Showing papers in "Annals of Clinical and Laboratory Science in 2000"

Review : Free radicals, antioxidants, and the immune system

Abstract: Oxygen-derived free radicals are important in both natural and acquired immunity. Neutrophil and macrophage phagocytosis stimulates various cellular processes including the "respiratory burst" whereby increased cellular oxygen uptake results in the production of the potent oxidant bactericidal agents, hypochlorous acid and hydroxyl radical. In addition, nitric oxide, a gaseous radical produced by macrophages, reacts with superoxide to form peroxynitrite, also a potent bactericidal agent. Conversely, oxidative stress may be detrimental in acquired immunity by activation of nuclear factor kappa B, which governs gene expression involving various cytokines, chemokines, and cell adhesion molecules, among others. However, antioxidant supplementation essentially reverses several age-associated immune deficiencies, resulting in increased levels of interleukin-2, elevated numbers of total lymphocytes and T-cell subsets, enhanced mitogen responsiveness, increased killer cell activity, augmented antibody response to antigen stimulation, decreased lipid peroxidation, and decreased prostaglandin synthesis.

Molecular pathology of cyclooxygenase-2 in neoplasia

Abstract: Cyclooxygenase (COX)-2 levels are elevated in several types of human cancer tissues. Nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit both the COX-1 and COX-2 protein, the two enzymes that convert arachidonic acids to prostaglandins. Regular use of such NSAIDs significantly reduces the risk and spread of some cancers. The objective of this study was to elucidate the molecular pathology of neoplasms that overexpress COX-2. Epidemiological data and clinical studies were analyzed and compared with results of studies of human tumor tissues, animal models, and cultured tumor cells. COX-2, but not COX-1, is highly expressed in human colon carcinoma, squamous cell carcinoma of the esophagus, and skin cancer. COX-2 is inducible by oncogenes ras and scr, interleukin-1, hypoxia, benzo[a]pyrene, ultraviolet light, epidermal growth factor, transforming growth factor beta, and tumor necrosis factor alpha. Dexamethasone, antioxidants, and tumor-suppressor protein p53 suppress COX-2 expression. COX-2 synthesizes prostaglandin E2 (PGE2) which stimulates bcl-2 and inhibits apoptosis, and induces interleukin-6 (IL-6) which enhances haptoglobin synthesis. PGE2 is associated with tumor metastases, IL-6 with cancer cell invasion, and haptoglobin with implantation and angiogenesis. Drastic reduction in polyp number results from COX-2 gene knockout as well as from selective COX-2 inhibition in a mouse model of human familial adenomatous polyposis. Nonselective NSAIDs, for instance aspirin, and selective COX-2 inhibitors such as celecoxib (SC-58635) and NS-398 suppress azoxymethane-induced colon carcinogenesis in rats. Aspirin, indomethacin, and ibuprofen decrease cultured lung cancer cell proliferation. Selective inhibition of COX-2 is preferable to nonselective inhibition. It reduces cancer cell proliferation, induces cancer cell apoptosis, and spares COX-1-induced cytoprotection of the gastrointestinal tract.

Effects of iron overload on the immune system

Abstract: Iron and its binding proteins have immunoregulatory properties, and shifting of immunoregulatory balances by iron excess or deficiency may produce severe, deleterious physiological effects. Effects of iron overload include decreased antibody-mediated and mitogen-stimulated phagocytosis by monocytes and macrophages, alterations in T-lymphocyte subsets, and modification of lymphocyte distribution in different compartments of the immune system. The importance of iron in regulating the expression of T-lymphocyte cell surface markers, influencing the expansion of different T-cell subsets, and affecting immune cell functions can be demonstrated in vitro and in vivo. The poor ability of lymphocytes to sequester excess iron in ferritin may help to explain the immune system abnormalities in iron-overloaded patients. Iron overload as seen in hereditary hemochromatosis patients enhances suppressor T-cell (CD8) numbers and activity, decreases the proliferative capacity, numbers, and activity of helper T cells (CD4) with increases in CD8/CD4 ratios, impairs the generation of cytotoxic T cells, and alters immunoglobulin secretion when compared to treated hereditary hemochromatosis patients or controls. A correlation has recently been found between low CD8+ lymphocyte numbers, liver damage associated with HCV positivity, and severity of iron overload in beta-thalassemia major patients. Iron overload, with its associated increases of serum iron levels and transferrin saturation, may cause a poor response to interferon therapy. Iron overload with hyperferremia is associated with suppressed functions of the complement system (classic or alternative types). High plasma ferritin content in patients with chronic, diffuse diseases of the liver (cirrhosis, chronic hepatitis), beta-thalassemia major, dyserythropoiesis, and hereditary hemochromatosis may induce the development of anti-ferritin antibodies with the production of circulating immune complexes. Increased body stores of iron in various clinical situations may tip the immunoregulatory balance unfavorably to allow increased growth rates of cancer cells and infectious organisms, and complicate the clinical management of preexisting acute and chronic diseases.

