Molecular diagnosis of X-linked chronic granulomatous disease in Iran

Chronic granulomatous disease (CGD) is an inherited disorder of pathogen killing by phagocytic leukocytes caused by mutations in NADPH oxidase subunits. Patients with CGD have life-threatening bacterial and fungal infections. Children’s Medical Center at Tehran University is the referral center for immunodeficiency in Iran. During 2 years of study, 11 non-consanguineous families with clinically diagnosed CGD were referred to this center. In functional assays performed on neutrophils from affected children and their mothers; no activity or strongly decreased oxidase activity was detected in the patients’ cells. In oxidase tests that scored this activity on a per-cell basis, a mosaic pattern was detected in the neutrophils from all 11 mothers. Western blot analysis revealed an X91° phenotype in all patients. Mutation screening in the CYBB gene encoding gp91^phox by SSCP analysis followed by sequencing showed nine different mutations, including two novel mutations. The present survey is the first study aimed to analyze the clinical features and the molecular diagnosis of X-CGD in Iranian patients.     

Repeated PR1 and WT1 peptide vaccination in Montanide-adjuvant fails to induce sustained high-avidity, epitope-specific CD8+ T cells in myeloid malignancies

Background

We previously showed that vaccination with one dose of PR1 and WT1 peptides induces transient anti-leukemia immunity. We hypothesized that maintenance of a sustained anti-leukemia response may require frequent boost injections.

Design and Methods

Eight patients with myeloid malignancies were enrolled in this phase II study, and 6 completed 6 injections of PR1 and WT1 peptides in Montanide-adjuvant with GM-CSF, every two weeks.

Results

Both high- and low-avidity PR1 or WT1-specific CD8⁺ T cells were detected in all evaluable patients after the first vaccine dose. Repeated vaccination led to selective deletion of high avidity PR1- and WT1-specific CD8⁺ T cells and was not associated with significant reduction in WT1-expression. Additional boosting failed to increase vaccine-induced CD8⁺ T-cell frequencies further and in all patients the response was lost before the 6^th dose. PR1- or WT1-specific CD8⁺ T cells were not detected in bone marrow samples, excluding their preferential localization to this site. Following a booster injection three months after the 6^th vaccine dose, no high-avidity PR1 or WT1-specific CD8⁺ T cells could be detected, whereas low-avidity T cells were readily expanded.

Conclusions

These data support the immunogenicity of PR1 and WT1 peptide vaccines. However, repeated delivery of peptides with Montanide-adjuvant and GM-CSF leads to rapid loss of high-avidity peptide-specific CD8⁺ T cells. These results may offer an explanation for the lack of correlation between immune and clinical responses observed in a number of clinical trials of peptide vaccination. New approaches are needed to induce long-term high-avidity memory responses against leukemia antigens. (ClinicalTrials.gov Identifier: NCT00499772)     

Repeated vaccination is required to optimize seroprotection against H1N1 in the immunocompromised host

Background

In 2009 the declaration by the World Health Organization of a global pandemic of influenza-H1N1 virus led to a vaccination campaign to ensure protection for immunocompromised patients. The goal of this study was to determine the efficacy of the 2009 H1N1 vaccine in patients with hematologic malignancies.

Design and Methods

We evaluated humoral and cellular immune responses to 2009 H1N1 vaccine in 97 adults with hematologic malignancies and compared these responses with those in 25 adult controls. Patients received two injections of vaccine 21 days apart and the controls received one dose. Antibody titers were measured using a hemagglutination-inhibition assay on days 0, 21 and 49 after injection of the first dose. Cellular immune responses to H1N1 were determined on days 0 and 49.

Results

By day 21 post-vaccination, protective antibody titers of 1:32 or more were seen in 100% of controls compared to 39% of patients with B-cell malignancies (P<0.001), 46% of allogeneic stem cell transplant recipients (P<0.001) and 85% of patients with chronic myeloid leukemia (P=0.086). After a second dose, seroprotection rates increased to 68%, (P=0.008), 73%, (P=0.031), and 95% (P=0.5) in patients with B-cell malignancies, after allogeneic stem cell transplantation and with chronic myeloid leukemia, respectively. On the other hand, T-cell responses to H1N1 vaccine were not significantly different between patients and controls.

