Calprotectin as potential novel biomarker in myasthenia gravis.

Myasthenia gravis (MG) is the most common autoimmune disease affecting the neuromuscular junction by specific autoantibodies. The etiology of MG and its heterogeneity in clinical courses are poorly understood, although it was recently shown that gut microbial dysbiosis plays a critical role. Since levels of Calprotectin (CLP) seem to correlate with level of dysbiosis, we hypothesize that CLP may serve as potential disease activity biomarker in MG. Sera from 251 patients with MG and 90 controls were analyzed in an explorative, cross-sectional design. Prospectively, we tested CLP levels in MG patients up to 3 years. Association of CLP levels with socio-demographics, disease activity (quantitative myasthenia gravis (QMG) score, myasthenia gravis-specific Activities of Daily Living scale (MG-ADL)), antibody (Abs) status, history of myasthenic crisis, treatment regime, and history of thymectomy were investigated using univariate analysis. Mean baseline serum levels of CLP were significantly higher in MG patients compared to controls (4.3 µg/ml vs. 2.1 µg/ml; p < 0.0001). Higher levels of CLP were associated with a higher clinical disease severity measured by MGFA classification and QMG score. Nevertheless, the only weak correlation of CLP with clinical outcome parameters needs confirmation in future studies. Currently, there are no validated blood biomarkers for MG. The significantly elevated CLP and mild correlation with parameters of disease activity suggests that CLP holds promise as a biomarker for measurement of individual disease severity.

Nutrient Intake and Status of German Children and Adolescents Consuming Vegetarian, Vegan or Omnivore Diets: Results of the VeChi Youth Study.

There is a lack of data on associations between modern vegetarian and vegan diets and health among children and adolescents. The aim of the Vechi Youth Study was to cross-sectionally examine anthropometry, dietary intakes and nutritional status in a sample of 149 vegetarian, 115 vegan and 137 omnivore children and adolescents (6-18 years old, mean age: 12.7 ± 3.9 years). Group differences of dietary intake (calculated from three-day dietary records), nutrient biomarker and blood lipid concentrations were assessed using an analysis of covariance, adjusted for sex, age and other covariates. The total energy intake did not differ significantly between groups, but intake of carbohydrates was higher among vegetarians and vegans than among omnivores (p = 0.0002, respectively). The median protein intake exceeded 0.9 g/kg body weight/day in all diet groups and was lowest among vegetarians (p < 0.02). There was no significant difference of haemoglobin, vitamin B2, 25-OH vitamin D3, HDL-C and triglycerides blood concentrations between diet groups. Vegan participants had higher folate concentrations than vegetarian participants (p = 0.0053). Ferritin concentration was significantly higher in omnivores than in vegetarians (p = 0.0134) and vegans (p = 0.0404). Vegetarians had lower concentrations of holotranscobalamin (p = 0.0042) and higher concentrations of methylmalonic acid (p = 0.0253) than omnivores. Vegans had the lowest non-HDL-C and LDL-C concentrations in comparison to vegetarians (p = 0.0053 and p = 0.0041) and omnivores (p = 0.0010 and p = 0.0010). A high prevalence (>30%) of 25-OH vitamin D3 and vitamin B2 concentrations below reference values were found irrespective of the diet group. In conclusion, the Vechi Youth Study did not indicate specific nutritional risks among vegetarian and vegan children and adolescents compared to omnivores.

Vitamin B-12-fortified toothpaste improves vitamin status in vegans: a 12-wk randomized placebo-controlled study.

