Resistance of cancers to immunologic cytotoxicity and adoptive immunotherapy via X-linked inhibitor of apoptosis protein expression and coexisting defects in mitochondrial death signaling.

The ability of cancers to evade immune surveillance and resist immunotherapy raises a fundamental question of how tumor cells survive in the presence of a competent immune system. Studies to address this question have primarily focused on mechanisms by which tumor cells avoid recognition by or induce tolerance in the immune system. However, little is known about whether cancer cells also acquire an intrinsic ability to resist killing by immune effectors. We find that cancer cells enhance their ability to withstand an attack by cytotoxic immune effector cells via acquisition of specific genetic alterations that interfere with the shared mitochondrial death signaling pathway entrained by granzyme B, IFN-gamma, and Apo2 ligand/tumor necrosis factor-related apoptosis inducing ligand (Apo2L/TRAIL), three key mediators of immunologic cell-mediated cytotoxicity. We show that the coexistence of specific mitochondrial signaling defects (either deletion of Bax, overexpression of Bcl-x(L), or deletion of Smac) with expression of X-linked inhibitor of apoptosis protein decreases the sensitivity of cancer cells to IFN-gamma/Apo2L/TRAIL- or granzyme B-induced apoptosis, lymphocyte-mediated cytotoxicity in vitro, and adoptive cellular immunotherapy in vivo. Conversely, negating X-linked inhibitor of apoptosis protein expression or function in tumor cells with defective mitochondrial signaling enables direct activation of caspase-3/-7 by granzyme B or Apo2L/TRAIL, and restores their susceptibility to immunologic cytotoxicity. These findings identify an important mechanism by which cancers evade elimination by immune effector cells and suggest that cancer immunotherapy might be improved by concurrent strategies to alleviate or circumvent the intrinsic mitochondrial death signaling defects that help cancer cells resist immunologic cytotoxicity.

Zinc homeostasis in premature infants does not differ between those fed preterm formula or fortified human milk.

The objectives of this study were to compare zinc homeostasis in premature infants enterally fed with either preterm infant formula or fortified human milk; to examine interrelationships of variables of zinc homeostasis; and to examine the findings in relation to estimated zinc requirements of preterm infants. Zinc homeostasis was studied in 14 infants (8 male), with mean gestational age of 31 wk and birth weight appropriate for gestational age, who were exclusively fed either preterm formula (n = 9) or own mother's milk with human milk fortifier (n = 5). Zinc stable isotopes were administered intravenously ((70)Zn) and orally as an extrinsic label ((67)Zn) over multiple feeds for determination of fractional absorption by dual isotope tracer ratio in urine; endogenous fecal zinc was determined by isotope dilution; and exchangeable zinc pool (EZP) size was estimated from linear regression of log-transformed urine (70)Zn enrichment data. Results indicated no significant differences in the variables of zinc homeostasis between the feeding groups; data for all subjects were thus combined. Mean (+/- SD) fractional absorption was 0.26 +/- 0.07; net absorbed zinc 0.43 +/- 0.25 mg/d (0.31 +/- 0.19 mg/kg/d). Mean EZP was 20 +/- 10 mg/kg, and was positively correlated with total absorbed zinc and with net absorbed zinc. Feeding type and total absorbed zinc were significantly related to daily weight gain (p = 0.003). Current zinc intakes from fortified human milk or formula are associated with acceptable weight gain, but whether the observed net zinc absorption was optimal in the human milk group cannot be definitively determined from these data.

Enumeration of lymphocyte subsets using flow cytometry: Effect of storage before and after staining in a developing country setting.

Lymphocyte subset estimations by flow cytometry in population-based studies require transportation of samples from the field site to the laboratory. As samples arrive late in the day they have to wait overnight before being processed. The effect of two possible approaches, sample storage for 24 h before staining and immediate staining with analysis after 24 h and 48 h were evaluated. Two sets of experiments were performed with EDTA (ethylenediamine tetra-acetate) anticoagulated peripheral blood. In the first experiment, after collection, each sample was divided into two portions. One portion was stained at the time of blood collection and the other 24 h later after keeping it at room temperature (38-45°C). In the second experiment, blood samples were stained within 1-2 h. Each sample was analyzed immediately upon completion of staining process and subsequently after 24 h and 48 h of storage at 4°C. Results suggest that blood collected in EDTA can be processed using whole blood lysis method, after storage at room temperature (38-45°C) for 24 h with some but not significant alteration in T-cell subsets. Storage at 4°C after staining for 24 h results in a lesser and insignificant loss of cells or alteration of T-cell subsets and may be the method of choice.