Assessment of Her-2/neu overexpression in primary breast cancers and their metastatic lesions: an immunohistochemical study

Abstract: Since the development of novel immunotherapy using Herceptin as the first agent specifically indicated for HER-2/neu overexpression in metastatic breast cancer, there has been interest in using HercepTest as a predictor of response to such therapy. There is debate whether it is justifiable to perform HercepTest on every newly diagnosed breast cancer, since only approximately 43% of the cases will have related metastatic disease, and Herceptin is indicated only for breast cancer with metastatic disease. It may be more cost-effective to limit HercepTest to the related metastatic lesions. Therefore, it is important to assess whether the pattern of HER-21neu overexpression of metastatic breast cancer is also present in the primary lesion. HercepTest was performed on formalin-fixed, paraffin-embedded tissue sections of 56 primary breast cancers and their corresponding metastatic lesions. The protocol and scoring guidelines recommended by the manufacturer were followed. Tissue sections (5 microm) of a primary and the metastatic lesion from the same case were placed parallel on a single glass slide. The pattern and intensity of HER-2/neu overexpression (32%) in the primary and metastatic lesions were found to be nearly identical. Heterogeneity was observed in only one case. The score of primary cancer was 3+, and the metastatic lesion was 2+. Both were reported as positive. Intratumor heterogeneity (1+ to 3+) was also noted in two (4%) cases. However, the same pattern was found in both the primary and related metastatic lesions. The nearly identical HercepTest results in the primary and metastatic lesions suggest the potentiality of limiting the HercepTest to breast cancer-related metastases. Currently, any superficial and most deep-seated metastatic lesions can be easily sampled by fine needle aspiration biopsy or core biopsy, providing adequate samples for HercepTest. Eliminating unnecessary use of the HercepTest may provide a cost-effective alternative approach to the management of breast cancer patients.

Review: Biology and relevance of C-reactive protein in cardiovascular and renal disease.

Abstract: C-reactive protein (CRP) is a member of the pentraxin family of proteins, which are characterised by a cyclic pentameric structure and radial symmetry. The five identical 24-kDa protomers consist of 206 amino acids, and are noncovalently linked. CRP binds to a range of substances such as phosphocholine, fibronectin, chromatin, histones, and ribonucleoprotein in a calcium-dependent manner. It is a ligand for specific receptors on phagocytic leukocytes, mediates activation reactions on monocytes and macrophages, and activates complement. Plasma CRP is the classical acute-phase protein, increasing 1,000-fold in response to infection, ischemia, trauma, burns, and inflammatory conditions. A growing number of studies suggest that CRP is an independent risk factor for atherosclerotic vascular disease. Plasma CRP concentrations in the highest quartile are associated, depending on the subject group, with 1.5- to 7-fold increases in relative risk. In the high-risk endstage renal failure population, a raised CRP is associated with up to 5.5-fold increased relative risk of CVD and 4.6-fold increased relative risk of death. This review examines the relationships between CRP, cardiovascular disease, and mortality, with special reference to renal disease.

Fluorescence in situ hybridization (FISH) for detection of HER-2/neu amplification in breast cancer: a multicenter portability study.

Abstract: Amplification and/or overexpression of HER-2/neu has been shown to be both a prognostic and predictive marker in breast cancer. Recent studies have also confirmed the efficacy of Herceptin (trastuzumab) as adjuvant therapy for patients with overexpression of HER-2/neu. Therefore, it is critical that precise and reproducible assays be used in the clinical laboratory setting for determination of the HER-2/neu status in patients with breast cancer. The objective of this study was to determine the portability (reproducibility between different institutions) of the PathVysion HER-2 fluorescence in situ hybridization (FISH) assay used for detection of amplification of the HER-2/neu gene in formalin-fixed, paraffin-embedded tissue sections of invasive ductal carcinoma of the breast. Study specimens consisted of one breast tumor with a normal HER-2/neu copy number, two tumors with a low level, and one tumor with a high level of HER-2/neu amplification. The PathVysion HER-2 assay was shown to be highly reproducible on different assay days (n = 3) and between different institutions (n = 5) in the detection of amplification of the HER-2/neu gene in routinely processed clinical specimens of breast carcinoma. In addition, this study examined the feasibility of enumerating FISH signals in 20 nuclei in contrast to 60 nuclei per specimen. Although a modest increase in variation was observed when analyzing 20 compared to 60 nuclei, the mean ratios were similar. Therefore, analysis of as few as 20 nuclei with this FISH HER-2/neu assay may be sufficient for determining the amplification level of the HER-2/neu gene.

Elevated serum chromogranin A is detectable in patients with carcinomas at advanced disease stages.

