Robin A. Roberts

Human Milk as a Potential Enteral Source of Erythropoietin

In addition to its content of traditional nutrients, milk is a rich source of hormones and peptides, which survive digestion in the neonatal gastrointestinal tract secondary to lower proteolytic activity and increased protein permeability. Previous studies have shown accelerated erythropoiesis or elevated serum erythropoietin (Epo) levels in neonatal (suckling) animals after maternal phlebotomy or maternal hypoxia exposure. We sought to determine whether significant quantities of Epo are present in human milk and whether Epo remains intact under physiologic digestion conditions. Immunoreactive Epo concentrations were determined in 409 human milk samples obtained from mothers of term and premature infants. Samples collected between birth and postpartum d 134 were divided into 11 postpartum day groups. Mean milk-borne Epo concentrations were within the normal range for plasma Epo concentrations and rose with postpartum day (F10,398 = 5.82, p < 0.0001). No differences were observed between milk collected from mothers of premature versus term infants. Estimated weekly human milk-borne Epo intakes approximated the lower range of published parenteral therapeutic doses. In simulated digestion at physiologic pH levels of 3.2, 5.8, and 7.4, milk-borne Epo resisted degradation at 1 and 2 h, compared with baseline. Therefore, we conclude that human milk contains considerable amounts of Epo which resist degradation after exposure to gastric juices at physiologic pH levels. These results support continued investigation into the fate and developmental roles of Epo in human milk.

Serum erythropoietin levels during infancy: associations with erythropoiesis.

OBJECTIVE To determine plasma erythropoietin levels and their association with hemoglobin and reticulocyte counts in healthy term infants. DESIGN We compared plasma erythropoietin levels measured in serial blood samples obtained every 4 weeks during the first 6 months of life with one another and with levels in term fetuses and healthy adults. Correlation analysis was applied to examine for associations of erythropoietin with hemoglobin and with reticulocyte count. RESULTS Plasma erythropoietin levels were lowest in the first and highest in the second postnatal months, a pattern reciprocal to that observed for hemoglobin during the period of physiologic anemia. The erythropoietin level was negatively correlated with hemoglobin (p < 0.0001) and positively correlated with reticulocytes (p < 0.0001). The slope of the inverse relationship of hemoglobin and plasma erythropoietin in infants was similar to those previously reported for anemic fetuses and premature infants, but much less steep than for anemic children and adults. CONCLUSION This study is the first to report simultaneous patterns of change observed in plasma erythropoietin, hemoglobin, and reticulocytes during normal infancy. These patterns are consistent with postnatal perturbations in tissue oxygenation and suggest a major role for erythropoietin in the regulation of erythropoiesis during normal infancy, but at a lower hemoglobin concentration than for older children and adults with pathologic anemia.

Quantitative nuclease protection assay in paraffin-embedded tissue replicates prognostic microarray gene expression in diffuse large-B-cell lymphoma

Gene expression profiling (GEP) has identified genes whose expression levels predict patient survival in diffuse large-B-cell lymphoma (DLBCL). Such discovery techniques generally require frozen samples unavailable for most patients. We developed a quantitative nuclease protection assay to measure expression levels of prognostic DLBCL genes using formalin-fixed, paraffin-embedded (FFPE) tissue. FFPE tissue was sectioned, permeabilized, denatured in the presence of specific probes, and hybridized to mRNA in situ. Nuclease subsequently destroyed non-hybridized probe. Alkaline hydrolysis freed mRNA-bound probes from tissue, which were transferred to ArrayPlates for probe capture and chemiluminescent quantification. We validated assay performance using frozen, fresh, and FFPE DLBCL samples, then used 39 archived DLBCL, previously microarray analyzed, to correlate GEP and ArrayPlate results. We compared old (>18 years) with new (<2 months) paraffin blocks made from previously frozen tissue from the original biopsy. ArrayPlate gene expression results were confirmed with immunohistochemistry for BCL2, BCL6, and HLA-DR, showing agreement between mRNA species and the proteins they encode. Assay performance was linear to ∼1 mg sample/well. RNase and DNase treatments demonstrated assay specificity for RNA detection, both fixed and soluble RNA detection. Comparisons were excellent for lysate vs snap-frozen vs FFPE (R2>0.98 for all comparisons). Coefficients of variation for quadruplicates on FFPE were generally <20%. Correlation between new and old paraffin blocks from the same biopsy was good (R2=0.71). Comparison of ArrayPlate to Affymetrix and cDNA microarrays showed reasonable correlations. Insufficient power from small sample size prevented successfully correlating results with patient survival, although hazard ratios trended the expected directions. We developed an assay to quantify expression levels of survival prediction genes in DLBCL using FFPE, fresh, or frozen tissue. While this technique cannot replace GEP for discovery, it indicates that expression differences identified by GEP can be replicated on a platform applicable to archived FFPE samples.

