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.     

Arg-Gly-Asp-dependent occupancy of GPIIb/IIIa by applaggin: evidence for internalization and cycling of a platelet integrin

Using indirect immunofluorescence microscopy we examined the distribution and cycling of GPIIb/IIIa after binding to applaggin, a high-affinity Arg-Gly-Asp (RGD)--containing ligand. Resting, unfixed platelets were incubated with applaggin for 30 minutes at 37 degrees C, and bound applaggin was detected by an affinity-purified rabbit anti- applaggin antibody. Examination of intact cells showed a rim pattern for applaggin, consistent with its binding to the platelet surface. Staining of Triton X-100--permeabilized cells showed an intracellular pool of applaggin. Competition of applaggin binding by either AP-2, an anti-GPIIb/IIIa monoclonal antibody (MoAb) that blocks fibrinogen binding, or the synthetic peptide RGDW eliminated both surface and intracellular staining, indicating that applaggin is binding to GPIIb/IIIa in an RGD-dependent manner. Inhibition of platelet activation by PGE1 and theophylline had no effect on the observed staining patterns, indicating that cellular activation is not required for surface binding and subsequent internalization. To evaluate whether occupancy of functional binding sites on GPIIb/IIIa is required for internalization, we used mAb15, an anti-GPIIIa antibody that neither blocks fibrinogen binding nor induces the expression of ligand-induced binding sites on GPIIb/IIIa. In these studies mAb15 was internalized in a manner analogous to both AP-2 and applaggin, showing that occupancy of the RGD binding site is not required to initiate receptor internalization. To estimate the size of the newly internalized pool of applaggin, 125I-applaggin--binding studies were performed. Displacement of bound 125I-applaggin by excess unlabeled applaggin or EDTA showed that at least 17% of bound applaggin was nondisplaceable when binding was performed under conditions permitting membrane flow and internalization. These data indicate that GPIIb/IIIa is internalized in unstimulated platelets independent of cellular activation or occupancy of the functional binding site(s) of GPIIb/IIIa by RGD-containing ligands. Thus, internalization of GPIIb/IIIa may represent a mechanism by which the surface expression of this adhesion receptor is regulated.     

Platelet turnover and kinetics in immune thrombocytopenic purpura: results with autologous 111In-labeled platelets and homologous 51Cr- labeled platelets differ

Mean platelet survival and turnover were simultaneously determined with autologous 111In-labeled platelets (111In-AP) and homologous 51Cr- labeled platelets (51Cr-HP) in ten patients with chronic immune thrombocytopenic purpura (ITP). In vivo redistribution of the 111In-AP was quantitated with a scintillation camera and computer-assisted image analysis. The patients were divided into two groups: those with splenic platelet sequestration (spleen-liver 111In activity ratio greater than 1.4), and those with diffuse sequestration in the reticuloendothelial system. The latter patients had more severe ITP reflected by pronounced thrombocytopenia, decreased platelet turnover, and prominent early hepatic platelet sequestration. Mean platelet life span estimated with 51Cr-HP was consistently shorter than that of 111In-AP. Platelet turnover determined with 51Cr-HP was thus over-estimated. The difference in results with the two isotope labels was apparently due to greater in vivo elution of 51Cr. Although the limitations of the techniques should be taken into account, these findings indicate that platelet turnover is not always normal or increased in ITP, but is low in severe disease. We suggest that this may be ascribed to damage to megakaryocytes by antiplatelet antibody. The physical characteristics in 111In clearly make this radionuclide superior to 51Cr for the study of platelet kinetics in ITP.     

Comparison of three methods for genotyping of prothrombotic polymorphisms

Several methods have been developed to detect common prothrombotic mutations, including factor V Leiden (G1691), prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677C. In this study, we compared the accuracy of three different molecular techniques, i.e.: (1) restriction enzyme digestion (RFLP), (2) real time with hybridization probes and final melting curve (Fluorescence Resonance Energy Transfer, FRET), and (3) real time with hydrolysis probes (TaqMan^®). Sequencing was used as the reference standard. Our data showed that RFLPs analysis for the detection of prothrombotic mutations, albeit easy-to-perform, had a limited reliability for assessing correct genotypes. FRET analysis displayed higher resolution than RFLPs. Additionally, FRET analysis was faster and less tedious than sequencing.     

Human dendritic cells following Aspergillus fumigatus infection express the CCR7 receptor and a differential pattern of interleukin-12 (IL-12), IL-23, and IL-27 cytokines, which lead to a Th1 response

Aspergillus fumigatus is the most prevalent airborne fungal pathogen and causes fatal invasive aspergillosis in immunocompromised patients. Given the essential role of dendritic cells (DC) in initiating and regulating immune responses, we investigated the impact of A. fumigatus conidial infection on human DC. A. fumigatus conidia were rapidly internalized and induced the release of tumor necrosis factor alpha within the first 8 h. After A. fumigatus infection, the majority of DC underwent full maturation, although CCR7 expression was observed only in DC that had internalized the conidia. Additionally, the analysis of regulatory cytokines showed that infected DC simultaneously produced interleukin-12p70 (IL-12p70) and significant amounts of IL-10. IL-10 neutralization was not able to further increase IL-12p70 production from infected DC. Whereas the central role of IL-12 in the generation of Th1 cells has long been appreciated, recently two other members of the IL-12 family, IL-23 and IL-27, were reported to play important roles in the regulation of gamma interferon (IFN-gamma) production from na?ve and memory T cells. A. fumigatus-infected DC were also able to express high levels of IL-23p19 and low levels of IL-27p28 at later stages of infection. According to this expression pattern, A. fumigatus-infected DC were able to prime IFN-gamma production of na?ve T cells. Thus, this study on the expression of the new IL-12 family members controlling the Th1 response sheds light on a novel aspect of the contribution of DC to anti-Aspergillus immunity.     