Abstract: Chromogranin A (CgA), a marker of neuroendocrine cells and an indicator for neuroendocrine differentiation, is associated with a poor prognosis when detected in tumor tissue, based on immunohistochemical techniques. We sought to determine whether it is possible to detect elevated serum CgA in patients with commonly occurring carcinomas of non-neuroendocrine origin. CgA was measured in both random and serial serum specimens, using a serum CgA assay developed in our laboratory. Elevated levels of serum CgA were detected in patients with carcinoma of the prostate, breast, ovary, pancreas, and colon. Serum CgA levels in patients with all types of carcinoma appeared to parallel the changes of serum dominant tumor markers and were found in sera containing highly elevated tumor markers. Based on these preliminary findings, perhaps we should monitor CgA, in addition to the routinely used tumor markers, during the treatment of patients with carcinomas to determine if CgA is useful as a prognostic marker in carcinomas other than prostatic cancer.

Differential Expression of Chemokines in a Mouse Model of Wound Healing

Abstract: Macrophages have a multifaceted role in wound healing. While their initial activity may be in the degradation and elimination of damaged tissue, macrophages also produce and secrete a variety of mediators that can participate in the repair process as well. To perform these functions, macrophages must be recruited to a wound site. Our purpose was to examine the temporal and spatial expression of macrophage chemoattracting cytokines (chemokines) at a surgical wound site. A surgical wound was prepared on the dorsal aspect of B6AF1/J mice. Biopsies were obtained from the wound and a comparable nonwounded area between 6 and 72 hr after wounding. The presence or absence of various chemokine mRNAs was detected by the reverse transcriptase-polymerase chain reaction (RT-PCR). Immunohistochemical staining and in situ RT-PCR determined localization of cells producing chemokines. In wounded tissue, both macrophage chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1) were detected; however, the time of expression differed for each molecule. MCP-1 mRNA was detected at 6 hr after wounding, with decreased expression at subsequent time periods. In contrast, MIP-1 messages were not observed until 24 hr after wounding, and steadily increased thereafter. MCP-1 and MIP-1 mRNA and protein were localized predominantly in keratinocytes. The rapid and strong expression of MCP-1 and MIP-1 messages within the wound site suggests a pivotal role for these chemokines in the repair process. The differences in appearance and level of expression over time, however, suggest distinctive functions for each chemokine and indicate that the local milieu, rather than a single cytokine, influences macrophage recruitment and/or activation.

Glutathione-S-Transferase as a selective inhibitor of oncogenic ras-p21-induced mitogenic signaling through blockade of activation of jun by jun-N-terminal kinase.

Abstract: We have identified the intracellular detoxification enzyme, glutathione-S-transferase (GST), as a potent inhibitor of the activation of jun by its kinase, jun-N-terminal kinase (JNK), in vitro. All three major isozymes (alpha, mu, and pi) bind to JNK-jun complexes and inhibit activation of jun by JNK. We now find that GST inhibits JNK-induced oocyte maturation in vivo and strongly inhibits oocyte maturation induced by oncogenic ras-p21 protein, but not by insulin-activated normal cellular p21 protein. These results correlate with the finding that oncogenic, but not insulin-activated normal, p21 induces high levels of activated JNK. GST also strongly blocks induction of oocyte maturation by protein kinase C (PKC) which is a critical downstream target of oncogenic but not normal ras-p21. Thus, we have established a new function for GST as a potent physiological inhibitor of the ras-JNK-jun pathway.

On the role of hydroxyl radical and the effect of tetrandrine on nuclear factor--kappaB activation by phorbol 12-myristate 13-acetate.

Abstract: Nuclear factor kappaB (NF-kappaB) is considered to be an important target for therapeutic intervention because of its role in the regulation of proinflammatory and profibrotic mediators. The present study examined the role of hydroxyl (*OH) radical and the effect of tetrandrine, an alkaloid extracted from the Chinese medicinal herb Stephania tetrandra, on NF-kappaB activation by a tumor promoter, phorbol 12-myristate 13-acetate (PMA) in human lymphoid T cells (ie, Jurkat cells). Exogenous superoxide dismutase (SOD) enhanced the NF-kappaB activation by PMA, while catalase blocked it. Formate, a scavenger of *OH radical, also was inhibitory, as was deferoxamine, a metal chelator. These data suggest an important role of *OH radical in PMA-induced NF-kappaB activation. Incubation of the cells with tetrandrine prior to the stimulation of the cells was found to inhibit PMA-induced NF-kappaB activation. Tetrandrine activity was so potent that 50 microM of tetrandrine was sufficient to inhibit activation of NF-kappaB completely. Electron spin resonance (ESR) spin trapping was used to investigate the antioxidant action of tetrandrine using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. Tetrandrine is an antioxidant for both *OH and superoxide (O2-)radicals. The reaction rate constant of tetrandrine with *OH is 1.4 x 10(10) M(-1)sec(-1), which is comparable with several well established antioxidants, such as ascorbate, glutathione, and cysteine. The Fenton reaction (Fe(II) + H2O2-->Fe(III) + *OH + OH-) and xanthine/xanthine oxidase were used as sources of *OH and O2- radicals. The free radical scavenging activity of tetrandrine is responsible for its inhibition of PMA-induced NF-kappaB activation.

