Recognition of activated CSF-1 receptor in breast carcinomas by a tyrosine 723 phosphospecific antibody.

The macrophage colony stimulating factor receptor (CSF-1R), the product of the c-fms proto-oncogene, plays an important role in regulating the normal proliferation and differentiation of macrophages and trophoblasts. However, the abnormal expression of CSF-1R transcripts and protein by human breast carcinomas has been shown to correlate with advanced stage and poor prognosis. Ligand activated CSF-1R dimers transphosphorylate several tyrosines in their cytoplasmic domains which provide recognition sites for various effector proteins in multiple signal transduction pathways. In cells transformed by the c-fms oncogene, one of the major CSF-1R phosphotyrosines, pTyr723 is important for phenotypic expression of anchorage-independent growth and metastasis. In order to investigate the relationship between receptor activation/phosphorylation and cellular phenotypes in vitro and in vivo, we prepared a CSF-1R phosphorylation-state specific antibody raised against a specific phosphopeptide of CSF-1R, which included phosphorylated tyrosine 723. On immunoblots of lysates from cells expressing CSF-1R, this antibody recognizes phosphorylated CSF-IR in CSF-1 stimulated cells but not in unstimulated cells. As an immunohistochemical reagent, this antibody stained 52% of invasive human breast tumors (72% of CSF-1R positive cases) in a sample of 114 cases and 38% of carcinoma in situ. This data represents the first direct evidence of in vivo phosphorylation of CSF-1R in human breast carcinomas.

Prognostic significance of colony-stimulating factor receptor expression in ipsilateral breast cancer recurrence.

The macrophage colony-stimulating factor receptor (CSF-1R), the product of the c-fms proto-oncogene, regulates normal proliferation and differentiation of macrophages and trophoblasts. Recent research found abnormal expression of CSF-1R in human carcinomas of the breast, endometrium, and ovary. Furthermore, activation of CSF-1R by its ligand has been shown to regulate invasiveness and anchorage-independent growth in breast carcinoma cells. To study the significance of CSF-1R expression in breast cancer, we designed a case-controlled immunohistochemical study. We chose 80 patients from a database of 1200 early stage I or II breast cancer patients treated with conservative surgery and radiation therapy. Expression of CSF-1R in the tumors of 40 patients who experienced an ipsilateral breast tumor recurrence (IBTR) as a primary site of relapse were compared with 40 patients who had not experienced an IBTR. The index and control patients were matched by age, clinical stage, nodal status, and follow-up. Paraffin-embedded sections were immunostained with antibodies directed toward CSF-1R. For the CSF-1R antibody, a total of 28 index cases (70%) demonstrated strong staining, whereas only 16 control cases (40%) demonstrated high immunoreactivity (P = 0.007). The CSF-1R antibody showed a positive correlation for local relapse, but no correlation was found between CSF-1R expression and distant metastasis. In summary, our findings provide evidence for the poor prognostic role of CSF-1R in IBTR.

Non-infectious complications of transfusion therapy.

Blood transfusion is considered safe when the infused blood is tested using state of the art viral assays developed over the past several decades. Only rarely are known viruses like HIV and hepatitis C transmitted by transfusion when blood donors are screened using these sensitive laboratory tests. However, there are a variety of transfusion risks which still remain that cannot be entirely eliminated, many of which are non-infectious in nature. Predominantly immune-mediated complications include the rapid intravascular or slow extravascular destruction (hemolysis) of transfused red cells or extravascular removal of platelets by pre-formed antibodies carried by the transfusion recipient. Alternatively, red cells can be damaged when exposed to excessive heat or incompatible intravenous fluids before or during the transfusion. Common complications of blood transfusion that at least partly involve the immune system include febrile non-hemolytic and allergic reactions. While these are usually not life-threatening, they can hamper efforts to transfuse a patient. Other complications include circulatory overload, hypothermia and metabolic disturbances. Profound hypotensive episodes have been described in patients on angiotensin-converting enzyme (ACE) inhibitors who receive platelet transfusions through bedside leukoreduction filters. These curious reactions appear to involve dysmetabolism of the vasoactive substance bradykinin. Products contaminated by bacteria during blood collection and transfused can cause life-threatening septic reactions. A long-term complication of blood transfusion therapy unique to chronically transfused patients is iron overload. Less common - but serious - reactions more specific to blood transfusion include transfusion-associated graft-versus-host disease and transfusion-associated acute lung injury. Many of these complications of transfusion therapy can be prevented by adhering to well-established practice guidelines. In addition, individuals who administer blood transfusions should recognize these complications in order to be able to quickly provide appropriate treatment.