Successful therapy of metastatic cancer using tumor vaccines in mixed allogeneic bone marrow chimeras.

A frequent outcome of allogeneic stem cell transplantation (alloSCT) in the treatment of leukemia is the destruction of the host hematolymphoid compartment and, thus, the malignancy, through the combined action of high-dose chemoradiotherapy and a T-cell-mediated graft-versus-host effect. Unfortunately, alloSCT is frequently limited by toxicity, including graft-versus-host disease (GVHD), and has not been successful in the treatment of tumors derived from solid organs. Here we report a novel cooperation between host and donor T cells in the response to a tumor cell vaccine given after a nonmyeloablative allogeneic stem cell transplantation (NST) protocol that achieves stable mixed bone marrow chimerism. Treatment of animals with NST, posttransplantation donor lymphocyte infusions (DLIs), and a vaccine, comprising irradiated autologous tumor cells mixed with a granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing bystander line, results in potent and specific antitumor immunity. This combined modality immunotherapy, administered after surgical removal of the primary tumor, cured metastatic mammary cancer in most animals without inducing GVHD. Cured animals contained tumor-specific T cells of both host and donor origin, but immunodeficient hosts could not be cured by NST, DLI, and vaccine administration. Thus, transfer of allogeneic donor T cells may help break functional tolerance of a host immune system to a solid tumor, thereby providing a rationale for the generation of mixed hematopoietic chimerism by NST prior to tumor cell vaccination.

Modifications in flow cytometric estimation of t cell subsets and b cells in peripheral blood to reduce the cost of investigation.

The development of monoclonal antibodies combined with flow cytometry has revolutionized the analysis of lymphocyte subsets. These newer methods using the Q-prep leucocyte preparation system require only 1-2 ml of blood as compared to 10 ml required traditionally. One of the main impediments in the use of this superior technology in Indian laboratories has been the high cost of reagents. This study evaluated methods to reduce the cost of assays. In the first experiment from 26 healthy subjects, 2ml venous blood samples in EDTA (ethylenediamine tetra-acetate) were obtained. Each sample was divided into two equal portions, one portion was stained using diluted monoclonal antibody, whereas the other portion was stained using standard concentrations of antibodies. In the second experiment, blood samples from 12 subjects were again divided into 2 portions; one portion of each pair was processed using commercial Q-prep reagents while the other portion was processed using our own reagents. In the first experiment, which evaluated use of a diluted antibody against the standard recommended concentrations, a 5-tube panel that estimated CD3, CD4, CD8, CD20 was used. In the second experiment CD3, CD4 and CD8 were estimated. The total cost per sample for a 5-panel estimation was however reduced from $39.11 to $1.10.Given the proven advantages of using a whole blood stain-lyse method for T cell subset estimations, its use should be encouraged in developing country settings. With the suggested methods the whole blood Q-prep could be performed at appreciably reduced costs, without loss in precision.

Zinc absorption and exchangeable zinc pool sizes in breast-fed infants fed meat or cereal as first complementary food.

BACKGROUND: The aims of this study were to compare the absorption efficiency of zinc from rice cereal and meat, with and without human milk, in 7-month-old breast-fed infants and to compare the size of exchangeable zinc pools in the infants according to the assigned complementary food. METHODS: Fractional absorption of zinc was measured in male infants using extrinsic labeling with a stable isotope of zinc in a test meal of either pureed beef (n = 9) or iron-fortified infant rice cereal (n = 9). The effect on fractional absorption of the addition of human milk to each complementary food was measured in each infant with a second oral zinc isotope. Fractional absorption was measured using fecal monitoring of isotope excretion, and exchangeable zinc pool size was calculated from isotopic enrichment in urine. RESULTS: Fractional absorption of zinc did not statistically differ between the beef (0.41 +/- 0.11) and cereal (0.36 +/- 0.05) test meals, although the trend showed that beef had higher fractional absorption than cereal. The higher intake of zinc from the beef versus cereal test meal resulted in a 16-fold greater amount of absorbed zinc ( P = 0.0002). The addition of human milk caused significant decreases in fractional absorption of zinc (0.07 +/- 0.02, P = 0.01) and absorbed zinc (0.04 +/- 0.01 mg, P < 0.0001). The size of the exchangeable zinc pool did not differ according to group but was strongly correlated with mean daily zinc intake ( r = 0.72, P = 0.003). CONCLUSIONS: These results confirm that meat as a complementary food for breast-fed infants can provide a rich source of dietary zinc that is well absorbed. The significant positive correlation between zinc intake and exchangeable zinc pool size suggests that increasing zinc intake positively affects metabolically available zinc.