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Differentiation of cytomegalovirus infection from acute rejection using renal allograft fine needle aspirates.

Cytomegalovirus (CMV) infection is an important cause of renal allograft dysfunction and may be difficult to distinguish from acute transplant rejection both clinically and histologically. To establish the early diagnosis of CMV infection, we used immunohistochemical staining with antibodies against CMV early nuclear protein (CMV-A) and histocompatibility leukocyte class II antigen (DR) in renal transplant fine needle aspirates. Fifty-eight aspirates from 27 patients were assessed, 53 for CMV-A and 53 for DR. Positive staining was defined as greater than or equal to 35% stained tubular cells for CMV-A and greater than or equal to 30% stained tubular cells for DR. Clinical diagnoses were made retrospectively without using the information obtained from aspirate diagnoses. CMV-A staining was negative in 44 aspirates, none at the time of CMV infection. CMV-A was positive in nine aspirates, seven during CMV infection (78%, P less than 0.00001 versus CMV-A negative). DR staining was never present in the absence of acute rejection. All aspirates performed during acute rejection had positive DR staining (P less than 0.00001 versus DR negative). Aspirates with acute rejection comprised 80% of all DR-positive aspirates, whereas those with CMV infection included only 13%. The percent CMV-A staining increased with CMV disease progression; DR staining decreased after successful treatment of acute rejection. These data demonstrate that CMV-A staining is associated with CMV infection whereas DR staining is not. DR staining is specifically related to acute rejection. CMV-A and DR staining of fine needle aspirates is a potentially valuable diagnostic tool to distinguish rapidly between CMV infection and acute transplant rejection as the etiology of renal allograft dysfunction.

Antigens, Viral↗

Distinct prognostic values of p53 mutations and loss of estrogen receptor and their cumulative effect in primary breast cancers.

A total of 76 primary breast cancers were screened for p53 mutations using the yeast p53 functional assay, and the mutations were determined by DNA sequencing. Clonal mutations of p53 were detected in 30 tumors (39%). Immunohistochemical staining for nuclear p53 accumulation performed on the yeast assay-positive cases clearly differentiated missense mutations in the DNA binding domain (contact mutant; 17 cases) as positive stain and nonsense-type mutations or missense mutations that may affect 3D-structure of p53 protein (structural mutant; 13 cases) as negative stain. Enzyme immunoassay revealed loss of estrogen receptor in 36 tumors (50%). Prognostic values of p53 mutation and loss of estrogen receptor were evaluated after a median follow-up period of 44 months. p53 mutations were associated with a short overall survival (log rank test, p = 0.0319), whereas it was not related to disease-free (recurrence-free) survival. Contact mutants were associated with slightly shorter survival compared with structural mutants. Inversely, loss of estrogen receptor was associated with early recurrence (p = 0.0461) but not with short overall survival. The patients with tumors harboring both p53 mutation and loss of estrogen receptor had the poorest outcome (p = 0.0019 and 0.0075 for overall and disease-free survivals, respectively), suggesting independent and additive effects of the 2 factors. The independent role of the 2 factors was confirmed by a multivariate analysis using the Cox proportional hazard model stratified according to clinical tumor stages. Although preliminary, due to the small number of patients studied and the relatively short follow-up time, our results suggest that p53 mutations and loss of estrogen receptor cooperatively affect the prognosis of primary breast cancer patients.

Adult↗

Immunohistochemical expression of beta-human chorionic gonadotropin in colorectal carcinoma.

The presence of human beta-chorionic gonadotropin (HCG) was immunohistochemically studied in 123 cases of primary colorectal carcinoma using the avidin-biotin-peroxidase complex method. Positive staining for HCG was recognized in 45 (36.6%) tumors and a statistical difference was observed between the HCG-positive (n = 45) and -negative (n = 78) groups concerning the frequency of blood vessel invasion in the primary tumor (P < 0.01). The prognosis for patients with HCG-positive carcinoma was thus significantly worse than that for patients with HCG-negative carcinoma (P < 0.05). A multivariate analysis using the Cox hazards model demonstrated the positive or negative staining of HCG to be one of the independent prognostic factors. The above findings show that, in addition to various other prognostic factors, the HCG staining status may thus also help in determining the prognosis of patients with primary colorectal carcinoma.

Aged↗

Aberrant p53 staining does not predict cisplatin resistance in locally advanced non-small cell lung cancer.