Plasma transferrin receptor levels and indices of erythropoiesis and iron status in healthy term infants.

Purpose: The goal of this study was to determine if the postnatal changes in plasma transferrin receptor (TfR) levels in healthy infants were associated with changes in erythropoiesis or iron status. Subjects and Methods Longitudinal blood samples were obtained monthly from healthy term infants fed iron-fortified formula for the first 7 months and analyzed for plasma TfR and indices of erythropoiesis and iron status. Results: Plasma TfR level rose during the first 2 months of life (p < 0.002). When examined for its association with indices of erythropoiesis, plasma TfR was negatively associated with hemoglobin (Hb) (p < 0.01), and positively associated with plasma erythropoietin (EPO) concentration (p < 0.005) and absolute reticulocyte count (p < 0.005). Plasma TfR was not associated with erythrocyte protoporphyrin. Although indices of iron status were not suggestive of iron deficiency, plasma TfR was negatively associated with plasma ferritin, Tf saturation, and plasma iron, and positively associated with total iron binding capacity (TIBC) (p < 0.0001 for all). Conclusions: Increases in plasma TfR levels were observed during normal infancy. The increases in plasma TfR levels correlate with increases in erythropoiesis without evidence for functional iron deficiency.

Immunohistochemical classification of de novo, transformed, and relapsed diffuse large B-cell lymphoma into germinal center B-cell and nongerminal center B-cell subtypes correlates with gene expression profile and patient survival

CONTEXT Diffuse large B-cell lymphoma (DLBCL) can be assigned to prognostic subgroups, including germinal center B-cell (GCB) and activated B-cell subgroups, by using gene expression profiling and, reportedly, immunohistochemistry for CD10, Bcl-6, and multiple myeloma-1/interferon regulatory factor-4 (MUM1/IRF4). OBJECTIVE To compare 2 commercial MUM1/IRF4 antibody formulations for accuracy in subtyping DLBCL against gene expression profiling, compare subtyping to patient survival, and evaluate the usefulness of GCB and non-GCB subtyping in relapsed and transformed DLBCL. DESIGN Evaluation of 2 commercial MUM1/IRF4 antibodies, ICSTAT/M17 and Mum-1p, by using 40 cases of de novo, relapsed, and transformed DLBCL; and comparison of the results obtained with gene expression profiling and survival. RESULTS Immunohistochemistry predicted the gene expression profiling subtype 71.8% and 69.2% of the time overall with use of the Mum-1p and ICSTAT/M17 antibodies, respectively, and 100% and 91.7% of the time when MUM1/IRF4 expression determined subtype. Gene expression profiling and immunohistochemistry revealed nearly identical 5-year overall survival rates for the GCB vs non-GCB subtypes (68.0% for GCB vs 24.7% for non-GCB with use of gene expression profiling [P = .03] and 70.2% vs 18.4%, respectively, with use of immunohistochemistry [P < .001]). When de novo, transformed, and relapsed cases were analyzed separately, 5-year overall survival rates were also significantly different. CONCLUSIONS Immunohistochemistry can be used to subclassify DLBCL, including a very small series of transformed and relapsed cases, into GCB and non-GCB subtypes and predict survival rates similar to those predicted by use of gene expression profiling. The 2 MUM1/IRF4 antibodies performed similarly.

Iron deprivation increases erythropoietin production in vitro, in normal subjects and patients with malignancy

Although tissue hypoxia is the major stimulus for erythropoietin (EPO) production, serum EPO (sEPO) levels at any given Hb in iron‐deficiency anaemia are relatively higher than in other anaemias. Iron chelators stimulate erythropoiesis in anaemia of chronic disease via unknown mechanisms. A recent study suggested that deferoxamine (DFO) regulates steady‐state EPO RNA. Here we report that altered intracellular iron balance regulates EPO production both in vitro and in two unique clinical trials. In vitro, both iron chelation with DFO and blockade of Tf‐mediated iron uptake with anti‐Tf receptor antibody 42/6, stimulated EPO production in serum‐deprived hepatoma cells. Conversely, iron repletion by haemin, inhibited EPO production in these cells. In clinical studies, sEPO levels rose in adult volunteers treated with DFO coupled to hydroxyethyl starch (HES‐DFO) and in patients with advanced malignancy treated with anti‐Tf receptor antibody 42/6, in a time‐ and dose‐dependent manner. These studies indicate intracellular iron balance regulates EPO production in humans.