Integration of a hematopoietic progenitor cell program using the ACT/DB database system.

Infusion of hematopoietic progenitor cells following high-dose chemotherapy is frequently used to treat patients with hematological malignancies and solid tumors. We have developed a comprehensive software system to monitor these patients once they are entered into an experimental protocol. The captured data encompasses all phases of progenitor cell therapy including progenitor cell mobilization and collection, stem cell processing, as well as cell infusion and engraftment kinetics. Particular attention was paid to the quality assurance and quality control functionality of the software during development of data entry forms and reports. The system was developed using the ACT/DB client-server database, which utilizes Microsoft Access as a front-end and accesses either an Oracle or SQL Server database. ACT/DB has been modified for deployment on the Internet in order to take advantage of Web-based technology. Information technology can help to integrate the diverse data requirements of complex therapeutic trials.

Effects of the submarine environment on renal-stone risk factors and vitamin D metabolism.

The effects of total sunlight deprivation on urinary risk factors for nephrolithiasis and vitamin D metabolism were studied in 20 healthy male subjects. Blood and 24-h urine samples were collected before submarine deployment and 68 days later while still at sea. No subject received sunlight exposure during the test interval. Significant decreases in daily urinary excretion of calcium, uric acid, sodium, sulfate, and phosphorus were found. The relative supersaturation ratio of monosodium urate also fell. There was no change in urinary citrate or urine volume. Mean serum levels of 25-hydroxyvitamin D [25(OH)D] declined from 31 to 19 pg/ml (P < 0.0001), parathyroid hormone increased from 22 to 30 pg/ml (P < 0.0001), and osteocalcin (GLA) increased from 2.7 to 3.3 ng/ml (P = 0.005). Mean serum levels of 1,25 dihydroxy-vitamin D were unchanged. Four subjects had 25(OH)D levels below 10 ng/ml by the end of the submarine patrol. These findings suggest that exposure to the submarine environment produces physiologic changes that decrease the risk for renal stone formation. The data are consistent with the role of vitamin D metabolism in sunlight deprivation and demonstrate that compensatory mechanisms are well established within 68 days.

The efficacy of the placental biopsy.

OBJECTIVE: Our purpose was to determine the sensitivity and specificity of pathologic diagnoses made from a placental biopsy specimen compared with diagnoses made from a complete placental examination. STUDY DESIGN: Biopsy was performed on 200 singleton placentas with a 16-gauge Rutner biopsy needle shortly after delivery. The biopsy specimens and placentas were evaluated by standard placental pathologic criteria. RESULTS: The presence of villous edema on the biopsy specimen led to the diagnosis of placental villous edema with a sensitivity of 51% and specificity of 86%, yielding a positive predictive value of 0.97. The sensitivity of the biopsy diagnosis of "increased syncytial knots" was 86%, whereas the specificity was 82%, yielding a positive predictive value of 0.90. CONCLUSIONS: Because a placental biopsy specimen after delivery is reasonably sensitive for diagnosing villous abnormalities that reflect acute and chronic stresses to the placenta, it may be useful to develop a placental biopsy that can be performed safely during pregnancy. Such a biopsy could be the basis for the rational treatment of some diseases of pregnancy.

Applying a novel statistical process control model to platelet quality monitoring.