Background: The oral application of vitamin B-12 may prevent its deficiency if the vitamin is absorbed via the mucosal barrier.Objectives: We studied the effect of the use of a vitamin B-12-fortified toothpaste on vitamin-status markers in vegans and assessed the efficiency of markers in the identification of vitamin-augmentation status.Design: In this 12-wk, double-blinded, randomized, placebo-controlled study, 76 vegans received either a placebo (n = 34) or vitamin B-12 (n = 42) toothpaste. Sixty-six subjects (n = 30 in the placebo arm; n = 36 in the vitamin B-12 arm) completed the intervention. Serum and plasma concentrations of vitamin B-12, holotranscobalamin, total homocysteine (tHcy), and methylmalonic acid (MMA) were measured before and after the intervention.Results: Both postintervention concentrations of vitamin B-12 and holotranscobalamin and their changes over 12 wk were higher in the vitamin B-12 group (mean ± SD change: 81 ± 135 pmol/L for vitamin B-12 and 26 ± 34 pmol/L for holotranscobalamin) than in the placebo group (-27 ± 64 and -5 ± 17 pmol/L, respectively) after adjustment for baseline concentrations. Postintervention concentrations of MMA and their changes differed significantly between groups (MMA changes: -0.169 ± 0.340 compared with -0.036 ± 0.544 µmol/L in vitamin B-12 and placebo groups, respectively; P < 0.001). After adjustment for baseline tHcy, postintervention concentrations of tHcy tended to be lower (P = 0.051), and the changes in tHcy (-0.7 ± 4.4 compared with 2.0 ± 5.6 µmol/L, respectively) were greater in the vitamin B-12 group than in the placebo group. Changes in vitamin B-12 markers were more prominent in vegans who reported that they had not taken vitamin B-12 supplements.Conclusion: Vitamin B-12 that is applied to the oral cavity via toothpaste enters the circulation and corrects the vitamin B-12 markers in the blood of vegans who are at higher risk of vitamin B-12 deficiency. This trial was registered at clinicaltrials.gov as NCT02679833.

CD34(+)-selected stem cell boost without further conditioning for poor graft function after allogeneic stem cell transplantation in patients with hematological malignancies.

We retrospectively analyzed outcomes of a CD34(+)-selected stem cell boost (SCB) without prior conditioning in 32 patients (male/22; median age of 54 years; range, 20 to 69) with poor graft function, defined as neutrophils ≤1.5 x 10(9)/L, and/or platelets ≤30 x 10(9)/L, and/or hemoglobin ≤8.5 g/dL). The median interval between stem cell transplantation and SCB was 5 months (range, 2 to 228). The median number of CD34(+) and CD3(+) cells were 3.4 x 10(6)/kg (.96 to 8.30) and 9 x 10(3)/kg body weight (range, 2 to 70), respectively. Hematological improvement was observed in 81% of patients and noted after a median of 30 days (range, 14 to 120) after SCB. The recipients of related grafts responded faster than recipients of unrelated grafts (20 versus 30 days, P = .04). The cumulative incidence of acute (grade II to IV) and chronic graft-versus-host disease (GVHD) after SCB was 17% and 26%, respectively. Patients with acute GVHD received a higher median CD3(+) cell dose. The 2-year probability of overall survival was 45%. We suggest that SCB represents an effective approach to improve poor graft function post transplantation, but optimal timing of SCB administration, anti-infective, and GVHD prophylaxis needs further evaluation.

Osmotic virial coefficients of hydroxyethyl starch from aqueous hydroxyethyl starch-sodium chloride vapor pressure osmometry.

Hydroxyethyl starch (HES) is an important industrial additive in the paper, textile, food, and cosmetic industries and has been shown to be an effective cryoprotectant for red blood cells; however, little is known about its thermodynamic solution properties. In many applications, in particular those in biology, HES is used in an aqueous solution with sodium chloride (NaCl). The osmotic virial solution thermodynamics approach accurately captures the dependence of osmolality on molality for many types of solutes in aqueous systems, including electrolytes, sugars, alcohols, proteins, and starches. Elliott et al. proposed mixing rules for the osmotic virial equation to be used for osmolality of multisolute aqueous solutions [Elliott, J. A. W.; et al. J. Phys. Chem. B 2007, 111, 1775-1785] and recently applied this approach to the fitting of one set of aqueous HES-NaCl solution data reported by Jochem and Körber [Cryobiology 1987, 24, 513-536], indicating that the HES osmotic virial coefficients are dependent on HES-to-NaCl mass ratios. The current study reports new aqueous HES-NaCl vapor pressure osmometry data which are analyzed using the osmotic virial equation. HES modifications were measured after dialysis (membrane cut off: 10,000 g/mol) and freeze-drying using vapor pressure osmometry at different mass ratios of HES to NaCl for HES up to 50% and NaCl up to 25% with three different HES modifications (weight average molecular weights [g/mol]/degree of substitution: 40,000/0.5; 200,000/0.5; 450,000/0.7). Equations were then fit to the data to provide a model for HES osmotic virial coefficient dependence on mass ratio of HES to NaCl. The osmolality data of the three HES modifications were accurately described over a broad range of HES-to-NaCl mass ratios using only four parameters, illustrating the power of the osmotic virial approach in analyzing complex data sets. As expected, the second osmotic virial coefficients increase with molecular weight of the HES and increase with HES-to-NaCl mass ratio.