Conclusions

These data demonstrate the efficacy of H1N1 vaccine in most patients with hematologic malignancies and support the recommendation for the administration of two doses of vaccine in immunocompromised patients. These results may contribute towards the development of evidence-based guidelines for influenza vaccination in such patients in the future.     

Use of fecal occult blood testing in hospitalized patients: Results of an audit

BACKGROUND:

The fecal occult blood test (FOBT), widely used as a colorectal cancer screening tool, continues to be used in hospitalized patients. However, the utility of this test for hospitalized patients is unclear.

OBJECTIVE:

To assess FOBT use in a large urban regional health authority.

METHODS:

Reports of all FOBTs performed between April 1, 2011 and March 30, 2012 from two academic and four community hospitals in Winnipeg (Manitoba) were extracted. Of 650 hospitalizations with a positive FOBT result and 1254 with a negative FOBT result, random samples of 230 and 97 charts, respectively, were reviewed. Information including demographics, admission diagnos(es), indication(s) for ordering the FOBT and clinical management was extracted.

RESULTS:

Thirty-four percent (650 of 1904) of hospitalizations with an FOBT had a positive FOBT result. Family medicine physicians ordered approximately one-half of the reviewed FOBTs. The most common indication for ordering an FOBT was anemia. Of those with a positive FOBT, 66% did not undergo further gastrointestinal investigations. Of those with a positive FOBT and overt gastrointestinal bleeding and/or melena who underwent endoscopy, 60% had their endoscopy performed before the FOBT result being reported while 38% underwent their endoscopy ≥3 days after the stool sample was collected. There were minimal differences in clinical practices between academic and community hospitals.

CONCLUSIONS:

The present study suggests that FOBT results in hospitalized patients may have little beneficial impact on clinical management. Hospital laboratories may be better served in directing resources to other tests.     

High-molecular-weight kininogen is exclusively membrane bound on endothelial cells to influence activation of vascular endothelium

An important biologic function of high-molecular-weight kininogen (HK) is to deliver bradykinin (BK) to its cellular receptors. Internalization and degradation of HK may provide a mechanism by which endothelial cells modulate the production of BK and control its activities. Therefore, we investigated the binding and subsequent distribution of biotinylated-HK (biotin-HK) associated with human umbilical vein endothelial cells (HUVEC). HUVEC bound 3 to 4 times more HK and with greater avidity at 1 to 3 hours at 37 degrees C than at 4 degrees C (Bmax = 1.0 +/- 0.02 x 10(7) molecules/cell, kd = 7 +/- 3 nmol/L v Bmax = 2.6 +/- 0.2 x 10(6) molecules/cell, kd = 46 +/- 8 nmol/L). However, there was no evidence that the difference was caused by internalization of HK at the higher temperature. First, the same amount of biotin-HK was associated with nonpermeabilized and permeabilized HUVEC using buffers containing 20 to 50 mumol/L zinc ion in the absence or presence of 2 mmol/L calcium ion. Second, binding of biotin-HK to HUVEC was approximately 92% reversible at 1 hour when the cells were maintained at both 37 degrees C and 4 degrees C. Third, neither chloroquine nor primaquine altered the amount of biotin-HK bound to HUVEC. Fourth, biotin-HK bound to HUVEC was almost completely removed by pronase. Fifth, the nonpermeable dye, crystal violet, almost completely quenched the fluorescence signal emitted by HUVEC-associated fluorescein isothiocyanate (FITC) HK. Finally, the localization of HUVEC-bound FITC-HK was restricted to the membrane as shown by laser scanning confocal microscopy. The expression of HK binding sites had an absolute requirement for metabolic energy, but was not dependent on new protein synthesis. Membrane-bound HK contributed to the anticoagulant nature of endothelial cells by blocking human alpha-thrombin binding and its resultant induction of prostacyclin formation. These studies indicate that HK is not internalized by HUVEC, but remains primarily on cell surfaces to be accessible for BK liberation and to modulate the binding and actions of alpha-thrombin.     