BACKGROUND: Many countries are implementing universal WBC reduction of blood components Thus, manufacturing procedures must include QC techniques to detect units that fail to meet established standards. STUDY DESIGN AND METHODS: A statistical process control model, based on the exponentially weighted moving average of the cumulative distribution function (CDF-EWMA), was developed to detect shifts in a mean and/or variance of a process. The model's parameters (weights) were optimized to maximize detection of an out-of-control process while minimizing sensitivity to autocorrelation. Validation was performed using a retrospective set of WBC-reduction data obtained from a blood bank. The WBC-reduction process was considered in control when there was 95-percent confidence that more than 95 percent of platelet concentrates would contain less than 1 x 10(6) WBCs (6.0 log WBC) as required by European standards. A sentry setting of 5.7 log WBCs was used to allow earlier detection of an out-of-control process. RESULTS: Graphic output of the CDF-EWMA model provided a continuous update of the probability that a WBC-reduction process was in control. Using the validation data, the model showed that the process was in control until Observation 332, at which point residual WBCs per unit increased. However, the first platelet concentrate to exceed specified criteria (Observation 346) occurred after the model detected that the process was out of control, demonstrating the forecasting value of this model. This deviation corresponded to an equipment failure in a single apheresis instrument. The Shewhart and EWMA techniques were similarly able to detect when the process was out of control using the test data. CONCLUSION: As a statistical process control model, the CDF-EWMA provides real-time estimation of the fraction of components meeting a regulatory limit. It is capable of detecting developing QC problems before units fail to meet regulatory requirements and is a potential alternative to other QC techniques for monitoring WBC reduction of blood components.

Removal of anaphylatoxins C3a and C5a and chemokines interleukin 8 and RANTES by polyester white cell-reduction and plasma filters.

BACKGROUND: A few bedside polyester white cell (WBC)-reduction filters have been shown to scavenge C3a anaphylatoxin from stored blood components. One has been shown to remove the chemokines interleukin (IL)-8 and RANTES, but not the proinflammatory cytokines IL-1, IL-6, and tumor necrosis factor alpha. Removal by any filter of the anaphylatoxin C5a or the soluble membrane attack complex (SC5b-9) has not been studied. Further, the ability of other filters to scavenge these biologic response modifiers (BRM) is not known. Four WBC-reduction filters and one plasma filter were studied for their ability to remove IL-8, RANTES, IL-1 beta, C3a, C5a, and SC5b-9. STUDY DESIGN AND METHODS: Plasma was obtained either as freshly thawed fresh-frozen plasma, fresh-frozen plasma thawed and stored for 5 days, or platelet-poor supernatant. Cell-poor plasma was obtained and samples were taken before and after filtration through the various filters Levels of IL-1 beta, IL-8, RANTES, C3a, and SC5b9 were quantitated by enzyme immunoassay. To evaluate filter scavenging of C5a, an in vitro model was developed to generate high levels of C5a in plasma by activating plasma with zymosan. RESULTS: Levels of C3a, C5a, IL-8, and RANTES were reduced by filtration through two bedside platelet WBC-reduction filters, a plasma filter, and a prestorage red cell WBC-reduction filter, but not following filtration through a prestorage platelet WBC-reduction filter. For some BRMs and filters, however, evidence of filter saturation was seen. IL-1 beta was not removed by any of the filters tested. CONCLUSION: Some, but not all, bedside polyester filters and prestorage polyester filters can remove IL-8, RANTES, C3a, and C5a from units of plasma or platelets. Improved biomaterial engineering of these and other filters could maximize scavenging of BRMs and potentially diminish the adverse reactions associated with their infusion during transfusion.

Liesegang rings and endometriosis.