Antigen-specific tolerance by autologous myelin peptide-coupled cells: a phase 1 trial in multiple sclerosis.

Multiple sclerosis (MS) is a devastating inflammatory disease of the brain and spinal cord that is thought to result from an autoimmune attack directed against antigens in the central nervous system. The aim of this first-in-man trial was to assess the feasibility, safety, and tolerability of a tolerization regimen in MS patients that uses a single infusion of autologous peripheral blood mononuclear cells chemically coupled with seven myelin peptides (MOG1-20, MOG35-55, MBP13-32, MBP83-99, MBP111-129, MBP146-170, and PLP139-154). An open-label, single-center, dose-escalation study was performed in seven relapsing-remitting and two secondary progressive MS patients who were off-treatment for standard therapies. All patients had to show T cell reactivity against at least one of the myelin peptides used in the trial. Neurological, magnetic resonance imaging, laboratory, and immunological examinations were performed to assess the safety, tolerability, and in vivo mechanisms of action of this regimen. Administration of antigen-coupled cells was feasible, had a favorable safety profile, and was well tolerated in MS patients. Patients receiving the higher doses (>1 × 10(9)) of peptide-coupled cells had a decrease in antigen-specific T cell responses after peptide-coupled cell therapy. In summary, this first-in-man clinical trial of autologous peptide-coupled cells in MS patients establishes the feasibility and indicates good tolerability and safety of this therapeutic approach.

Assessment of physiologic natural killer cell cytotoxicity in vitro.

Here, we describe an improved (51)chromium release assay (CRA) to compare donor natural killer (NK) cell activity. To validate the assay, we analyzed sample preparation, incubation, and cryopreservation of NK cells. The effector-to-target ratio was corrected for the percentage of NK cells. A logarithmic curve was fitted to the data of the CRA for calculation of the maximum activity. The specific lysis was standardized to a reference sample and normalized to the mean specific lysis of the reference. We found that a longer time span involved with both the addition and the removal of DMSO increased the recovery of NK cell activity. Freezing and thawing reduced the cytotoxicity of NK cells but sustained the relative differences that were seen between freshly prepared NK cells. In contrast, medium incubation of thawed cells markedly increased the cytotoxic potential but also deranged these relative differences. Those were widely equalized when cells were stimulated with IL-2. In conclusion, we established a standardized assay with cryopreserved peripheral blood mononuclear cells as an appropriate tool for investigation of individual physiologic NK cell activity. This assay may help to predict donor NK cell activity in vivo, to reconcile conflicting data about NK cells obtained in transplantation studies.

Bioequivalence comparison of a new freezing bag (CryoMACS(®)) with the Cryocyte(®) freezing bag for cryogenic storage of human hematopoietic progenitor cells.

BACKGROUND AIMS: We investigated two different plastic freezing bags, namely the most recently U.S. Food and Drug Administration (FDA)-approved CryoMACS(®) freezing bag (200-074-402) from Miltenyi Biotec and the familiar Cryocyte(®) freezing bag (R4R9955) from (Baxter Healthcare, Deerfield, IL, United States) for the cryogenic storage of human hematopoietic progenitor cells (HPC). METHODS: The study material consisted of 12 frozen HPC pairs (= 24 transplant units) that were no longer needed for autologous treatment of patients. After thawing, one unit of a pair was transferred into the Miltenyi (M) bag; the other unit remained in the original Baxter (B) bag. After refreezing both units, all units were stored again under cryogenic conditions either partially immersed in liquid nitrogen (n = 22) or in the vapor phase over liquid nitrogen, n = 2, <-170°) before thawing. RESULTS: The correlation coefficients (r) between the results obtained from the two bag types were high for white blood cells (WBC) content (r = 0.98), mononuclear cells (MNC) (r = 0.97), lymphocytes (r = 0.98), monocytes (r = 0.96), membrane integrity (r = 0.93), concentration of 'free' hemoglobin (r = 0.97) and hemolysis rate (r = 0.95). With regard to clonogenicity, there were no significant differences (Student's paired t-test) for the three parameters investigated [i.e. total number of colonies, including the numbers of burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte-macrophage (CFU-GM) colonies, respectively). CONCLUSIONS: The CryoMACS freezing bag 200-074-402 is bioequivalent to the Cryocyte freezing container R4R9955. An advantageous feature of the CryoMACS is that its double-sterile wrapping provides additional safety regarding potential cross-contamination during cryogenic storage.