Cisplatin based chemotherapies have increased the survival in nonsmall cell lung cancer. A mechanism for identifying tumors resistant to cisplatin would be useful in avoiding unnecessary toxicity of platinum regimens. Mutation of p53 has been shown to induce chemotherapy resistance in vitro. We hypothesized that tumors staining for p53 would be resistant to cisplatin. In Cancer and Leukemia Group B protocol 8935, patients with stage IIIA (N2 node positive) nonsmall cell lung cancer received chemotherapy followed by surgery, then post-operative chemotherapy and/or radiation. All patients underwent pre-treatment staging mediastinoscopy. Twenty-five out of forty-nine pre-treatment mediastinal lymph node specimens stained positively for p53. Positive staining did not correlate with response to chemotherapy or survival. It did predict a slightly higher complete or partial resection rate compared to negative staining (76 vs. 45%) (p = 0.042). A trend toward longer median survivals was seen in patients with positive p53 staining. This study does not support the ability of p53 staining to predict chemotherapy resistance.

Antibodies, Monoclonal↗

Diagnostic value of microtubule-associated protein-2 in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare, aggressive cutaneous carcinoma with neuroendocrine differentiation and a propensity for early spread to regional lymph nodes. Since surgical resection is the mainstay of treatment of MCC, differentiation of MCC from malignant lymphoma, metastatic small cell carcinoma, basal cell carcinoma, and malignant melanoma is very important and is sometimes challenging with routine histologic examination. Immunohistochemical studies may be required to differentiate MCC from other primary and metastatic skin neoplasms. Previously, the authors reported that microtubule-associated protein-2 (MAP-2) is a sensitive and specific marker for pulmonary neoplasms with neuroendocrine differentiation. Because MCC is also a neuroendocrine carcinoma, the authors hypothesized that MAP-2 may be expressed in MCC and therefore may be a useful marker in establishing an accurate diagnosis. MAP-2 staining was demonstrated in all 14 MCCs with diffuse (10 cases) to focal (4 cases) patterns of immunoreactivity. No MAP-2 immunoreactivity was observed in any lymphoma (14 cases), basal cell carcinoma (20 cases), or squamous cell carcinoma (14 cases). CK20 reactivity was present in 12 of 14 cases with focal (2 cases) to diffuse (10 cases) staining having the characteristic perinuclear dot-like pattern. NSE was positive in 13 of 14 cases, SYN was positive in all 14 cases, CHR was positive in 8 of 14 cases, CK7 was positive in 4 of 14 cases, and CD99 was focally positive in 2 cases and diffusely positive in 3 cases. MAP-2 showed a diffuse or focal staining of MCC with a +1 to +4 intensity in most cases. MAP-2 was positive in two cases of MCC that were negative for CK20 and CHR and negative or only slightly positive for SYN and NSE. Therefore, MAP-2 may be a valuable ancillary study in skin tumors suspicious for neuroendocrine origin with faint or negative staining with the antibodies traditionally used for diagnosing MCC. The authors believe this is the first study to demonstrate the utility of MAP-2 in the immunohistochemical workup of MCC. The authors recommend that MAP-2 be added to immunohistochemical panels to confirm the diagnosis of MCC.

Aged↗

Immunoperoxidase study of the secretory immunoglobulin system and lysozyme in normal and diseased gastric mucosa.

Using an immunoperoxidase technique the distribution of secretory component, IgA, and lysozyme has been investigated in normal, inflamed, dysplastic, and carcinomatous gastric mucosa. Apart from pyloric glands which contain lysozyme, normal gastric mucosa stains negatively for all three antigens. In gastric mucosa neck cells appear to adapt by synthesising secretory component and lysozyme and transporting IgA. Intense staining for the three antigens is seen in dysplastic gastric epithelium and in well-differentiated intestinal type carcinomas. With progressive de-differentiation the tumours lose the ability to synthesise secretory component and lysozyme. Carcinomas of the diffuse type stain positively for secretory component and lysozyme and individual cells appear to take up IgA even in the absence of surrounding IgA containing plasma cells. These functional properties are retained in lymph node metastases. It is suggested that secretory component synthesising malignant cells might take up circulating dimeric IgA and that this could be a reflection of an important physiological mechanism.

Gastric Mucosa↗

Induction of metastasis by S100P in a rat mammary model and its association with poor survival of breast cancer patients.