Liesegang rings (LRs) are acellular, ringlike structures that may from within and around inflamed or necrotic tissue. LRs are most commonly found within the kidneys, synovium, and eyelid and in association with pelvic inflammatory disease and other infectious processes. LRs are only rarely found within the female genital tract, usually within endometriotic cysts or around areas of chronic inflammation. Three additional patients with LRs associated with endometriosis are described. In one of them, LRs occurred at the edge of an endometriotic cyst adjacent to a well-differentiated endometrioid adenocarcinoma of the ovary. All cases were characterized by the presence of multiple eosinophilic, sharply demarcated ringlike structures that were highlighted by the periodic acid-Schiff method. LRs within the female genital tract, which appear to be closely related to endometriosis, should be distinguished from both benign or malignant processes.

Inducible nitric oxide synthase with transitional cell carcinoma of the bladder.

PURPOSE: Nitric oxide (NO) plays a critical role as both a cell signaling molecule and as a cytotoxic/cytostatic mediator. Nitric oxide synthase (NOS) present in macrophages and neutrophils produces NO in response to immune stimulation. We evaluated NO production in both bladder tissue and urine from patients with transitional cell carcinoma (TCC) of the bladder. MATERIALS AND METHODS: Inducible NOS (iNOS) RNA and protein were evaluated in bladder tissue from patients with and without TCC. Human iNOS-RNA products were identified with the reverse transcriptase-polymerase chain reaction (RT-PCR). Western blot analysis using a polyclonal antibody directed against iNOS recognized immunoreactive iNOS protein. Using the same iNOS antibody, the distribution of iNOS was examined in formalin-fixed, paraffin embedded samples of various grades of TCC. NOS activity was measured in the urine particulate fraction from patients with TCC and from controls by the conversion of [14C]-L-arginine to [14C]-L-citrulline. RESULTS: Inducible NOS-RNA products and iNOS specific proteins were found in bladder tissue that contained TCC but not in control bladder tissue. Inducible NOS was uniformly localized in inflammatory cells within the carcinomas. Scattered tumor cells expressed iNOS in 8 of 12 specimens. There was no clear relationship between tumor immunoreactivity and tumor grade. NOS activity in urine from patients with TCC was not significantly elevated or decreased in comparison with control urine. CONCLUSIONS: Inducible NOS is expressed by cells comprising and surrounding human bladder tumors. It is primarily localized to inflammatory cells, but also is demonstrated within individual tumor cells.

Effects of methylene blue-treated plasma on red cells and stored platelet concentrates.

BACKGROUND: Photochemical methods can effectively inactivate extracellular viruses and bacteria found in blood components. Treatment of plasma with methylene blue (MB), a phenothiazine dye, and visible light inactivates enveloped viruses including HIV-1. The effects of MB-treated plasma on cellular components stored in vitro have not been well characterized. STUDY DESIGN AND METHODS: MB-treated plasma (83 microg MB/250 mL plasma) was added to single-donor platelets, stored AS-1 red cells (RBCs), irradiated RBCs, and frozen-deglycerolized RBCs. In vitro platelet assays performed after 1 and 5 days of storage in MB-treated plasma included pH, pO2, pCO2, HCO3, platelet number, lactate dehydrogenase, glucose, osmotic recovery, and CD62 expression. RBC components were examined at specific intervals for leakage of potassium, plasma hemoglobin level, and percentage of hemolysis. Direct antiglobulin tests, osmotic fragilities, and RBC antigen stability tests were also performed on RBCs stored in MB-treated plasma. Components stored with autologous plasma or nontreated allogeneic plasma served as controls. RESULTS: Similar storage-induced changes in pH, glucose, and platelet numbers, as well as increases in lactate dehydrogenase, CD62 expression, and lactate were seen in single-donor platelets stored with MB-treated and control plasma. Platelet morphology scores and osmotic recoveries were not altered. Plasma hemoglobin and potassium and percentage of hemolysis increased equally in the various RBC components stored with MB-treated or nontreated plasma. Osmotic fragility and RBC antigen stability were not appreciably altered by MB-treated plasma. CONCLUSION: Plasma treated by MB photoinactivation can be used for in vitro resuspension and storage of platelets or RBCs, because of the lack of influence of MB-treated plasma on a variety of in vitro platelet and RBC assays.