Abnormal function of the bone marrow microenvironment in chronic myelogenous leukemia: role of malignant stromal macrophages

The bone marrow microenvironment supports and regulates the proliferation and differentiation of hematopoietic cells. Dysregulated hematopoiesis in chronic myelogenous leukemia (CML) is caused, at least in part, by abnormalities in CML hematopoietic progenitors leading to altered interactions with the marrow microenvironment. The role of the microenvironment itself in CML has not been well characterized. We examined the capacity of CML stroma to support the growth of long-term culture-initiating cells (LTC-IC) obtained from normal and CML marrow. The growth of normal LTC-IC on CML stroma was significantly reduced compared with normal stroma. This did not appear to be related to abnormal production of soluble factors by CML stroma because normal LTC- IC grew equally well in Transwells above CML stroma as in Transwells above normal stroma. In addition, CML and normal stromal supernatants contained similar quantities of both growth-stimulatory (granulocyte colony-stimulating factor (CSF), interleukin-6, stem cell factor, granulocyte-macrophage CSF, and interleukin-1 beta) and growth- inhibitory cytokines (transforming growth factor-beta, macrophage inflammatory protein-1 alpha, and tumor necrosis factor-alpha). The relative proportion of different cell types in CML and normal stroma was similar. However, polymerase chain reaction and fluorescence in situ hybridization studies showed the presence of bcr-abl-positivo cells in CML stroma, which were CD14+ stromal macrophages. To assess the effect of these malignant macrophages on stromal function, CML and normal stromal cells were separated by fluorescence-activated cell sorting into stromal mesenchymal cell (CD14-) and macrophage (CD14+) populations. CML and normal CD14- cells supported the growth of normal LTC-IC equally well. However, the addition of CML macrophages to normal or CML CD14- mesenchymal cells resulted in impaired progenitor support. This finding indicates that the abnormal function of CML bone marrow stroma is related to the presence of malignant macrophages. In contrast to normal LTC-IC, the growth of CML LTC-IC on allogeneic CML stromal layers was not impaired and was significantly better than that of normal LTC-IC cocultured with the same CML stromal layers. These studies demonstrate that, in addition to abnormalities in CML progenitors themselves, abnormalities in the CML marrow microenvironment related to the presence of malignant stromal macrophages may contribute to the selective expansion of leukemic progenitors and suppression of normal hematopoiesis in CML.     

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)     

Effect of the inserted active-site-covering lid loop on the catalytic activity of a mutant B. subtilis γ-glutamyltransferase (GGT)

γ-Glutamylpeptides are compounds derived from the acylation of an amino acid or a short peptide by the γ-carboxyl carbon of the side chain of glutamic acid. Due to their altered chemico-physical and organoleptic properties, they may be interesting substitutes or precursors of parent compounds used in pharmaceutical, dietetic and cosmetic formulations. Some of them are naturally occurring flavor enhancers or are endowed with biological activities. Enzymatic approaches to the synthesis of γ-glutamyl derivatives based on the use of γ-glutamyltransferases (GGTs, EC 2.3.2.2) have been proposed, which should be able to alleviate the problems connected with the troublesome and low-yielding extraction from natural sources or the non-economical chemical synthesis, which requires protection/deprotection steps. With the aim of overcoming the current limitations in the use of GGTs as biocatalysts, a mutant GGT was investigated. The mutant GGT was obtained by inserting the active-site-covering lid loop of the E. coli GGT onto the structure of B. subtilis GGT. With respect to the wild-type enzyme, the mutant showed a more demanding substrate specificity and a low hydrolase activity. These results represent an attempt to correlate the structural features of a GGT to its different activities. However, the ability of the mutant enzyme to catalyze the subsequent addition of several γ-glutamyl units, inherited by the parent B. subtilis GGT, still represents a limitation to its full application as a biocatalyst for preparative purposes.

A mutant γ-glutamyltransferase with improve transpeptidase activity was obtained by inserting the active site-covering lid loop on an enzyme naturally lacking it.     