Persistent intracellular binding of mitoxantrone in a human colon carcinoma cell line

Incubation of human carcinoma cells with mitoxantrone resulted in an intracellular distribution of the drug into cytoplasmic, nuclear and cytoskeletal compartments occurring within 1 min of drug treatment. Incubation of the cells in drug-free medium resulted in an efflux of the drug such that 80% of the intracellular drug was eliminated from the cells by 72 hr. Approximately 20% of the initial intracellular drug concentration remained in the cells after the drug had been removed from the medium. The majority of the persistent intracellular drug was associated with soluble cytoplasmic proteins and fractions enriched in nucleic acid. Approximately 10% of the persistent drug binding was associated with cellular structures that had been depleted of soluble cytoplasmic protein and nucleic acid. During the persistent drug binding, the cells enlarged at least 2-fold as determined by microscopic examination. An increasing percentage of the cells was also observed to contain a DNA content consistent with a G2 cell cycle arrest. Taken together, these data suggest that the persistent intracellular binding of mitoxantrone results in a G2 cell cycle arrest and cellular damage.

Presence of Erythropoietin in Human Milk. |[diams]| 491

In addition to its content of traditional nutrients, milk is a rich source of hormones and peptides which survive digestion in the neonatal gut secondary to lower proteolytic activity and increased protein pemeability. Erythropoietin (Epo), the primary hormone regulating erythropoiesis, is present in rat and human milk, and has erythropoietic effects in suckling rats.

COMPARISON OF LATEX AGGLUTINATION AND ELISA MONO-CLONAL ANTIBODY ASSAYS FOR DETECTION OF HUMAN ROTAVIRUS (HRV)

Rotavirus is an important cause of childhood gastroenteritis resulting in dehydration, hospital admission and nosocomial infections. False negative antigen tests may lead to further unnecessary diagnostic tests and therapy. We compared a latex agglutination assay (Rotalex; Medical Technology Corp.) with an ELISA procedure (Pathfinder; Kallestad Laboratories) for the diagnosis of HRV. The latex agglutination test used fresh stool suspended in 10 mls of buffer which was mixed with reactive and non-reactive latex particles for 2 minutes and observed for agglutination. The 1 1/2 hour direct ELISA utilized diluted stool mixed with a monoclonal antibody and added to tubes precoated with antibody. Both assays were visually interpreted. From 11/85 to 4/86, 109 stool specimens were tested by both methods; 15 (14%) were Rotalex (+) for HRV and 48 (44%) were ELISA (+) for HRV. Thirty-five specimens were ELISA (+)/ Rotalex (-). Of these, 22 of 30 available specimens were confirmed by a blocking assay as true positives. Sensitivity and specificity of Rotalex was 37% and 98%, respectively, when compared with the confirmed ELISA. The Rotalex latex assay appears to be less sensitive than the Pathfinder ELISA assay for detecting HRV. The benefits of the very rapid latex test must be weighed against its insensitivity. The latex test may best be used as a screening procedure.

COMPARISON OF SHELL VIAL CENTRIFUGATION, STANDARD CULTURE AND FLUORESCENT ANTIBODY (FA) TECHNIQUES FOR DIAGNOSIS OF RESPIRATORY SYNCYTIAL VIRUS (RSV)

Respiratory syncytial virus (RSV) is an important cause of respiratory disease in young children. We evaluated a centrifugation culture techniques ability to provide rapid culture diagnosis. Between 11/85 & 3/86, 365 respiratory specimens were processed by standard culture &/or direct immunofluorescent antibody (FA). Of these, 183 specimens (74% nasal washes) were inoculated with 0.2 ml onto HEp-2 shell vials (SV), centrifuged at 35°C, 2500 × g for one hour & incubated for 24 & 48 hours. Coverslips were acetone fixed & stained with RSV Fitc-conjugated monoclonal antibody. For standard cell cultures, the time to positive cytopathic effect (CPE) was seven days. Thirty of 140 (22%) were standard cell culture positive for RSV, 38 of 111 (34%) were FA positive for RSV & 34 of 183 (34%) were FA positive for RSV by shell vial. Sensitivity & specificity by shell vial were, respectively, 70% & 96%, compared with culture, & 63% & 96% compared with FA. Specificity for SV is 99% if specimens positive on FA & SV are considered false negatives on routine culture. This centrifugation culture technique, as applied to RSV, provided specific results five days sooner than standard culture. Only 10% of specimens negative by both FA & SV were positive on culture. A combination of FA & shell vial methods could provide a rapid, specific & sensitive diagnosis for RSV while allowing virus isolation.