IgG Fc-receptor polymorphisms in Guillain-Barré syndrome

The authors studied immunoglobulin G (IgG) Fc receptor (FcgammaR) IIA, IIIA, and IIIB polymorphisms in 62 patients with Guillain-Barré syndrome (GBS) and in 89 healthy controls. The FcgammaR genotypes and allele frequencies did not differ significantly between the patients with GBS and the controls. Patients homozygous for the FcgammaRIIIB neutrophil antigen (NA) 1 allele had a significantly less severe disease than patients heterozygous or homozygous for the NA2 allele. The FcgammaRIIIB NA1/NA1 genotype has high affinity for IgG1 and IgG3, and clearance of circulating autoantibodies and immune complexes may therefore be of importance in the pathogenesis of GBS.     

Isolation and characterization of propagable cell lines (HUNC) from the androgen-sensitive Dunning R3327H rat prostatic adenocarcinoma

The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC- E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-alpha and the TGF-betas, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma.      

Endometrial evaluation with transvaginal US and hysterosonography in asymptomatic postmenopausal women with breast cancer receiving tamoxifen

PURPOSE: To determine performance characteristics of transvaginal ultrasonography (US) and hysterosonography for diagnosing endometrial abnormality in asymptomatic postmenopausal women with breast cancer receiving tamoxifen. MATERIALS AND METHODS: The authors prospectively examined 138 women receiving tamoxifen by using transvaginal US, hysterosonography, and office hysteroscopy. The combined hysteroscopic-histopathologic diagnosis was the reference standard. Sensitivity, specificity, positive and negative predictive values, and likelihood ratios of transvaginal US and hysterosonography were calculated. RESULTS: All 138 women underwent transvaginal US; 104, successful hysterosonography; and 117, successful hysteroscopy. Uterine abnormality was present in 47 (40.2%) of 117 women: 45 with polyps and two with submucosal fibroids. Receiver operating characteristic curve analysis revealed 6 mm to be the optimal endometrial thickness cutoff for diagnosing endometrial abnormalities. When a thickness greater than 6 mm or a focal endometrial finding was considered abnormal, transvaginal US had a sensitivity of 85.1% and a specificity of 55.7%. In 92 women who completed transvaginal US, hysterosonography, and hysteroscopy, hysterosonography was more specific (79.2%; P =.008) but not significantly more sensitive (89.7%; P =.508) than transvaginal US. When women with abnormal transvaginal US findings were further examined with hysterosonography, the sequential combination of transvaginal US and hysterosonography was more specific (77.1%) than transvaginal US alone (P <.001), without a significant decrease in sensitivity (78.7%; P =.25). CONCLUSION: In asymptomatic postmenopausal women receiving tamoxifen, 6 mm is the optimal endometrial thickness cutoff for diagnosing endometrial abnormalities with transvaginal US. Further examination with hysterosonography can improve specificity by reducing the high false-positive rate of transvaginal US.     

Combination Therapy Reverses Hyperglycemia in NOD Mice With Established Type 1 Diabetes

An increasing number of therapies have proven effective at reversing hyperglycemia in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D), yet situations of successful translation to human T1D are limited. This may be partly due to evaluating the effect of treating immediately at diagnosis in mice, which may not be reflective of the advanced disease state in humans at disease onset. In this study, we treated NOD mice with new-onset as well as established disease using various combinations of four drugs: antithymocyte globulin (ATG), granulocyte-colony stimulating factor (G-CSF), a dipeptidyl peptidase IV inhibitor (DPP-4i), and a proton pump inhibitor (PPI). Therapy with all four drugs induced remission in 83% of new-onset mice and, remarkably, in 50% of NOD mice with established disease. Also noteworthy, disease remission occurred irrespective of initial blood glucose values and mechanistically was characterized by enhanced immunoregulation involving alterations in CD4⁺ T cells, CD8⁺ T cells, and natural killer cells. This combination therapy also allowed for effective treatment at reduced drug doses (compared with effective monotherapy), thereby minimizing potential adverse effects while retaining efficacy. This combination of approved drugs demonstrates a novel ability to reverse T1D, thereby warranting translational consideration.     

Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+Thy-1+Lin- primitive progenitor cells

Thrombopoietin (TPO) or MpI ligand is known to stimulate megakaryocyte (MK) proliferation and differentiation. To identify the earliest human hematopoietic cells on which TPO acts, we cultured single CD34+Thy- 1+Lin- adult bone marrow cells in the presence of TPO alone, with TPO and interleukin-3 (IL-3), or with TPO and c-kit ligand (KL) in the presence of a murine stromal cell line (Sys1). Two distinct growth morphologies were observed: expansion of up to 200 blast cells with subsequent differentiation to large refractile CD41b+ MKs within 3 weeks or expansion to 200-10,000 blast cells, up to 25% of which expressed CD34. The latter blast cell expansions occurred over a 3- to 6-week period without obvious MK differentiation. Morphological staining, analysis of surface marker expression, and colony formation analysis revealed that these populations consisted predominantly of cells committed to the myelomonocytic lineage. The addition of IL-3 to TPO-containing cultures increased the extent of proliferation of single cells, whereas addition of KL increased the percentage of CD34+ cells among the expanding cell populations. Production of multiple colony- forming unit-MK from single CD34+Thy-1+Lin- cells in the presence of TPO was also demonstrated. In limiting dilution assays of CD34+Lin- cells, TPO was found to increase the size and frequency of cobblestone areas at 4 weeks in stromal cultures in the presence of leukemia inhibitory factor and IL-6. In stroma-free cultures, TPO activated a quiescent CD34+Lin-Rhodamine 123lo subset of primitive hematopoietic progenitor cells into cycle, without loss of CD34 expression. These data demonstrate that TPO acts directly on and supports division of cells more primitive than those committed to the MK lineage.     