Assessment of a daily online implanted fiducial marker‐guided prostate radiotherapy process

The aims of this study were to investigate whether intrafraction prostate motion can affect the accuracy of online prostate positioning using implanted fiducial markers and to determine the effect of prostate rotations on the accuracy of the software‐predicted set‐up correction shifts. Eleven patients were treated with implanted prostate fiducial markers and online set‐up corrections. Orthogonal electronic portal images were acquired to determine couch shifts before treatment. Verification images were also acquired during treatment to assess whether intrafraction motion had occurred. A limitation of the online image registration software is that it does not allow for in‐plane prostate rotations (evident on lateral portal images) when aligning marker positions. The accuracy of couch shifts was assessed by repeating the registration measurements with separate software that incorporates full in‐plane prostate rotations. Additional treatment time required for online positioning was also measured. For the patient group, the overall postalignment systematic prostate errors were less than 1.5 mm (1 standard deviation) in all directions (range 0.2–3.9 mm). The random prostate errors ranged from 0.8 to 3.3 mm (1 standard deviation). One patient exhibited intrafraction prostate motion, resulting in a postalignment prostate set‐up error of more than 10 mm for one fraction. In 14 of 35 fractions, the postalignment prostate set‐up error was greater than 5 mm in the anterior–posterior direction for this patient. Maximum prostate rotations measured from the lateral images varied from 2° to 20° for the patients. The differences between set‐up shifts determined by the online software without in‐plane rotations to align markers, and with rotations applied, was less than 1 mm (root mean square), with a maximum difference of 4.1 mm. Intrafraction prostate motion was found to reduce the effectiveness of the online set‐up for one of the patients. A larger study is required to determine the magnitude of this problem for the patient population. The inability in the current software to incorporate in‐plane prostate rotations is a limitation that should not introduce large errors, provided that the treatment isocentre is positioned near the centre of the prostate.     

An ultrasonographic study of Peyronie's disease

A study was undertaken to determine the usefulness of ultrasonography as an investigative tool, and its role in deciding the management of Peyronie's disease. Fifteen patients with Peyronie's disease were studied by ultrasonography. The plaque could be demonstrated in all patients. The dimensions of the plaque varied from less than 1 cm to more than 7cm in length and 2-4mm in thickness. The disease was active in 26% of the patients, as indicated by the presence of hypoechoic areas around a central region of hyperechoism. Ultrasonogram was more accurate than clinical assessment in delineating the extent of lesions. In one-third of the patients, sonography demonstrated the plaques to be more extensive than had been detected by clinical examination. Calcification and activity of disease (which are clearly defined by ultrasonogram) are determining factors in the management of Peyronie's disease. This information allows the surgeon to select the modality of treatment, the timing of surgery and extent of excision. Thus, ultrasonography plays a vital role in the preliminary investigation and management of Peyronie's disease.     

Radioimmunologic assay of PRL,E2 and prosterone,before and after pregnancy termination by metergoline and sulpiride

The authors have assayed plasma prolactin, estradiol, and progesterone in 30 women undergoing voluntary termination of pregnancy by vacuum aspiration under general anesthesia, in order to study in more detail, the correlations between PRL and sexual steroid hormones during the 1st trimester of pregnancy. Results obtained in the various plasma samples taken at intervals until 5 hours following vacuum curettage show a marked increase in PRL in the control group, from 80 to 3000 ng/ml, with a progressive decrease at the 5th hour. A much slighter increase was found in patients treated with Metergoline, compared to that of the former group, with an increase from 90 to 180 ng/ml, while in those patients treated with Sulpiride, the maximum increase ranges between 160-340 ng/ml. The plasma level of E2 decreased significantly and progressively after the sample taken at 30 minutes, with a similar pattern in the 3 groups. The plasma progesterone concentrations showed a similar patern in the groups treated with Metergoline and Sulpiride, with a progessive decrease in comparison with basal levels. On the contrary, the increase was found in the control groups, in comparison with basal values, in samples taken at 30 minutes, with a slow, progressive decrease in subsequent samples. It can be concluded from these results that the stress caused by vacuum curettage under anesthesia causes a stronger stimulation of PRL than the inhibition of this hormone caused by the fall of E2 and P concentrations; the inhibitory effect of Metergoline on PRL did not completely annul the effect of anesthesia and vacuum curettage on PRL production. While E2 shows a progressive decrease compared to basal values, with a similar behavior in all 3 groups, P, which decreases in the 2 groups that have undergone pharmacological treatment, shows a peak increase in the sample taken at 30 minutes. THe significance of this observation is not yet clear, and will be the subject of further study. (author's modified)     