Antioxidant properties of pyrrolidine dithiocarbamate and its protection against Cr(VI)-induced DNA strand breakage.

Abstract: Pyrrolidine dithiocarbamate (PDTC) is considered an antioxidant and is frequently used to study the role of free radical reactions in various biological processes and against free radical-induced cellular injuries. However, its antioxidant properties are not characterized. In this study, electron spin resonance (ESR) was used to investigate the antioxidant potential of PDTC with hydroxyl radical ( . OH) and superoxide anion radicals (O 2 .- ). The Fenton reaction [Fe(II) + H 2 O 2 → Fe(II) + . OH + OH - )] and xanthine and xanthine oxidase were used as sources of . OH and O 2 .- radicals, respectively. The results show that PDTC effectively scavenges . OH radicals with a reaction rate constant of approximately 2.73 x 10 10 M -1 s -1 , which is comparable to other efficient . OH radical scavengers, such as ascorbate and glutathione. PDTC is also able to scavenge O 2 .- radicals. Through its antioxidant properties, PDTC protects against Cr(VI)-induced DNA strand breakage.

Prognostic factors in malignant gastrointestinal stromal tumors

Abstract: Gastrointestinal stromal tumors are a heterogeneous group of mesenchymal neoplasms of the gastrointestinal tract in which routine histopathological evaluation fails to reveal definitive evidence of differentiation. Given the heterogeneity in clinical presentation and the frequent morphological overlap, the biological behavior of these neoplasms is difficult to predict. We have evalauated, by Cox Proportional Hazards Regression Analysis, the clinicopathological features of 51 malignant gastrointestinal stromal tumors to identify predictors of survival. In the univariate analysis, survival inversely correlated with size, number of mitoses, and patient's age. In the multivariate analysis, only the degree of necrosis and phenotypic differentiation toward smooth muscle were found to be indicators of poor prognosis. Based on these results, a simple classification scheme for gastrointestinal stromal tumors is proposed. This classification appears to have great prognostic value for these tumors, and may be useful in guiding therapeutic management.

Understanding angiogenesis and its clinical applications.

Abstract: Angiogenesis is the growth of new vessels from pre-existing blood vessels. Angiogenesis is critical during embryogenesis but occurs minimally in healthy adults, except in wound repair, inflammation, female reproductive organs, and pathologic conditions. Various growth factors and proteins, elements of the extracellular matrix, components of the coagulation/fibrinolytic system, and platelets interact with the endothelial cells and pericytes of blood vessels to regulate angiogenesis. Characterization of angiogenic factors has revealed that remodeling of the extracellular matrix occurs during angiogenesis, mediated by integrins that are found on the endothelial cell surface membrane. Counter-regulatory antiangiogenic proteins and molecules that show an intricate balance in the regulation of angiogenesis have also been characterized. Components of the coagulation/ fibrinolysis cascade also play a critical role in angiogenesis. Elucidation of the mechanisms of angiogenesis has led to better understanding of certain disease states. Ongoing studies are evaluating the stimulation of angiogenesis to treat ischemic disorders, and the inhibition of angiogenesis to prevent abnormal proliferation in malignant and non-malignant disorders.

Modification of screening immunoassays to detect sub-threshold concentrations of cocaine, cannabinoids, and opiates in urine: Use for detecting maternal and neonatal drug exposures

Abstract: Testing for drugs of abuse in urine is commonplace in emergency departments and neonatal units. However, the clinical sensitivity of immunochemical screening methods is limited by the threshold concentrations used to distinguish between positive and negative specimens. Immunochemical screening methods for cocaine metabolite (benzoylecgonine), cannabinoids, and opiates in urine were recalibrated to detect drugs at lower threshold concentrations. The precision and linearity of the signals at the modified thresholds were verified by diluting drug-positive urine specimens to concentrations below the conventional cutoff concentration and measuring the rate signals in triplicate. To assess the clinical performance of the modified methods, specimens that tested negative using the unmodified assays were re-screened at the lower threshold, and specimens that re-screened positive were submitted for gas chromatographic/mass spectrometric (GC/MS) confirmation. Reproducibility of sub-threshold measurements was comparable to the unmodified assays, and rate separations between successive dilutions were sufficient to give semi-quantitative results. Using the lower thresholds, drugs were detected in 4-5% of the subjects that had screened negative at the conventional threshold concentration. GC/MS analysis confirmed the presence of cannabinoids and cocaine metabolite in 74% and 84%, respectively, of urine specimens that re-screened positive. Morphine, codeine, hydromorphone, or hydrocodone was detected by GC/MS analysis in 31% of opiate-positive re-screens.

Chromatographic measurements of hemoglobin A2 in blood samples that contain sickle hemoglobin.

