intervention arms: high fibre supplement (HFS); high fibre diet (HFD) or placebo. RESULTS: During the intervention stage, intake of HFS increased significantly between week 1 and week 4 (p = 0.04) and in the placebo group between week 1 and week 3 (p = 0.02). There was no significant increase in fibre intake for those on the HFD. Laxative dose appeared to decrease in the HFS group (38%) and the HFD group (16%) but these changes were not significant when compared to the placebo. CONCLUSION: This study has confirmed the prevalence of laxative use amongst patients on PD and shown that fibre use can confer improvements in bowel function without affecting biochemistry.

Prolonged pancytopenia in a gene therapy patient with ADA-deficient SCID and trisomy 8 mosaicism: a case report.

A patient with adenosine deaminase-deficient severe combined immune deficiency (ADA-SCID) was enrolled in a study of retroviral-mediated ADA gene transfer to bone marrow hematopoietic stem cells. After the discontinuation of ADA enzyme replacement, busulfan (75 mg/m2) was administered for bone marrow cytoreduction, followed by infusion of autologous, gene-modified CD34+ cells. The expected myelosuppression developed after busulfan but then persisted, necessitating the administration of untransduced autologous bone marrow back-up at day 40. Because of sustained pancytopenia and negligible gene marking, diagnostic bone marrow biopsy and aspirate were performed at day 88. Analyses revealed hypocellular marrow and, unexpectedly, evidence of trisomy 8 in 21.6% of cells. Trisomy 8 mosaicism (T8M) was subsequently diagnosed by retrospective analysis of a pretreatment marrow sample that might have caused the lack of hematopoietic reconstitution. The confounding effects of this preexisting marrow cytogenetic abnormality on the response to gene transfer highlights another challenge of gene therapy with the use of autologous hematopoietic stem cells.

Selective survival of peripheral blood lymphocytes in children with HIV-1 following delivery of an anti-HIV gene to bone marrow CD34(+) cells.

Two HIV-1-infected children on antiretroviral therapy were enrolled into a clinical study of retroviral-mediated transfer of a gene that inhibits replication of HIV-1, targeting bone marrow CD34+ hematopoietic stem/progenitor cells. Two retroviral vectors were used, one encoding a "humanized" dominant-negative REV protein (huM10) that is a potent inhibitor of HIV-1 replication and one encoding a nontranslated marker gene (FX) to serve as an internal control for the level of gene marking. Peripheral blood mononuclear cells (PBMC) containing the huM10 gene or FX gene were detected by quantitative PCR at frequencies of approximately 1/10,000 in both subjects for the first 1-3 months following re-infusion of the gene-transduced bone marrow, but then were at or below the limits of detection (<1/1,000,000) at most times over 2 years. In one patient, a reappearance of PBMC containing the huM10 gene, but not the FX gene, occurred concomitant with a rise in the HIV-1 viral load during a period of nonadherence to the antiretroviral regimen. Unique clones of gene-marked PBMC were detected by LAM-PCR during the time of elevated HIV-1 levels. These findings indicate that there was a selective survival advantage for PBMC containing the huM10 gene during the time of increased HIV-1 load.

Gene therapy for inborn and acquired immune deficiency disorders.

Gene therapy has been under development as a way to correct inborn errors for over 20 years. Immune deficiencies are favorable candidates for gene therapy because of the potential selective advantage of genetically corrected cells in these conditions. Gene therapy for immune deficiencies has been the only application to show incontrovertible benefit in clinical trials to date. Despite the success in treating the underlying disease, there have been two cases of insertional oncogenesis reported in one of these early phase trials. Gene therapy approaches and clinical trials for several inborn as well as acquired immune deficiencies will be reviewed.