Control of aerostasis in pulmonary metastasectomy by precision resection. Standard technique versus fibrin glue modification

The control of aerostasis after performing non-anatomical pulmonary resections constitutes a serious problem. The presence of an air leak in the postoperative period requires a prolonged thoracic drainage and consequently a longer hospital stay. The aim of our study was to evaluate the usefulness of fibrin glue and its effectiveness in the prevention of air leaks. At the Department of Thoracic Surgery of the National Cancer Institute of Milan, we conducted a case-control study in 90 patients submitted to metastasectomy for secondary lung cancer, removing multiple small nodules < or = 1.5 cm using the precision resection technique. We divided the patients into two groups, both of 45 subjects: group 1 treated with fibrin glue and group 2 submitted to cauterization of the pulmonary parenchyma. The patient characteristics were well matched for age, type of approach and operation, number of resections performed and type of pathology. The assessment parameters investigated were the duration of the air leak, expected complications, drainage time and length of hospital stay. In group 1 we performed fewer than 5 precision resections in 21 cases, from 5 to 10 in 16, and more than 10 in 8. In group 2 we executed fewer than precision resections in 21 cases, from 5 to 10 in 17, and more than 10 in 7. In group 1 the duration of the air leak was 2.93 +/- 1.91 days as against 6.95 +/- 7.01 days in group 2 (p = 0.000). In group 1 we had one complication (2%) (a long-term air leak lasting > 10 days), while in group 2 we had a long-term air leak in 11 cases (24%) (p = 0.000). Mean thoracic drainage time was 4.22 +/- 1.43 days in group 1, and 8.13 +/- 7.37 in group 2 (p = 0.000). The mean postoperative hospital stay was 6.22 +/- 1.43 days in group 1 compared to 10.13 +/- 7.37 days in group 2 (p = 0.000). In the group of patients treated with fibrin glue we obtained a significant reduction in drainage time, complications and postoperative hospital stay. The results of our experience show that the use of fibrin glue in non-anatomical resections with a high risk of developing air leakage is effective in reducing the expected complications, with a favourable impact also on the quality of life of patients with metasases.     

Gold standard for sternectomies and plastic reconstructions after resections for primary or secondary sternal neoplasms

Background Between January 1980 and December 1999, 88 patients underwent thesurgical resection of sternal tumors: 30 primary malignant tumors, 28 local relapses or metastases from breast carcinomas, 16 other types of tumor, and 14 radionecroses. Methods The sternectomies were total in 8 cases, subtotal in 32, and partial in 48. Prosthetic materials covered by flaps of myocutaneous or muscle tissue were used in 55 patients, prosthetic material alone in 13, myocutaneous or muscle flaps alone in 5, and other techniques in the remaining 15. The resection was radical in 78 cases and palliative in the other 10 Results Forty-eight of the subjects who underwent radical surgery were alive and disease free at the end of the follow-up period. The expected 10-year survival of the patients treated for primary tumors is approximately 85% (Kaplan-Meier), and that of the patients with relapsing breast carcinomas is the same as after 5 years (41.8%). Conclusions In our experience, the treatment of neoplasms by means of a broad sternal resection followed by a reconstruction based on the use of prosthetic materials is an effective and safe solution that considerably improves the quality of life and makes it possible to perform curative broad radical resections in the case of primary sternal resections. Presented at the 54th Annual Cancer Symposium of the Society of Surgical Oncology, Washington, DC, March 15–18, 2001.     

Clinical evaluation of biomarkers in Gaucher disease

Novel or candidate biomarkers require thorough evaluation to establish their utility in a clinical setting. This paper describes an evaluation of several established enzyme markers of Gaucher disease and a newly-described chemokine, pulmonary and activation-regulated chemokine (PARC). The ability of the biomarkers to rank patients with Gaucher disease in order of disease severity and organ bulk, and to reflect changes in key clinical parameters in response to enzyme replacement therapy were evaluated. PARC concentrations were found to be reliably correlated with visceral disease and with key clinical responses to enzyme replacement in an unbiased manner. Unlike chitotriosidase and serum angiotensin-converting enzyme activity, genetic variation in serum PARC did not appear to influence its utility as a biomarker.
Conclusion: For each new candidate biomarker of lysosomal storage diseases, a similar clinical evaluation will be required, though the approach will need to be modified according to the clinical features and natural history of each disorder.