Age and number of lesions predict chalazion recurrence.

PURPOSE: Periocular lesions in pediatric patients usually require general anesthesia for surgical intervention. The US Food and Drug Administration (FDA) warns against multiple exposures to anesthesia in children younger than 3 years due to the increased risk of learning disabilities in this population. This study aimed to evaluate risk factors associated with chalazion recurrence after surgery. METHODS: A retrospective chart review over a five-year period identified 649 patients at our institution undergoing surgical intervention for chalazion. The primary outcomes examined were as follows: (1) return to the operating room for additional surgical intervention and (2) recurrence of chalazion during convalescence from surgery and follow-up. RESULTS: Fewer than one-third of patients suffered a recurrence after surgery. Multivariate logistic regression found younger age (p = 0.01), female sex (p = 0.01), and a greater number of chalazia drained (p < 0.001) were significantly correlated with recurrence of chalazia after surgery. CONCLUSIONS: Patients presenting at a younger age and with a greater number of chalazion were statistically more likely to have a recurrence of chalazion after surgery. Given recurrence is more likely in younger children, reconciling this with the risk-benefit ratio with regard to FDA guidelines on anesthesia in children under three years is a critical consideration for ophthalmologists.

Characterizing bioavailable phosphorus concentrations in an agricultural stream during hydrologic and streambed disturbances.

In freshwater ecosystems, phosphorus (P) is often considered a growth-limiting nutrient. The use of fertilizers on agricultural fields has led to runoff-driven increases in P availability in streams, and the subsequent eutrophication of downstream ecosystems. Isolated storms and periodic streambed dredging are examples of two common disturbances that contribute dissolved and particulate P to agricultural streams, which can be quantified as soluble reactive P (SRP) using the molybdate-blue method on filtered water samples, or total P (TP) measured using digestions on unfiltered water reflecting all forms of P. While SRP is often considered an approximation of bioavailable P (BAP), research has shown that this is not always the case. Current methods used to estimate BAP do not account for the role of biology (e.g., NaOH extractions) or require specialized platforms (e.g., algal bioassays). Here, in addition to routine analysis of SRP and TP, we used a novel yeast-based bioassay with unfiltered sample water to estimate BAP concentrations during two storms (top 80% and > 95% flow quantiles), and downstream of a reach where management-associated dredging disturbed the streambed. We found that the BAP concentrations were often greater than SRP, suggesting that SRP is not fully representative of P bioavailability. The SRP concentrations were similarly elevated during the two storms, but remained consistently low during streambed disturbance. In contrast, turbidity and TP were elevated during all events. The BAP concentrations were significantly related to turbidity during all disturbance events, but with TP only during storms. The novel yeast assay suggests that BAP export can exceed SRP, particularly when streams are not in equilibrium, such as the rising limb of storms or during active dredging.

High frequency of fetal cells within a primitive stem cell population in maternal blood.

BACKGROUND: During pregnancy, fetal cells enter the maternal bloodstream resulting in fetal cell microchimerism. The fetal cells persist in the mother for decades and colonize a variety of maternal organs. They are associated with maternal autoimmune diseases and may also participate in tissue repair. The identity of the microchimeric cells is not certain but they must be able to persist long-term and have potential for multitissue differentiation. METHODS AND RESULTS: Here we tested the hypothesis that the fetal microchimeric cells are primitive stem cells, represented by CD34+ adherent cells, which have a wide potential for differentiation. We isolated these stem cells from the blood of pregnant females (n = 25) and detected fetal cells of the correct gender, using fluorescence in situ hybridization, in a high proportion (71% male fetuses and 90% female fetuses; false positive rate 11%, false negative rate 29%) of cases. By RT-PCR, we demonstrated that the cells express Oct-4, Nanog and Rex-1. No fetal cells were detected in the mononuclear or total CD34+ cell populations but high frequencies (mean 11.8%) of fetal cells were detected in the adherent CD34+ cell population. CONCLUSIONS: These results identify adherent CD34+ stem cells as candidate fetal microchimeric cells, which are capable of sustaining the fetal cell population in the long term and have the ability to colonize multiple tissues and organs.

In vitro effects of interferon-gamma and tumor necrosis factor-alpha on CD34+ bone marrow progenitor cells from aplastic anemia patients and normal donors.