Hematopoietic growth factors for graft failure after bone marrow transplantation: a randomized trial of granulocyte-macrophage colony- stimulating factor (GM-CSF) versus sequential GM-CSF plus granulocyte- CSF

Delay in hematologic recovery after bone marrow transplantation (BMT) can extend and amplify the risks of infection and hemorrhage, compromise patients' survival, and increase the duration and cost of hospitalization. Because current studies suggest that granulocyte- macrophage (GM) colony-stimulating factor (CSF) may potentiate the sensitivity of hematopoietic progenitor cells to G-CSF, we performed a prospective, randomized trial comparing GM-CSF (250 micrograms/m2/d x 14 days) versus sequential GM-CSF x 7 days followed by G-CSF (5 micrograms/kg/d x 7 days) as treatment for primary or secondary graft failure after BMT. Eligibility criteria included failure to achieve a white blood cell (WBC) count > or = 100/microL by day +21 or > or = 300/microL by day +28, no absolute neutrophil count (ANC) > or = 200/microL by day +28, or secondary sustained neutropenia after initial engraftment. Forty-seven patients were enrolled: 23 received GM-CSF (10 unrelated, 8 related allogeneic, and 5 autologous), and 24 received GM- CSF followed by G-CSF (12 unrelated, 7 related allogeneic, and 5 autologous). For patients receiving GM-CSF alone, neutrophil recovery (ANC > or = 500/microL) occurred between 2 and 61 days (median, 8 days) after therapy, while those receiving GM-CSF+G-CSF recovered at a similar rate of 1 to 36 days (median, 6 days; P = .39). Recovery to red blood cell (RBC) transfusion independence was slow, occurring 6 to 250 days (median, 35 days) after enrollment with no significant difference between the two treatment groups (GM-CSF: median, 30 days; GM-CSF+G- CSF; median, 42 days; P = .24). Similarly, platelet transfusion independence was delayed until 4 to 249 days (median, 32 days) after enrollment, with no difference between the two treatment groups (GM- CSF: median, 28 days; GM-CSF+G-CSF: median, 42 days; P = .38). Recovery times were not different between patients with unrelated donors and those with related donors or autologous transplant recipients. Survival at 100 days after enrollment was superior after treatment with GM-CSF alone. Only 1 of 23 patients treated with GM-CSF died versus 7 of 24 treated with GM-CSF+G-CSF who died 16 to 84 days (median, 38 days) after enrollment, yielding Kaplan-Meier 100-day survival estimates of 96% +/- 8% for GM-CSF versus 71% +/- 18% for GM-CSF+G-CSF (P = .026). These data suggest that sequential growth factor therapy with GM-CSF followed by G-CSF offers no advantage over GM-CSF alone in accelerating trilineage hematopoiesis or preventing lethal complications in patients with poor graft function after BMT.(ABSTRACT TRUNCATED AT 400 WORDS)     

Early human thymocyte proliferation is regulated by an externally controlled autocrine transforming growth factor-beta 1 mechanism

Early thymocytes undergo extensive proliferation after their entry into the thymus, but cellular interactions and cytokines regulating this intrathymic step remain to be determined. We analyzed the effects of various T-cell growth factors and cellular interactions on in vitro proliferation of early CD2+CD3/TCR-CD4-CD8- (triple negative [TN]) human thymocytes. Freshly isolated TN cells were then assayed for their growth capacity after incubation with CD2I+III-monoclonal antibody (MoAb), recombinant human interleukin-2 (IL-2), IL-7, and/or IL-4. These cells displayed significant proliferative responses with IL-4, IL- 7, or CD2-MoAb+IL-2. The addition of recombinant transforming growth factor beta (TGF beta) or autologous irradiated CD3+CD8+CD4- cells to TN cell cultures dramatically decreased their growth responses to IL-2 and IL-7, whereas IL-4-induced proliferation was less sensitive to growth inhibition. We thus asked whether the CD8+ cell-derived inhibitory effect was due to TGF beta. The addition of neutralizing anti-TGF beta MoAb completely abolished CD8+ cell-derived inhibition of TN cell growth. Analysis of CD8+ cell-derived supernatants indicated that these cells had low TGF beta 1 production capacity, whereas TN cells secrete significantly high levels of TGF beta 1. Cell fixation studies showed that TN cells were the source of the TGF beta. TGF beta 1 released from TN cells was in the latent form that became the active inhibitory form through interaction of TN cells with CD8+ cells. Together, these data suggest a role for TGF beta 1 as an externally controlled, autocrine inhibitory factor for human early thymocytes, with a regulatory role in thymic T-cell output.     