S100P, an EF-hand calcium-binding protein, has been reported to be associated with the progression of many types of cancers. Transfection of an expression vector for S100P into a benign, nonmetastatic rat mammary cell line causes a 4- to 6-fold increase in its level in all four transformant cell clones. When the resultant transformant cell lines are introduced in turn into the mammary fat pads of syngeneic Furth-Wistar rats, there is a significant 3-fold increase in local muscle invasion and a significant induction of metastasis in 64% to 75% of tumor-bearing animals. In a group of 303 breast cancer patients followed for up to 20 years, antibodies to S100P immunocytochemically stain 161 primary tumors. Survival of patients with S100P-positive carcinomas is significantly worse by about 7-fold than for those with negatively stained carcinomas. There is also a significant association between the class level of immunocytochemical staining of the carcinoma cells and decreased patient survival. Positive staining for S100P is significantly associated with that for two other metastasis-inducing proteins, S100A4 and osteopontin. Patients with tumors that stained positively for both S100P and S100A4 have a significantly reduced survival of 1.1% over patients with either S100 protein alone. Multivariate regression analysis identifies S100P, S100A4, and osteopontin as the most significant independent indicators of death in this group of patients. These results suggest that stratification of patients into groups according to expression of multiple metastasis-inducing proteins may lead to a more accurate prediction of patient survival.

Adult↗

Immunohistochemical study on superoxide dismutases in spinal cords from autopsied patients with amyotrophic lateral sclerosis.

A role mutations in the superoxide dismutase (SOD)-1 gene in the pathogenesis of amyotrophic lateral sclerosis (ALS) has been discussed. To investigate immunohistochemical alterations of SOD in the spinal cord affected with the disease, we examined 3 patients with SOD1 mutation-associated family with ALS, 20 patients with sporadic ALS and 10 control individuals. Lewy body-like hyaline inclusions (LBHIs) were seen in the anterior horn cells of all the familial patients and 10 of the 20 sporadic patients, while skein-like inclusions (SIs) and Bunina bodies (BBs) were present in the 20 sporadic patients but not in the familial patients. The primary antibodies used for immunostaining were rabbit antisera raised against human SOD1 and SOD2. The anti-SOD1 antibody reacted strongly with all LBHIs of each familial patient and with some LBHIs of each sporadic patient. The cytoplasm of morphologically intact and degenerated spinal cord neurons as well as spheroids seen in the cases examined was only weakly stained by the antibody to SOD1 or not at all. The reactive astrocytes displayed weak to moderate staining for SOD1. The anti-SOD2 antibody strongly immunolabeled the reactive astrocytes and microglia. LBHIs of both familial and sporadic ALS were negatively stained for SOD2. Spinal cord neurons and spheroids of each case exhibited no significant SOD2 immunoreactivity. Neither antibodies reacted with SIs nor BBs. These results indicate that SOD1 may be involved in the formative process of LBHIs especially in familial ALS but not always in that of SIs or BBs, and imply that SOD2 may have no connection with any of these ALS-related abnormal structures.

Adult↗

[Experiment study of c-fos expression on myocardial acute ischemia/reperfusion injury in rats].

OBJECTIVE: To investigate the changes of c-fos mRNA induced by myocardial ischemia/reperfusion(I/R) during acute period. METHODS: The model of I/R was established, and the rats was divided normal control, ischmia and I/R groups. Insitu hybridization (ISH) and computerized image analysis method was used to observe alteration of c-fos mRNA in cardiac myocytes. RESULTS: After 10 min ischemia and 30 min reperfusion, the area of reperfusion showed a part of cardiac myocytes weak staining, and get to peak at I-60 min/R-30 min. The myocardium in normal control and ischemia groups less than 60 min showed negative staining. No changes were found in all groups by HE staining. CONCLUSION: c-fos mRNA detection may become an important method for diagnosis of I/R.

Animals↗

[An immunohistochemical study on three aldolase isozymes in human lung cancer].

We investigated three aldolase isozymes (aldolase A, B, and C) in human lung cancer by using an indirect peroxidase labeled antibody method. We used 27 tissue samples obtained at surgical operations which were fixed in periodate-lysine-4% paraformaldehyde (PLP) solution, and embedded in optimum cutting temperature (OCT) compound. They were 11 adenocarcinomas, 9 squamous cell carcinomas, 3 large cell carcinomas, 3 small cell carcinomas, and 1 adenosquamous carcinoma. Aldolase A and C expressed intensely positive stainings in the cytoplasm of cancer cells compared with normal lung tissues, and its positivities were 81% respectively. However, Aldolase B showed almost negative staining, and its positivities were only 41%. These rates had no relation to the histological types or pathological stages of lung cancers, and suggested that human lung cancer contained increased levels of aldolase A, and C.

Adenocarcinoma↗

[Preparation, identification and functional studies on liposome (ApoA-I:PC)].