Abstract: In the sickle cell syndromes, Hb A2 measurements aid in the differential diagnosis of sickle cell anemia from sickle-beta-thalassemia. The purpose of this study is to assess the Hb A2 levels in samples containing sickle hemoglobin (Hb S) by the use of an automated high performance liquid chromatography system (HPLC-Variant beta-thalassemia Short Program). The blood samples analyzed were from individuals of African descent living in the state of Tennessee who had either sickle cell trait (Hb AS), sickle cell disease (Hb SS), or sickle cell-hemoglobin C disease (Hb SC). Interestingly, the Hb A2 levels determined by HPLC were found elevated in samples containing Hb S. The Hb A2 mean in Hb AS samples (n=146) is 4.09% (SD +/- 0.42, range 2.20 to 5.20%); in Hb SS samples (n=33) it is 3.90% (SD +/- 1.08, range 0.60 to 5.90%); and in Hb SC samples (n=27) it is 4.46% (SD +/- 0.70, range 2.30 to 5.91%). The Hb A2 mean by HPLC in normal individuals (Hb AA, n=70) is 2.57% (SD +/- 0.25, range 2.1 to 3.0%), and the Hb A2 range in beta-thalassemia carriers is 4 to 9%. Our results show that the Hb A2 levels in Hb S-containing samples partially overlap with those expected from beta-thalassemia carriers. The hemoglobinopathy laboratory should be aware of this apparent elevation in Hb A2 levels determined by HPLC in individuals carrying Hb S. Other factors, such as family history and clinical symptoms, should be taken into account before a diagnosis of sickle cell trait, sickle-beta-thalassemia, or sickle cell anemia is made.

Adenoviral-mediated gene therapy with Ad5CMVp53 and Ad5CMVp21 in combination with standard therapies in human breast cancer cell lines

Abstract: Our objective was to determine the efficacy of adenoviral-mediated gene therapy with wild-type p53 or p21 in human breast cancer cells and investigate interactions with radiation and chemotherapy. Two human breast cancer cell lines, MDA-MB-231 and MDA-MB-435, both with p53 mutations, were transduced with adenoviral vectors containing wild-type p53 (Ad5CMV-p53) or p21/WAF1/Cip1 (Ad5CMV-p21), and the effects on growth were determined. Infection was combined with low-dose (1.4 - 3.7 Gy) irradiation to see if this would improve transduction efficiency and enhance numbers of cells killed. Transduction with either vector resulted in expression of p21WAF1/cip1 and growth inhibition, although Ad5CMV-p53 transduction produced greater growth inhibition than did Ad5CMV-p21. The cell lines differed in sensitivity to the vectors. The Ad5CMV-p53 vector in a multiplicity of infection (MOI) of 125 resulted in 50% to 80% inhibition of MDA-MB-231, while MOI 250 of the same vector resulted in 27% inhibition of MDA-MB-435. Infection with Ad5CMV-p21 produced modest growth inhibition in both cell lines (< or = 40% at MOI 200), although protein expression was detected at lower viral doses. Low dose gamma-irradiation (1.4 to 3.7 Gy) was used to try and improve the rate of gene transfer. Modest increases in transduction efficiency and duration of expression of a vector containing beta-galactosidase occurred in irradiated breast cancer cells. Radiation 24 hr before transduction with Ad5CMV-p53 increased the proportions of apoptotic MDA-MB-231 cells. The cells transduced with Ad5CMV-p21 were arrested in G1, yet when they were irradiated before adenoviral transduction, the overexpression of p21 protected the cells from the cytotoxic effects of the radiation. Clonogenic assays showed that Ad5CMV-p21 reduced the sensitivity of MDA-MB-231 to VP-16 and paclitaxel. Combining these drugs with Ad5CMV-p53 did not consistently or significantly decrease clonogenic survival.

Effect of aerobic training on diabetic nephropathy in a rat model of type 2 diabetes mellitus

Abstract: The effect of aerobic exercise intervention on the renal functional and ultrastructural changes associated with diabetes mellitus were studied in the obese Zucker rat, a rat model of type 2 diabetes. The obese Zucker rats began training at 18 wk of age (n=8) and were compared to obese sedentary controls (n=12) and lean sedentary nondiseased littermates (n=10). Body weight, kidney weight, serum creatinine, urine creatinine, creatinine clearance, urine IgG, urine IgG/creatinine ratio, urine total protein, urine albumin, urine albumin/creatinine ratio, glycated hemoglobin, serum fructosamine, fasting serum glucose, serum insulin, serum total cholesterol, serum triglycerides, blood pressure, and morphometric analyses of cortical glomeruli by light microscopy and electron microscopy were performed to evaluate renal function, structure, and metabolic control. The exercise training consisted of treadmill running, 5 da/wk for 1 hr/da. Exercise intervention lowered the body weight (p <0.05), reduced the percentage of glycated hemoglobin (p <0.05), and diminished the urine albumin concentration (p <0.05), compared to the obese sedentary controls. Exercise intervention did not significantly affect morphometric indices of renal ultrastructure. This study shows that aerobic exercise intervention significantly improved metabolic control and reduced albuminuria in a rat model of type 2 diabetes.

Autoantibodies to p53 in sera of patients with autoimmune thyroid disease.

