Evaluation of osteopontin expression in chronic wounds: a potential prognostic and therapeutic biomarker.

OBJECTIVE: Osteopontin (OPN) is abundantly expressed during tissue repair, acting as a powerful chemokine that recruits inflammatory cells such as neutrophils, macrophages, and Langerhans cells. The role of OPN in chronic wounds has not been explored. In this study, we assess the expression levels of OPN in chronic wounds to assess its potential contribution to the exacerbated inflammation seen in chronic ulcers, which is thought to contribute to poor healing. METHODS: This retrospective study included archived biopsies of chronic wounds from several aetiologies. Immunohistochemical staining and blind analysis of OPN expression were carried out. RESULTS: We assessed biopsies from venous leg ulcers (n=5), diabetic foot ulcers (n=5), pyoderma gangrenosum (n=5), squamous cell carcinoma ulcers (n=4), and calciphylaxis ulcers (n=3). The data revealed that all these sets of chronic ulcers expressed high levels of OPN. CONCLUSION: This study provides strong histopathologic evidence that OPN expression is significantly increased in chronic wounds, suggesting that its upregulation could contribute to the exacerbated inflammation. Furthermore, further characterisation of the role of OPN in wound healing could aid the development of specific and efficient anti-OPN therapies for the treatment of chronic wounds.

Casein Protein Block to Optimize Immunohistochemistry Detection of Surface Immunoglobulin Heavy Chain Expression in Lymphoid Cells.

We describe our recent experience of studying expression of immunoglobulin (Ig) heavy chain (IgG, IgM, and IgA) in lymphoid cells comprising a research set of formalin-fixed, paraffin-embedded human diffuse large B-cell lymphoma samples. We found that using typical clinical automated immunohistochemistry protocols and usual buffers as blocking agents, the extent of undesirable staining was extreme and impaired our ability to interpret heavy chain Ig expression by individual lymphoid cells. We were not able to optimize this with serial dilutions in antibody concentration or time of primary antibody exposure. We therefore developed an added step of casein protein block, which solved the problem. We are not aware of other such reports in clinical or human research tissue sets and believe this solution may be useful when clinical pathologists or researchers encounter similar technical issues.

Facial Nerve Function and Quality of Resection in Large and Giant Vestibular Schwannomas Surgery Operated By Retrosigmoid Transmeatal Approach in Semi-sitting Position with Intraoperative Facial Nerve Monitoring.

INTRODUCTION: Large and giant vestibular schwannomas pose a real problem in their management. The preservation of facial nerve function may limit tumor resection despite the use of intraoperative monitoring of the facial nerve. In Algeria, vestibular schwannomas represent 5% of all intracranial tumors operated on, 80.5% of which are large or giant. METHODS: From January 2010 to December 2015, 151 large and giant vestibular schwannomas were operated in our department. Tumor diameter was between 30 and 60 mm. The most common presenting symptom was hearing loss, which was observed in 41.66% of all our patients. All patients were operated in the semi-sitting position with opening of the posterior wall of the internal auditory canal and under continuous intraoperative facial nerve function monitoring. RESULTS: Tumor resection was total in 126 patients. Anatomic preservation of the facial nerve was the reason for nontotal resection in 25 patients. The facial nerve was anatomically preserved in 149 patients. Two years after surgery, the facial nerve function was grade I-II House-Brackmann (H-B) score in 124 cases (82%), grade III-IV H-B score in 21 cases (14%), and grade V-VI H-B score in 06 cases (04%). The status and the improvement of postoperative facial nerve function depend on 4 factors: anatomic preservation of nerve, stimulation threshold, cystic form, and the presence of train activity. CONCLUSIONS: The development of anesthesia techniques and microsurgery and the systematic use of intraoperative monitoring of the facial nerve have allowed us to move from a life preservation era to another era of preservation of function.

Distribution and physical properties of BCA200, a Mr 200,000 glycoprotein selectively associated with human breast cancer.

Of 122 mouse monoclonal antibodies selective for human breast cancer, 13 immunoprecipitated an acidic glycoprotein from SK-Br-3 and ZR-75-30 human breast cancer cells. The antigen (BCA200) migrates with an apparent molecular weight of 200,000 on reducing and 180,000 on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a single polypeptide chain with a folded domain stabilized by a disulfide bond. Cross-blocking and sandwich immunoassays detected at least three distinct antigenic determinants on BCA200. Scatchard experiments measured 1,000,000 to 5,000,000 antigen copies per SK-Br-3 cell. The tissue distribution of BCA200 was studied using two monoclonals to different epitopes. Neither antibody stained any cells in human blood. When frozen sections of 20 normal human tissues were immunoperoxidase stained, the only positive structures were mucinous glands of colon, transitional epithelium of bladder, sweat glands of skin, and acinar epithelium of breast. Antibody 454C11 stained 16 of 21 breast tumor frozen sections and 9 of 12 breast cancer cell lines, while antibody 520C9 stained 5 of 20 breast tumors and 4 of 10 breast cancer lines. Cross-reaction was observed with lung, prostatic, pancreatic, endometrial, and ovarian cancer, but not with lymphoma, melanoma, colon, stomach, bladder, or esophageal cancer. When conjugated to ricin A chain, 10 of 13 antibodies produced immunotoxins selectively cytotoxic to SK-Br-3 breast cancer cells.