Charlson Co‐morbidity Index and albumin significantly associated with fracture risk in peritoneal dialysis patients

Published literature on fracture in dialysis patients seldom addressed the effect of co‐morbidity and malnutrition. In this study, we reported the incidence and risk factors for fracture in peritoneal dialysis patients. Peritoneal dialysis patients who had fractures between 2006 and 2011 were recruited. Demographic data, details of fracture, Charlson Co‐morbidity Index (CCI) and biochemical parameters were also collected. Non‐fracture controls, matched for age, gender and duration of dialysis, were also recruited at ratio 1:1 for fracture risk analysis. The incidence of fracture was 1 in 37 patient‐years. The commonest site of fracture was neck of femur (n = 16, 55.2%). Twenty‐four patients (82.8%) developed fracture after slip and fall injury. Eight out of 17 self‐ambulatory patients (47.1%) became non‐ambulatory after fracture. Infection was the commonest complication during hospitalization. Univariant analysis demonstrated high CCI (P = 0.001), hypoalbuminaemia (P < 0.001), loss of self autonomy (P = 0.006) and non‐ambulatory state (P = 0.011) significantly associated with increased fracture risk. However, only CCI (odds ratio (OR) 1.373, P = 0.028) and albumin (OR 0.893, P = 0.025) increased fracture risk significantly on multivariant analysis. Bone profile and parathyroid hormone were not significant risk factors. To conclude, fracture associated with adverse outcome in peritoneal dialysis patients. High CCI score and hypoalbuminaemia significantly increase risk of fracture.     

Surgical delivery of drug releasing poly(lactic-co-glycolic acid)/poly(ethylene glycol) paste with in vivo effects against glioblastoma

Introduction

The median survival of patients with glioblastoma multiforme (astrocytoma grade 4) remains less than 18 months despite radical surgery, radiotherapy and systemic chemotherapy. Surgical implantation of chemotherapy eluting wafers into the resection cavity has been shown to improve length of survival but the current licensed therapy has several drawbacks. This paper investigates in vivo efficacy of a novel drug eluting paste in glioblastoma.

Methods

Poly(lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG) self-sintering paste was loaded with the chemotherapeutic agent etoposide and delivered surgically into partially resected tumours in a flank murine glioblastoma xenograft model.

Results

Surgical delivery of the paste was successful and practical, with no toxicity or surgical morbidity to the animals. The paste was retained in the tumour cavity, and preliminary results suggest a useful antitumour and antiangiogenic effect, particularly at higher doses. Bioluminescent imaging was not affected significantly by the presence of the paste in the tumour.

Conclusions

Chemotherapy loaded PLGA/PEG paste seems to be a promising technology capable of delivering active drugs into partially resected tumours. The preliminary results of this study suggest efficacy with no toxicity and will lead to larger scale efficacy studies in orthotopic glioblastoma models.     

Optimizing dose and scheduling of filgrastim (granulocyte colony- stimulating factor) for mobilization and collection of peripheral blood progenitor cells in normal volunteers [see comments]

To define an optimal regimen for mobilizing and collecting peripheral blood progenitor cells (PBPC) for use in allogeneic transplantation, we evaluated the kinetics of mobilization by filgrastim (recombinant met- human granulocyte colony-stimulating factor [r-metHuG-CSF]) in normal volunteers. Filgrastim was injected subcutaneously for up to 10 days at a dose of 3 (n = 10), 5 (n = 5), or 10 micrograms/kg/d (n = 15). A subset of volunteers from each dose cohort underwent a 7L leukapheresis on study day 6 (after 5 days of filgrastim). Granulocyte-macrophage colony-forming cell (GM-CFC) numbers in the blood were maximal after 5 days of filgrastim; a broader peak was evident for CD34+ cells between days 4 and 6. The 95% confidence intervals (CI) for mean number of PBPC per milliliter of blood in the three dose cohorts overlapped on each study day. However, on the peak day, CD34+ cells were significantly higher in the 10 micrograms/kg/d cohort than in a pool of the 3 and 5 micrograms/kg/d cohorts. Mobilization was not significantly influenced by volunteer age or sex. Leukapheresis products obtained at the 10 micrograms/kg/d dose level contained a median GM-CFC number of 93 x 10(4)/kg (range, 50 x 10(4)/kg to 172 x 10(4)/kg). Collections from volunteers receiving lower doses of filgrastim contained a median GM- CFC number of 36 x 10(4)/kg (range, 5 x 10(4)/kg to 204 x 10(4)/kg). The measurement of CD34+ cells per milliliter of blood on the day of leukapheresis predicted the total yield of PBPC in the leukapheresis product (r = .87, P < .0001). Assuming a minimum GM-CFC requirement of 50 x 10(4)/kg (based on our experience with autologous PBPC transplantation), all seven leukapheresis products obtained at the 10 micrograms/kg/d dose level were potentially sufficient for allogeneic transplantation purposes. We conclude that in normal donors, filgrastim 10 micrograms/kg/d for 5 days with a single leukapheresis on the following day is a highly effective regimen for PBPC mobilization and collection. Further studies are required to determine whether PBPC collected with this regimen reliably produce rapid and sustained engraftment in allogeneic recipients.     

Administration of pegylated recombinant human megakaryocyte growth and development factor to humans stimulates the production of functional platelets that show no evidence of in vivo activation

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.