Abstract: Mutations in the tumor suppressor gene, p53, lead to intracellular accumulation of abnormal p53 protein and are associated with p53 autoantibodies. p53 also accumulates in autoimmune diseases and Hashimoto's thyroiditis, but it is unknown if p53 autoantibodies occur in the latter. We measured p53 autoantibodies in the sera of 93 patients with thyroid disease and 19 patients without thyroid disease. Anti-p53 antibodies were detected in the sera from 4.2% (2/48) of patients with autoimmune thyroid disease, including one patient with Hashimoto's thyroiditis (3.7%, 1/27) and one with Graves' disease (4.8%, 1/21). A third patient with pseudohypoparathyroidism, but without thyroid disease, was also positive (1/19; 5.2%). None of 19 patients with differentiated thyroid cancer had anti-p53 antibodies. We conclude that anti-p53 antibodies can be detected in the sera from approximately 4% of patients with autoimmune thyroid disease. This finding suggests that increased DNA damage and apoptosis may be associated with autoimmune thyroid disease.

Acute renal artery and vein thrombosis after renal transplant, associated with a short partial thromboplastin time and factor V Leiden mutation.

Abstract: Renal graft thrombosis is a rare but devastating complication of renal transplantation. It accounts for one-third to one-half of early graft losses. We report a patient with acute renal artery and vein thrombosis associated with abnormally short activated partial thromboplastin time (aPTT) and factor V Leiden mutation. Vascular thrombosis developed on the ninth post-transplant day and led to a graft loss. Before transplantation, the patient had three episodes of thrombosis of arteriovenous access for hemodialysis. Our case illustrates the importance of investigating pretransplant patients for hypercoagulable states, particularly those with short aPTT.

How effective are screening tests for microalbuminuria in random urine specimens

Abstract: The effectiveness of four urine screening tests-microalbumin (MAlb), total protein (TProt), total protein/creatinine ratio (TProt/Cr R), and dipstick (DPalb) test for albumin-were evaluated for the detection of MAlb in random urine specimens. The following criteria were used to assess the effectiveness of each urine screening test: 100% specificity (no false positive results); cost effectiveness; rapidity and ease of performing the screening test; and increased laboratory efficiency. A "gold standard" for presence of MAlb in random urine samples was defined as a microalbumin/creatinine ratio (MAlb/Cr R) of > or = 30 mg/g. The least costly urine screening test was the DPalb, which, if assigned a value of 1.0, allowed a cost ranking order for the screening tests-DPalb (1.0) or = 15% for overt nephropathy. The TProt/Cr R ratio would only be effective in populations with prevalence rates of > or = 30%. Of the four urine screening tests, only DPalb would significantly streamline the process of measuring urine MAlb. The dipstick test is inexpensive, easy and rapid to perform, does not delay measuring the ratio, since there is no wait for the screening test result, and can be used by referring laboratories to screen urine specimens before they are submitted to a central laboratory, thereby reducing laboratory workload.

Urine Protein Electrophoresis and Immunofixation Electrophoresis Supplement One Another in Characterizing Proteinuria

Abstract: Urine protein electrophoresis (UPE) is often considered to have limited usefulness in evaluating proteinuria that is not associated with gammopathies. Unusual protein bands that are detected by UPE are commonly characterized by immunofixation electrophoresis (IFE). In this paper, electrophoretic gel patterns are shown to illustrate the greater sensitivity of IFE, compared to UPE. However, UPE remains useful for three applications: (1) UPE provides distinctive patterns that can indicate the source of proteinuria and is useful in assessing renal diseases that are independent of gammopathy; (2) combined use of UPE and IFE can avoid misinterpretations and repeated analyses of urine proteins, and (3) UPE can be used in conjunction with IFE to improve the quantitation of Bence-Jones proteinuria (BJP).

Cell typing the sensitized transfusion-dependent patient.

Abstract: Extended red cell typing is required for the management of transfusion-dependent patients to confirm the identity of suspected alloantibodies or determine the specificity of potential additional antibodies that may be formed in the future. Typing may be complicated by the presence of circulating allogeneic cells or a positive direct antiglobulin test. Phenotyping such individuals by hemagglutination is dependent on the separation of a reticulocyte-enriched fraction by differential centrifugation. Flow cytometric typing of reticulocytes is also possible. The effectiveness of these techniques is limited in those who are heavily transfused or have low reticulocyte counts. Heavily transfused patients with sickle cell anemia may be typed, however, following hypotonic lysis of allogeneic cells. In patients with a positive direct antiglobulin test, sensitized cells are usually typed with either direct agglutinating antisera and/or IgG antisera following elution of the autoantibody. Inactivation of some antigens during the elution process or the lack of some antisera specificities limit such typing. By designing appropriate oligonucleotide primers, polymerase chain reaction (PCR) amplification of target gene sequences for most blood group systems and the identification of a large number of their allelic specificities is now possible. Peripheral blood leukocytes can be used as the DNA source. Restriction fragment length polymorphism determination is widely adopted for the identification of allelic specificity of the amplified target sequence. Alternate strategies, including allele-specific PCR, are often employed if the genetic basis of the polymorphism is more complex than a single nucleotide substitution, or if it does not create or ablate a restriction endonuclease cleavage site. These techniques may permit genotyping of sensitized transfusion-dependent patients, and can improve transfusion safety and efficacy.