Liposome (ApoA-1: PC) was prepared by the deoxycholic acid dialysis method and identified by agarose gel electrophoresis, two dimensional thin-layer chromatography and electron microscopy. The purified liposomes manifested one band in Amino black 10B staining and no color in Oil red O staining due to the presence of phospholipid. Only one brown spot appeared in two dimensional TLC, and electron microscopy with 1% sodium phosphotungstate negative staining revealed discoidal shapes stuck together. All of the above indicate that the liposome consisted of ApoA-1 and PC. Liposomes were added to the medium of smooth muscle cell cultures and then LDL receptor expression and cholesterol efflux from the cells were observed. The results suggest that the ApoA-1:PC can enhance the expression of LDL receptors indirectly by stimulating cholesterol efflux from the cells.

Animals↗

Studies on the binding of proteins to the human platelet surface: relation to platelet activation.

Several antibody fractions and sera from patients with rheumatoid arthritis, systemic lupus erythematosus and chronic idiopathic thrombocytopenic purpura were examined for their ability to bind to normal platelets using immunofluorescent staining techniques. Platelet aggregometry was used to study the activating capacity of the samples. Both C1q, C1s, C1 inactivator, fibrinogen, factor VIII-related antigen, alpha 1-acid glycoprotein, alpha 1-antitrypsin, beta 2-microglobulin and isoantigens A and B, as well as fibronectin and plasminogen were found on the platelet surface. Only antibodies to C1q, C1s and beta 2-microglobulin were able to induce platelet aggregation. Sera containing immune complexes or platelet autoantibodies revealed positive surface staining for IgG, or for IgG and IgM. There sera also induced aggregation of platelets. Sera not containing immune complexes or autoantibodies gave negative staining and aggregation results. Thus, only some of the ligand receptor interactions were able to induce platelet aggregation.

Antibodies↗

Progressive multifocal leukoencephalopathy. A case report and review of the literature.

Progressive multifocal leukoencephalopathy developed in a 70-year-old man with hepatoma treated with oncostatica. The brain showed multiple demyelinated lesions in the upper half of the cerebral white matter. The lesions consisted of proliferation of hypertrophied astrocytes and glial cells with abnormally large nuclei. These nuclei were shown to be filled with DNA by DAPI staining and reacted specifically with anti-JC antibody by immunofluorescence. Papova-like virions were confirmed on both the sections and negatively-stained brain extract under electron microscopy. The sixteen cases so far reported in Japan were reviewed.

Aged↗

Immunohistochemical localization of amyloid beta-protein deposits in extracerebral tissues of patients with Alzheimer's disease.

An autopsy study was conducted on patients with Alzheimer's disease in order to explore the possibility of amyloid beta-protein deposits in tissues other than the brain. Immunoperoxidase staining techniques were employed using an amyloid beta-protein antiserum as primary antibody; paraffin sections of kidney, liver, heart, lung, spleen, bone marrow, colon, stomach, adrenal, thyroid, and brain were examined. Our autopsy cases were divided into two groups. The first group comprised 10 patients with Alzheimer's disease. The second group consisted of eight control cases, of age-matched individuals, that died of unrelated causes and showed no clinicopathologic evidence of Alzheimer's disease. Identification was made of strong, although focal, positive staining in two kidneys and one lung of three different patients with Alzheimer's disease. All other tissues, including the control cases, showed negative staining.

Aged↗

Immunohistochemical staining of precursor forms of prostate-specific antigen (proPSA) in metastatic prostate cancer.

Precursors of prostate-specific antigen (proPSA) have been previously shown to be more concentrated in prostate cancer tissue. This study characterizes the immunohistochemical staining (IHS) of proPSA forms in metastatic prostate cancer compared with prostate specific antigen (PSA) and prostatic acid phosphatase (PAP). A tissue microarray, consisting of 74 cases of metastatic prostate carcinoma and control tissues, was used. IHS, using monoclonal antibodies against proPSA with a truncated proleader peptide containing 2 amino acids ([-2]pPSA), native ([-5/-7]pPSA), PSA, and PAP, was analyzed. The monoclonal antibodies were specific for both benign and malignant prostatic glandular tissue. IHS with [-5/-7]pPSA showed the least number of cases with negative staining (3%), and the most number of cases with moderate or strong staining (76%). In the 60 cases where all 4 stains could be evaluated, none of them were negative for proPSA and positive for PSA or PAP, and all 7 cases that were negative for both PSA and PAP showed IHS to proPSA. [-5/-7]pPSA (native proPSA) may be a better marker than PSA and PAP in characterizing metastatic prostate adenocarcinoma, with most of the cases showing positivity for the marker. Even cases that were negative for PSA and PAP, were reactive for proPSA. Such enhanced detection is particularly important in poorly differentiated carcinomas involving metastatic sites where prostate carcinoma is a consideration. A panel of markers, including proPSA, should be performed when metastatic prostate carcinoma is in the differential diagnosis.