Acquired aplastic anemia is characterized by loss or dysfunction of hematopoietic stem and progenitor cells. The proinflammatory cytokines Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) may be responsible for the immune-mediated pathology observed in some patients. The CD34+ population of bone marrow mononuclear cells contains primitive cells responsible for hemopoiesis. We investigated the response of CD34+ cells from aplastic anemia patients to a combination of IFN-gamma and TNF-alpha, and compared them to cells from normal volunteer donors. This was to determine whether aplastic CD34+ cells are more sensitive than normal cells to IFN-gamma/TNF-alpha-mediated effects, and whether cytokine-induced CD95 expression can explain the high levels of apoptosis observed in CD34+ cells from aplastic patients. CD34+38- cells were most affected by overnight incubation with these cytokines, their proportion and numbers being reduced in both normal donors and patients. There was no evidence for increased apoptosis, suggesting that this effect may be due to differentiation. IFN-gamma/TNF-alpha induced upregulation of CD95 on both normal and aplastic CD34+ cells, although the basal level of CD95 expression was increased in aplastic cells. However, CD95 induction did not make cells from normal donors or aplastic anemia patients susceptible to induction of apoptosis by agonistic anti-CD95 antibodies, soluble CD95 ligand, or membrane-bound CD95L. In vivo CD95L is required for CD95 induced apoptosis. No forms of this protein were detectable in lymphocytes from aplastic patients. We conclude that increased apoptosis in aplastic CD34+ cells is not due to increased sensitivity to IFN-gamma/TNF-alpha. We further show that normal and aplastic CD34+ cells are resistant to CD95 apoptosis, even in the presence of mCD95L.

Characterization and clinical application of human CD34+ stem/progenitor cell populations mobilized into the blood by granulocyte colony-stimulating factor.

A phase I study was performed to determine the safety and tolerability of injecting autologous CD34(+) cells into five patients with liver insufficiency. The study was based on the hypothesis that the CD34(+) cell population in granulocyte colony-stimulating factor (G-CSF)-mobilized blood contains a subpopulation of cells with the potential for regenerating damaged tissue. We separated a candidate CD34(+) stem cell population from the majority of the CD34(+) cells (99%) by adherence to tissue culture plastic. The adherent and nonadherent CD34(+) cells were distinct in morphology, immunophenotype, and gene expression profile. Reverse transcription-polymerase chain reaction-based gene expression analysis indicated that the adherent CD34(+) cells had the potential to express determinants consistent with liver, pancreas, heart, muscle, and nerve cell differentiation as well as hematopoiesis. Overall, the characteristics of the adherent CD34(+) cells identify them as a separate putative stem/progenitor cell population. In culture, they produced a population of cells exhibiting diverse morphologies and expressing genes corresponding to multiple tissue types. Encouraged by this evidence that the CD34(+) cell population contains cells with the potential to form hepatocyte-like cells, we gave G-CSF to five patients with liver insufficiency to mobilize their stem cells for collection by leukapheresis. Between 1 x 10(6) and 2 x 10(8) CD34(+) cells were injected into the portal vein (three patients) or hepatic artery (two patients). No complications or specific side effects related to the procedure were observed. Three of the five patients showed improvement in serum bilirubin and four of five in serum albumin. These observations warrant further clinical trials.

Fidelity and reproducibility of antisense RNA amplification for the study of gene expression in human CD34+ haemopoietic stem and progenitor cells.

Microarrays provide a powerful tool for the study of haemopoietic stem and progenitor cells (HSC). Because of the low frequency of HSC, it is rarely feasible to obtain enough mRNA for microarray hybridizations, and amplification will be necessary. Antisense RNA (aRNA) amplification is reported to give high-fidelity amplification, but most studies have used only qualitative validation. Before applying aRNA amplification to the study of HSC, we wished to determine its fidelity and reproducibility, and whether statistically significant results can be obtained. We found that aRNA amplification introduced biases into relative RNA abundance. However, these biases were extremely consistent, and valid comparisons could be made, if amplified RNA was compared with amplified RNA. By applying this method to the effect of interferon-gamma and tumour necrosis factor-alpha on normal primary CD34+ HSC, biologically significant differences could be detected, including potential mechanisms for resistance of CD34+ cells to CD95-mediated apoptosis and evidence of the differentiating effects of the cytokines. Differences of twofold or less were detected, and most of these differences attained statistical significance after triplicate experiments. These data demonstrate that aRNA amplification can be used with microarray hybridization to study the transcriptional profiles of small numbers of primary CD34+ HSC.

Direct binding of antithymoctye globulin to haemopoietic progenitor cells in aplastic anaemia.

Antithymocyte globulin (ATG) is widely used in the treatment of aplastic anaemia (AA) and a response occurs in 60-80% of patients. However, its exact mechanism of action in the treatment of AA has yet to be determined. Previously, we have shown that ATG increases colony growth from purified bone marrow CD34+ cells of AA patients in vitro, and decreases stem cell apoptosis and the expression of soluble Fas receptor after ATG therapy in vivo. The aim of this study was to further examine the association of ATG with AA haemopoietic progenitor cells. We describe here that ATG bound directly to CD34+ cells. Forty-six patients and 20 normal control subjects were studied. ATG bound to CD34+ cells in normal control subjects (mean 90.38%) as determined by flow cytometry. The mean percentage of CD34+ cells binding to ATG was 59.90% in untreated aplastic patients, 83.24% in partial responders, 58.3% in non-responders and 62.73% in relapsed patients. In completely recovered patients, ATG binding was indistinguishable from control subjects. The functionality of AA patients' haemopoietic progenitor cells was assessed using colony assays. These results demonstrate the direct binding of ATG to CD34+ cells and suggest that differences in its binding to AA CD34+ cells could reflect functional differences in the haemopoietic stem cell compartment throughout the disease process.