The ‘Draw‐the‐Family Test’ in the preoperative assessment of patients with anorectal diseases and psychological distress: a prospective controlled study

Aim Some benign anorectal diseases may have psychosomatic aetiology, but patients often refuse direct psychological counselling. The Draw‐the‐Family Test (DFT) is a simple indirect investigation. The aim of this study was to evaluate the DFT in patients with psychological problems undergoing surgery for benign anorectal disease and to correlate the results with surgical outcome. Method DFT was administered prior to surgery to 62 patients with benign anorectal disease who admitted psychological problems at the time of the outpatient visit. Of these 18 (29%) had functional disease, mainly obstructed defecation (OD) while 44 (71%) had organic disease (haemorrhoids, fissures, pilonidal sinus or fistula). DFT was also administered to 40 healthy control subjects. Each DFT was judged as normal or pathological according to 10 parameters. Patients were followed up for a median of 12 months (range 3–64) and divided into two outcome groups, success (n = 58) and failure (n = 12) according to the results of a questionnaire. The DFT of all patients was then correlated with the outcome. Results None of the patients refused DFT. All DFT parameters but one (animal/things instead of human beings) were more frequent in patients compared with controls (P < 0.05). When comparing separately organic or functional disease patients with controls, one parameter (absence of patient in the drawing) was only pathological in the organic disease group (P < 0.05). Another parameter (schematic figures) was more frequently altered in the organic disease group compared with the functional disease group (P = 0.01). Eight out of 10 parameters were more frequently pathological in patients who failed after treatment, but none reached statistical significance. Conclusion Results of DFT in patients with anorectal disease admitting to psychological problems are markedly different from healthy controls. Patients with organic disease and those with functional bowel disease have different DFT profiles. In our study group, DFT had an excellent compliance but could not predict the outcome of surgery.     

Percutaneous fluoroscopic removal of a knotted Swan–Ganz catheter in a patient with a persistent left‐sided superior vena cava

Knotting of intravascular catheters is an uncommon but a well‐recognized occurrence. The Swan–Ganz catheter (SGC) is the one that knots most commonly. A case of a knotted SGC is described in a patient with a persistent left‐sided superior vena cava, and we propose that the presence of a left‐sided superior vena cava is a risk factor for knot formation not previously reported. We review the published work on the risk factors for knot formation and on the techniques used to remove knotted SGC. We describe a technique using a gooseneck snare and Omni Flush catheter (Angiodynamics, Queensbury, NY, USA) to loosen and untie a knotted SGC.     

Early clinical and radiological pulmonary complications following breast cancer radiation therapy: NTCP fit with four different models.

OBJECTIVE: To fit four different NTCP (Normal Tissue Complication Probability) models to prospectively collected data on short-term pulmonary complications following breast cancer radiotherapy (RT). MATERIALS/METHODS: Four hundred and seventy-five breast cancer patients, referred to the Radiotherapy Department at Stockholm S?der Hospital (1994-1998) for adjuvant post-operative RT were prospectively followed for pulmonary complications 1, 4 and 7 months after the completion of RT. Eighty-seven patients with complete dose-volume histogram (DVH) of the ipsilateral lung were selected for the present analysis. Mean dose to the ipsilateral lateral lung ranged from 2.5 to 18Gy (median 12Gy). Three different endpoints were considered: (1) clinical pneumonitis scored according to CTC-NCIC criteria: asymptomatic (grade 0) vs grade 1 and grade 2; (2) radiological changes assessed with diagnostic chest X-ray: no/slight radiological changes vs moderate/severe; (3) radiological changes assessed with CT: no/slight vs moderate/severe. Four NTCP models were used: the Lyman model with DVH reduced to the equivalent uniform dose (LEUD), the Logit model with DVH reduced to EUD, the Mean Lung Dose (MLD) model and the Relative Seriality (RS) model. The data fitting procedure was done using the maximum likelihood analysis. The analysis was done on the entire population (n=87) and on a subgroup of patients treated with loco-regional RT (n=44). RESULTS: 24/87 patients (28%) developed clinical pneumonitis; 28/81 patients (35%) had radiological side effects on chest X-rays and 11/75 patients (15%) showed radiological density changes on Computed Tomography (CT). The analysis showed that the risk of clinical pneumonitis was a smooth function of EUD (calculated from DVH using n=0.86+/-0.10, best fit result). With LEUD, the relationship between EUD and NTCP could be described with a D(50) of 16.4Gy+/-1.1Gy and a steepness parameter m of 0.36+/-0.7. The results found in the overall population were substantially confirmed in the subgroup of patients treated with loco-regional RT. CONCLUSIONS: A large group of prospective patient data (87 pts), including grade 1 pneumonitis, were analysed. The four NTCP models fit quite accurately the considered endpoints. EUD or the mean lung dose are robust and simple parameters correlated with the risk of pneumonitis. For all endpoints the D(50) values ranged in an interval between 10 and 20Gy.