Acid Phosphatase↗

Scrapie associated fibril detection from formaldehyde fixed brain tissue in natural cases of ovine scrapie.

The medulla oblongata of the brains of 71 scrapie-suspect cases were routinely fixed in 10 per cent formal saline and assessed for vacuolation on HE-stained sections. A pool of fresh brain material was also dissected from each animal and extracts prepared for the routine detection of scrapie-associated fibrils by negative stain transmission electron microscopy. The remaining formaldehyde fixed medulla samples, which were not used for the histological examination, were coded and subjected to a pretreatment with sodium borohydride and then processed using the routine fibril detection procedure. Of the 71 samples tested 46 were considered positive by all three test procedures. Sixteen samples were negative for all three tests. Four samples were positive by histopathological examination and positive for fibrils using fresh tissue, but fibrils could not be detected in the fixed tissue preparations. Conversely, there were five fixed samples in which fibrils could be detected which were negative for the other two tests. The fibrils observed in fixed preparations were indistinguishable from those observed in fresh tissue extracts. The sensitivity of the test for fibril detection using fixed tissue was 92 per cent and the specificity 76 per cent. It is concluded that scrapie-associated fibrils can be recovered from formaldehyde fixed tissue, as presented for routine histopathological examination, and therefore the method has potential in the retrospective analysis of archived brain tissue where only fixed material was stored.

Animals↗

The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes.

The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes were studied. The Cu2+ exposure of mussels caused severe perturbations in haemocyte actin and fibronectin organization with respect to non-exposed organisms. Cytoskeletal actin was analysed by indirect immunofluorescence, using an antitotal actin monoclonal antibody, and by rhodamine-conjugated phalloidin. The majority of haemocytes from Cu(2+)-exposed mussels displayed a round morphology, with short and blunt filopodia; they lacked the polarized phenotype which was typical in control samples. The cytoskeleton alteration, more evident after phalloidin staining, resulted in the disappearance of filamentous actin. The actin cortical meshwork also appeared disorganized. The cytoskeletal morphology studied by transmission electron microscopy after negative staining of Triton X-100-treated haemocytes confirmed these observations. The structural organization of actin when analysed by Western blotting showed a larger number of Triton-soluble actin pools in treated mussel haemocytes. Fibronectin was studied by indirect immunofluorescence using a polyclonal antiserum directed against mussel fibronectin. In treated mussels, fibronectin appeared to be strongly disorganized and its levels decreased in both haemocytes and haemolymph. The mechanism(s) of the copper-induced alterations on actin and fibronectin organization in mussel immunocytes is discussed.

Actins↗

Overexpression of BMP-2/4, -5 and BMPR-IA associated with malignancy of oral epithelium.

The aim of the present study was to determine the relationships between bone morphogenetic proteins (BMPs), BMP receptor type IA and carcinogenesis of oral epithelium. A retrospective study was performed on material obtained from oral mucosa, including nine cases of normal mucosa (NB), eight cases of nonspecific chronic inflammation (NCI), seven cases of hyperkeratosis (HK), five cases of squamous cell papilloma (SCP), 29 cases of squamous cell carcinoma (SCC) with various grades of differentiation and 10 cases of epithelium adjacent to carcinoma (EAC). Six cases of NB from hard palate (NHP) were chosen as a control group. The benign groups consisted of NCI, HK and SCP. The antibodies against BMP-2/4, -5, receptor BMPR-IA and purified bovine BMP (bBMP-McAb) were utilised using an immunocytochemical method. The results demonstrated that the immunostaining of BMP-2/4, BMP-5, BMPR-IA and bBMP-McAb was weak and not consistent in normal and benign groups. The immunoreactivity level was independent of the clinical and pathological grading of SCC. All cases of SCC showed positive staining for BMP-2/4, BMP-5, BMPR-IA and bBMP-McAb except for three cases and one case of SCC which negatively stained for BMP-2/4 and BMP-5, respectively. The staining intensity and proportion of the positively stained cells were markedly increased in SCC when compared with that of the normal and benign groups except for EAC. The metastatic carcinoma cells in lymph nodes were strongly and positively stained for BMP-2/4 and BMP-5 when compared with the primary lesions. Our results indicate that there was an overexpression of BMP-2/4, BMP-5, bBMP-McAb and BMPR-IA in the high-risk premalignant and malignant lesions of oral epithelium. Our findings suggest that BMP-2/4 and BMP-5 but not BMPR-IA might be involved in the metastasis of oral carcinoma cells.

Analysis of Variance↗