Expression of stress proteins HSP 72 & HSP 32 in response to endotoxemia

Abstract: Pretreatment with heat decreases mortality and acute lung injury in the rat septic shock model, presumably by the production of heat shock proteins (HSP). However, endotoxin, a severe cell stresser, has not been shown to induce HSP 70. We investigated the effects of severe endotoxemia on the expression of specific protective stress proteins, including HSP 72 (inducible HSP 70), HSP 32 (heme oxygenase-1), and HSP 90. Fifteen rats received intravenously either 3 mg/kg of endotoxin (E. coli O127:B8 lipopolysaccharide, LPS) (n=9) or saline (n=6). Two hr later the spleen was removed and splenocytes were separated into three groups and analyzed for specific HSP by Western blot. In Group 1, both endotoxin-treated and saline-treated splenocytes were incubated for 3 hr at 37 degrees C. In Group 2, the splenocytes were washed twice, then heat shocked for 30 min at 42 degrees C and subsequently incubated for 2.5 hr at 37 degrees C. In Group 3, splenocytes were washed twice, then incubated for 3.0 hr at 37 degrees C. HSP 90 & HSP 70c (constitutive) were present in all groups. Consistent with observations by others, HSP 72 was not induced in Group 1. HSP 72 was induced in both the saline-treated and endotoxin-treated splenocytes after heating (Group 2). However, in the absence of heat stress, HSP 72 was present in endotoxin-treated but not in saline-treated splenocytes after incubation (Group 3). Conversely, HSP 32, while present in Group 1 splenocytes, was not detected in the endotoxin-treated splenocytes of Group 2 and Group 3, but was present in the saline-treated cells. In conclusion, endotoxemic shock results in induction of HSP 72 and depletion of HSP 32, but only after the cells have been washed and further incubated.

Hemolysis during leukocyte-reduction filtration of stored red blood cells

Abstract: Hemolysis has been reported in red blood cells (RBCs) that have undergone leukocyte-reduction filtration. This study investigated whether the age of RBCs or the filter type affected hemolysis. One hundred eighty units of RBCs (adenine-saline added) were leukocyte-reduced by filtration. At each of the 6 weeks of shelf life, 10 units were filtered with the "BPF4" filter, 10 units with the "Purecell RCQ" filter, and 10 units with the "Sepacell" filter. Filtration was performed with strict adherence to the manufacturers' directions. Pre- and post-filtration samples were assayed for plasma hemoglobin by measuring the plasma absorbances at 578 nm and 562 nm. The increase of plasma hemoglobin concentration following filtration was significantly greater (p < 0.05) in older units, compared to fresher units, when the Sepacell and BPF4 filters were used. For example, the increase of plasma hemoglobin at week 6 (83.47 mg/dl:Sepacell, 128.93 mg/dl BPF4) was significantly greater than at week 1 (7.07 mg/dl Sepacell, 4.77 mg/dl BPF4) (Sepacell: p=0.008; BPF4: p=0.006). For units stored 1, 2, 4, 5, or 6 weeks, the increase of plasma hemoglobin concentration post-filtration was significantly greater with the BPF4 filter, compared to the Purecell RCQ filter (p <0.045); for units stored 5 weeks, the increase in plasma hemoglobin concentration post-filtration was significantly greater with the BPF4 filter compared to the Sepacell filter (p = 0.009). Mean filtration times were significantly longer in older units compared to fresh units. This study shows that increased storage duration of RBCs (adenine-saline added) is attended by greater hemolysis during leukocyte-reduction filtration and by prolongation of the filtration time. In addition, the amount of hemolysis may be influenced by the type of filter.

Serum lipid concentrations change with serum alkaline phosphatase activity during pregnancy.

Abstract: To investigate the relationship between serum lipids and alkaline phosphatase during normal pregnancy, we measured triglyceride, total cholesterol, HDL-cholesterol, and LDL-cholesterol concentrations and alkaline phosphatase activity in serum samples from 546 apparently healthy pregnant, postpartum, and nonpregnant women. Serum HDL-cholesterol levels did not change significantly during pregnancy, but serum triglyceride, total cholesterol, LDL-cholesterol, and alkaline phosphatase levels increased gradually as pregnancy proceeded, reached maximum values in the third trimester, and returned to nonpregnant levels by 20-24 wk postpartum. The serum alkaline phosphatase activity averaged 2.1-fold higher in the late third trimester than in the first trimester; the serum triglyceride concentration averaged 2.3-fold higher in the late third trimester than in the first trimester. Compared to the peak values during pregnancy, serum alkaline phosphatase activity averaged 45% lower and serum triglyceride level averaged 47% lower at 12-16 wk postpartum. The serum alkaline phosphatase activity was correlated with the serum concentrations of total cholesterol (r = 0.68, p < 0.01) and triglyceride (r = 0.71, p < 0.01). In short, this study shows that serum triglyceride and total cholesterol levels change in parallel with serum alkaline phosphatase activity during and after normal pregnancy.