Behavioral Similarities and Differences among Alcohol-Preferring and -Nonpreferring Rats: Confirmation by Factor Analysis and Extension to Additional Groups

Thirteen behavioral variables from six tasks were measured in alcohol-preferring (AA, FH, and P) and -non-preferring (ANA, FRL, and NP) rat lines/strains and subjected to Factor Analysis. Four independent factors accounted for >90% of the variance. Defecation in the open field and ultrasonic vocalizations after an air puff were negatively correlated with alcohol intake and preference, whereas the increase in daily fluid intake in the presence of saccharin was positively correlated. Other factors could be labeled Activity, Emotionality, and Immobility Factors, and each was independent of the Alcohol Factor. When an additional alcohol-preferring rat line (HAD) and two additional non-preferring groups (LAD and ACI) were tested, they were found to differ on most behaviors that were associated with alcohol intake and preference in the Factor Analysis: vocalizations and saccharin-induced increase in fluid intake, but not defecation. A new Factor Analysis was then performed incorporating these three new groups and including five new behavioral measures. The following measures had high loadings on the Alcohol Factor: alcohol intake under choice conditions; alcohol preference; forced alcohol intake; alcohol acceptance (forced alcohol intake/basal water intake × 100); ultrasonic vocalization; saccharin intake; saccharin-induced increase in daily fluid intake; defecation in the open field test; and immobility in a modified forced swim test. These findings indicate that there are indeed certain behavioral characteristics that are common among alcohol-preferring rat lines/strains, but there are also substantial group differences on other behavioral measures. For those behavioral measures reflecting emotionality (defecation and ultrasonic vocalization) that loaded highly on the Alcohol Factor, the alcohol-preferring rats had lower scores.     

Selective Inhibition of Alcohol Intake in Diverse Alcohol-Preferring Rat Strains by the 5-HT2A Antagonists Amperozide and FG 5974

The present studies sought to elucidate the role of 5-HT_2A receptor antagonists in suppressing alcohol intake by comparing the effects of amperozide and FG 5974 on alcohol, food, and water intake in strains of alcohol-preferring rats: P, Alko Alcohol (AA), and Fawn-Hooded (FH). Both amperozide and FG 5974 have 5-HT_2A receptor antagonist properties, but FG 5974 also shows presynaptic 5-HT_1A receptor agonist activity. After establishment of stable baselines for intake measures in a two-bottle continuous access paradigm, rats ( n = 10) were injected with 1 of 5 doses (0, 1.0, 2.5, 5.0, and 10.0 mg/kg, sc) of amperozide or FG 5974 at weekly intervals. Amperozide dose-dependently reduced alcohol intake, total fluid intake, and alcohol preference in all three strains under continuous access conditions, whereas FG 5974 was less effective. Food intake was also suppressed by amperozide at higher doses, whereas it was increased by FG 5974. Amperozide also dose-dependently reduced alcohol intake when it was available for only 1 hr/day, but FG 5974 tended to increase it. After oral administration, amperozide was also more effective than FG 5974 in reducing alcohol intake. Despite these differences in efficacy in suppressing alcohol intake, both compounds produced taste aversion to a novel saccharin solution. These complex findings suggest that biochemical properties other than 5-HT_2A receptor antagonism (e.g., 5-HT_1A receptor agonism) may be involved in the effects of amperozide and FG 5974 on alcohol intake and other consummatory behaviors.