Mobilization of early hematopoietic progenitor cells with BB-10010: a genetically engineered variant of human macrophage inflammatory protein- 1 alpha

BB-10010 is a genetically engineered variant of human macrophage inflammatory protein-1 alpha with improved solution properties. We show here that it mobilizes stem cells into the peripheral blood. We investigated the mobilizing effects of BB-10010 on the numbers of circulating 8-day spleen colony-forming units (CFU-S8), CFU-S12, and progenitors with marrow repopulating ability (MRA). A single subcutaneous dose of BB-10010 caused a twofold increase in circulating numbers of CFU-S8, CFU-S12, and MRA 30 minutes after dosing. We also investigated the effects of granulocyte colony-stimulating factor (G- CSF) and the combination of G-CSF with BB-10010 on progenitor mobilization. Two days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA progenitors by 25.7-, 19.8-, and 27.7-fold. A single administration of BB-10010 after 2 days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA even further to 38-, 33-, and 100- fold. Splenectomy resulted in increased circulating progenitor numbers but did not change the pattern of mobilization. Two days of treatment with G-CSF then increased circulating CFU-S8, CFU-S12, and MRA by 64-, 69-, and 32-fold. A single BB-10010 administration after G-CSF treatment further increased them to 85-, 117-, and 140-fold, respectively, compared with control. We conclude that BB-10010 causes a rapid increase in the number of circulating hematopoietic progenitors and further enhances the numbers induced by pretreatment with G-CSF. BB- 10010 preferentially mobilized the more primitive progenitors with marrow repopulating activity, releasing four times the number achieved with G-CSF alone. Translated into a clinical setting, this improvement in progenitor cell mobilization may enhance the efficiency of harvest and the quality of grafts for peripheral blood stem cell transplantation.     

A Sensitive Microtitre Plate Enzyme Immunoassay of Oestradiol-17β in the Cow and Mare

Abstract

Microtitre plates were coated with antiserum against oest-radiol-17β-6-(0-carboxymethyl)-oxime bovine serum albumin raised in sheep. The plasma samples (0.2–1.0 ml) were extracted with peroxide-free dlethyl ether prepared daily by treatment with Al₂O₃. The enzyme conjugate was prepared by coupling oestradiol-17β-6-(0-carboxymethyl)-oxime to horse-radish peroxidase. The conjugate was chromatographed on a Sephadex G-25 column. The standard curve ranged from 0.37 to 18.40 fmol/well of oestradiol-17β. The amount of oestradiol-17β causing a 50% reduction of maximum binding was 4.4 fmol/well.

Standards and samples were Incubated overnight at 4°C. The conjugate solution was added followed by further incubation for 2 h at 4°C. Tetramethylbenzidine was used as a chromogen, and the optical density was measured at 450 nm. The patterns of oestradiol-17β during a normal oestrus cycle in the cow and mare are presented.     

Sleep disturbances in women with polycystic ovary syndrome

Sleep disturbances in women with Polycystic Ovary Syndrome (PCOS) have been reported in recent years. The majority of published studies are related to Obstructive Sleep Apnea (OSA) while not many researches have analyzed any other causes of sleep disturbances. A group of ninety five women with Polycystic Ovary Syndrome were enrolled into the study. Sleep disturbances were assessed using validated questionnaires. On the grounds of Athens Insomnia Scale (AIS) evaluation a clinically significant insomnia was ascertained in 12.6% of women with PCOS, while according to Insomnia Severity Index (ISI) in 10.5%. Clinically significant insomnia according to both AIS and ISI, occurred significantly more often in women with PCOS than in women without PCOS based on the chi-square test. The Mann–Whitney U test revealed statistically significant difference between women with and without PCOS based on total values of ISI. An excessive daytime sleepiness occurred at 7.4% of women with PCOS. Statistically significant dependance between: clinically significant insomnia in both AIS and ISI and excessive daytime sleepiness indicated by Epworth Sleepiness Scale (ESS) was observed. Sleep disorders are common in women with PCOS. Screening assessment of sleep disturbances should be a part of medical diagnostics in women with PCOS.     

Multivariate Analysis of Complications After Simultaneous Pancreas and Kidney Transplantation

Objective

Identification of factors that have an impact on postoperative complications after simultaneous pancreas and kidney transplantation (SPKTx) could help overcome limitations of this kind of treatment.

Methods

Postoperative complications among 112 SPKTx recipients were divided into 3 groups: related to transplanted pancreas (n = 66), related to transplanted kidney (n = 23) and general surgical complications (n = 31) 120 refers to complications among 112 recipients. According to the modified Clavien-Dindo scale, complications were classified according to their severity for each group. Risk factors for complication development related to donor, recipient, surgical technique, and immunosuppression were included to establish the multivariable model using logistic regression.

Results

Multiple regression analysis showed the following independent factors influenced mortal complications due to transplanted pancreas: age of donor (OR, 1.07; P < .04), duration of vascular pancreas anastomosis above 35 minutes (OR, 3.94; P < .04) and duration of recipient dialysis above 24 months before transplantation (OR, 0.14; P < .01). Area under receiver operating characteristic curve for this model was 0.8.

Conclusion

To improve results, the following modification of identified risk factors should be assumed: selection of donor in term of age, shortening of the second warm ischemia time, and adjustment of the waiting list to avoid prolongation of recipient dialysis before SPKTx.     

Experimental support for the "E pathway hypothesis" of coupled transmembrane e- and H+ transfer in dihemic quinol:fumarate reductase

Reconciliation of apparently contradictory experimental results obtained on the quinol:fumarate reductase, a diheme-containing respiratory membrane protein complex from Wolinella succinogenes, was previously obtained by the proposal of the so-called "E pathway hypothesis." According to this hypothesis, transmembrane electron transfer via the heme groups is strictly coupled to cotransfer of protons via a transiently established pathway thought to contain the side chain of residue Glu-C180 as the most prominent component. Here we demonstrate that, after replacement of Glu-C180 with Gln or Ile by site-directed mutagenesis, the resulting mutants are unable to grow on fumarate, and the membrane-bound variant enzymes lack quinol oxidation activity. Upon solubilization, however, the purified enzymes display approximately 1/10 of the specific quinol oxidation activity of the wild-type enzyme and unchanged quinol Michaelis constants, K(m). The refined x-ray crystal structures at 2.19 A and 2.76 A resolution, respectively, rule out major structural changes to account for these experimental observations. Changes in the oxidation-reduction heme midpoint potential allow the conclusion that deprotonation of Glu-C180 in the wild-type enzyme facilitates the reoxidation of the reduced high-potential heme. Comparison of solvent isotope effects indicates that a rate-limiting proton transfer step in the wild-type enzyme is lost in the Glu-C180 --> Gln variant. The results provide experimental evidence for the validity of the E pathway hypothesis and for a crucial functional role of Glu-C180.     

Modern approaches to the treatment of breast cancer

Breast cancer is the most common cause of cancer death in women in this country. Until recently, the traditional treatment has been radical surgery with or without radiation therapy for patients with primary breast cancer, and palliative endocrine therapy followed by chemotherapy for patients with advanced disease. These treatments have met with limited effectiveness in terms of eradicating the disease. Studies in the past decade have given cause for optimism for breast cancer patients. Adjuvant systemic therapy after local treatment appears promising for certain subsets of patients with primary breast cancer. The development of estrogen receptor assays has markedly changed our approach to the disease and improved patient care. Estrogen receptor is an important prognostic factor and is useful in planning appropriate therapy for patients with primary breast cancer as well as those with advanced disease. Further research is urgently needed to improve the dismal survival of certain women with this common malignancy.