Purification of CD4+ T cells for adoptive immunotherapy after allogeneic hematopoietic stem cell transplantation.

Donor lymphocyte infusions (DLIs) are used for adoptive immunotherapy to prevent or treat relapse and infectious complications after allogeneic hematopoietic stem cell transplantation (HSCT). Unmanipulated DLIs are associated with a risk of graft-versus-host disease (GVHD), probably related to CD8(+) T cell activity. We investigated an automated clinical-scale human-CD4(+)-cell purification method to deplete CD8(+) cells. Twenty-four stem cell recipients received a total of 24 leukapheresis products being enriched for CD4(+) cells using magnetic associated cell sorting (MACS) with an automated device (CliniMACS(®)) before DLIs. MACS resulted in a mean CD4(+) cell count of 16 × 10(6)/kg bw corresponding to 3.4-fold CD4(+) cell enrichment. Mean yield and purity of CD45(+)CD3(+)CD4(+)CD14(-)7AAD(-) were 74% ± 23% and 82% ± 11%, respectively. Median initial dose of DLIs was 1.1 × 10(6) CD4(+)/kg. During a median follow-up of 25 months, 7 (30%) patients experienced GVHD (acute II-IV: n = 4, 17%; acute III-IV: n = 2, 8%; chronic limited: n = 2, 8%; chronic extensive: n = 1, 4%). Thirteen of 21 further evaluable patients (62%) showed measurable clinical response, 2 patients with therapy refractory infectious complications (HSV) showed remarkable immunologic improvement. Automated enrichment of CD4(+) by magnetic cell sorting provides an efficient and rapid method for processing donor lymphocytes. Additional studies should further investigate this approach in terms of efficacy and the risk of GVHD.

Turning CD34 Non-Mobilizers into Mobilizers: A Case Report Involving Plerixafor (AMD3100).

SUMMARY: OBJECTIVE: In a significant proportion of patients with hematologic malignancies (5-30%) poor mobilization of hematopoietic stem cells (HSC) is observed. This compromises the application of effective and potentially curative high-dose chemotherapy (HDC) treatment. CASE REPORT: Here we report the case of a 38-year-old female patient who was treated for recurrent follicular B-cell non-Hodgkin's lymphoma grade III. In this patient, we failed twice to mobilize stem cells using chemotherapy followed by granulocyte-colony stimulating factor (G-CSF). Recently a new chemokine receptor CXCR4 antagonist, AMD3100 (plerixafor), was introduced which can be combined with G-CSF mobilization and has been reported to increase the number of harvested stem cells significantly. Using this protocol, we were able to harvest a HSC product. This product was transplanted 3 weeks after the harvest (after HDC), and the patient had an uncomplicated recovery of granulopoiesis (day 11 after transplantation of autologous HSC). CONCLUSION: Plerixafor has the potency to become an important tool in mobilizing HSC, especially in those patients in whom HSC cannot be mobilized by the combination of G-CSF and chemotherapy alone.

Cryopreservation of red blood cells and platelets.

Blood cells can be regarded as a classical field of application of low-temperature biology. Cryopreservation methods have been developed for different categories of blood cells namely red blood cells (RBCs) (erythrocytes), platelets (thrombocytes), mononuclear cells (i.e., lymphocytes, monocytes), and hematopoietic progenitor cells. This chapter outlines the four most commonly applied techniques for RBCs and two for platelets.

The glass transition temperature of mixtures of trehalose and hydroxyethyl starch.

Although mixtures of HES and sugars are used to preserve cells during freezing or drying, little is known about the glass transition of HES, or how mixtures of HES and sugars vitrify. These difficulties may be due to the polydispersity between HES samples or differences in preparation techniques, as well as problems in measuring the glass transition temperature (T(g)) using differential scanning calorimetry (DSC). In this report, we examine the T(g) of mixtures of HES and trehalose sugar with <1% moisture content using DSC measurements. By extrapolating these measurements to pure HES using the Gordon-Taylor and Fox equations, we were able to estimate the T(g) of our HES sample at 44 degrees C. These results were additionally confirmed by using mixtures of glucose-HES which yielded a similar extrapolated T(g) value. Our approach to estimating the glass transition temperature of HES may be useful in other cases where glass transitions are not easily identified.