The incidence and significance of pseudoparaproteins in a community hospital.

Abstract: Pseudoparaproteins were observed in 129 (10.5%) of 1,229 high resolution protein electrophoretic fractionations of serum (N = 847), urine (N = 368), or cerebrospinal fluid (N = 14) performed in this laboratory during a 12-month period. The pseudoparaproteins identified in serum electrophoretic patterns included fibrinogen, C-reactive protein, hemoglobin-haptoglobin complex, elevated beta-globulins (transferrin and C3), lysozyme (muramidase), and an extended migration artifact. In the electrophoretic patterns of urine, the pseudoparaproteins consisted of nonspecific gamma zone bands of varying intensity. Gamma zone trace protein in the cerebrospinal fluid was often of sufficient intensity to cause potential confusion with oligoclonal bands. Awareness of the characteristic electrophoretic migration positions of these pseudoparaproteins helps to avoid unnecessary ancillary testing and expense.

Commentary: Iron metabolism in hepatitis C infection

Abstract: Hepatitis C virus (HCV) infections have started to decline, but up to 10,000 deaths each year are the consequence of chronic liver disease, following the infection. Laboratory testing identifies HCV-infected individuals using positive recombinant immunoblot assays to detect the presence of the antibody; the diagnosis is confirmed by detecting HCV RNA in serum. HCV-infected patients who have large accumulations of hepatic iron have not responded well to interferon therapy, compared to patients with normal hepatic iron stores. Physicians who treat patients infected with HCV should be aware of the detrimental effect of excess liver iron on interferon therapy. The degree of hepatic iron overload should be assessed and the reason for the excess iron should be investigated. Phlebotomy is the most practical method for iron removal and is well tolerated by patients with HCV infection.

Cytogenetics as an aid in the diagnosis of lymphomas.

Abstract: Multiple classifications of lymphomas are available. Generally, distinctions are made to identify low, intermediate, and high-risk groups. Histopathologic differentiation is at times difficult. The revised European-American lymphoma classification (REAL) uses histology, clusters of differentiation markers, histochemistry, and cytogenetics for definitive identification. This work reviews the karyotypic and FISH (fluorescent in situ hybridization) findings in some common lymphomas. B-Cell lymphomas, which make up approximately 85-90% of lymphomas, are associated with cytogenetic changes of +12, 13q14, 14q32, 2p11, and 22q13. Translocations help to support the diagnosis of follicular cell lymphoma t(14;18),(q32;q21), mantle cell lymphoma t(11;14)(q13;q32), and Burkitt's lymphoma t(2;8),t(8;14) and t(8;22). T-Cell lymphomas may show changes in 14q11,7p or 7q. Many of the lymphomas are characterized by complex karyotypic changes. Specific FISH probes are useful in determining characteristic or identifying marker chromosomes. Cytogenetic and FISH studies aid in the diagnosis, correct classification, and evaluation of therapy for a variety of lymphomas.

Castleman's disease confined to the leptomeninges.

Abstract: We report a rare case of the plasma cell variant of Castleman's disease confined to the leptomeninges in a 42-year-old female. Flow cytometry demonstrated a minor monoclonal kappa light chain population, and conventional Southern blotting confirmed clonal rearrangement of the J H immunoglobulin heavy-chain gene. Polymerase chain reaction for Epstein-Barr virus and Kaposi's sarcoma-associated herpes virus was negative. The patient is disease-free five years after surgical resection. To our knowledge, clonal gene rearrangement has not been previously reported in the plasma cell variant of localized intracranial Castleman's disease.

Troponin I: an update on clinical utility and method standardization.

Abstract: Cardiac troponin I (cTnI) is now widely recognized as one of the preeminent biochemical markers for the diagnosis of myocardial injury. The biochemical specificity of this biomolecule for cardiac tissue has forced a reevaluation of the diagnostic criteria for non-Q-wave acute myocardial infarction, unstable angina, acute coronary artery disease, and minor myocardial injury. Further, its use by clinicians has revolutionized the way that chronic and acute heart diseases are both diagnosed and managed. Unfortunately, the standardization of cardiac troponin I assays is problematic. Up to 20-fold variation of serum cTnI mass determinations may be observed for a given patient sample when measured by different assay systems. As a result, significant ambiguity often exists in the clinical interpretation of serum cTnI concentrations. Recent efforts have been directed toward the biochemical standardization of cTnI assays. However, the heterogeneous nature and biochemical complexity of the serum forms of cTnI and differences of the epitope recognition by the various methods have hindered the harmonization of serum cTnI assays.