Antimicrobial activity of UMFix tissue fixative.

AIMS: The aim of this study was to determine the antimicrobial effects of UMFix, an alcohol based tissue fixative, on various microorganisms. The UMFix solution was compared with 10% neutral buffered formalin. METHODS: Standard methods to determine microorganism colony counts were performed after exposure of the microorganisms to UMFix and 10% neutral buffered formalin. RESULTS: After a short exposure, UMFix rapidly killed vegetative bacteria, yeasts, moulds, and viruses. Bacterial spores were resistant to killing by UMFix. All organisms were killed by the 10% neutral buffered formalin preparation. CONCLUSIONS: UMFix was microbicidal for vegetative bacteria, yeasts, and aspergillus species after a short exposure, although it was not active against spore forming bacillus species. The methanol content of the fixative was responsible for the killing effect of this fixative. No killing was seen when polyethylene glycol was used alone.

Pathophysiology of apolipoprotein E deficiency in mice: relevance to apo E-related disorders in humans.

Apolipoprotein E (apo E) deficiency (or its abnormalities in humans) is associated with a series of pathological conditions including dyslipidemia, atherosclerosis, Alzheimer's disease, and shorter life span. The purpose of this study was to characterize these conditions in apo E-deficient C57BL/6J mice and relate them to human disorders. Deletion of apo E gene in mice is associated with changes in lipoprotein metabolism [plasma total cholesterol (TC) (>+400%), HDL cholesterol (-80%), HDL/TC, and HDL/LDL ratios (-93% and -96%, respectively), esterification rate in apo B-depleted plasma (+100%), plasma triglyceride (+200%), hepatic HMG-CoA reductase activity (-50%), hepatic cholesterol content (+30%)], decreased plasma homocyst(e)ine and glucose levels, and severe atherosclerosis and cutaneous xanthomatosis. Hepatic and lipoprotein lipase activities, hepatic LDL receptor function, and organ antioxidant capacity remain unchanged. Several histological/immunohistological stainings failed to detect potential markers for neurodegenerative disease in the brain of 37-wk-old male apo E-KO mice. Apo E-KO mice may have normal growth and development, but advanced atherosclerosis and xanthomatosis may indirectly reduce their life span. Apo E plays a crucial role in regulation of lipid metabolism and atherogenesis without affecting lipase activities, endogenous antioxidant capacity, or appearance of neurodegenerative markers in 37-wk-old male mice.

Immunohistochemistry of tissue prepared by a molecular-friendly fixation and processing system.

A recently introduced histologic fixative (Universal Molecular Fixative [UMFIX]) has been shown to preserve macromolecules in tissue at ambient temperature. When UMFIX-exposed tissues are processed by a formalin-free, microwave-assisted rapid processing system, the resulting paraffin blocks retain good histomorphology and intact nucleic acids suitable for expression microarray analysis. Because UMFIX may be used as an alternative to formalin, the authors set out to study the effect of this new fixation and processing system on immunohistochemistry (IHC) by analyzing a range of human neoplastic and non-neoplastic specimens. Parallel slices from surgically removed specimens were fixed in formalin and UMFIX and processed in a rapid microwave-assisted tissue processor. IHC was performed following routine procedures. The staining for those antibodies that normally required antigen retrieval was carried out with and without that step. The intensity and pattern of reactions were compared in 144 tissue samples fixed by the two methods using 70 monoclonal and polyclonal antibodies. The intensity of IHC reactions for most cytoplasmic antigens was generally equal or stronger in UMFIX tissues. This was particularly true with intermediate filaments and HercepTest, where the antigen retrieval step became unnecessary. Conversely, there was a decrease in the intensity of reactions for HepPar1, bcl-2, and three nuclear antigens (Ki-67, TTF-1, and estrogen receptor). Increasing their exposure times optimized the sensitivity of the latter four antibodies. The study shows that IHC staining results of tissues fixed in UMFIX and processed by the microwave-assisted system are comparable to those obtained on formalin-fixed, similarly processed specimens. There is an enhancement of the sensitivity of few antibodies in UMFIX-exposed tissue, rendering antigen retrieval unnecessary. This increased sensitivity may be due to the effect of eliminating formalin from fixation and processing or the microwave energy.

Cytologic characteristics of meningeal carcinomatosis: increased diagnostic accuracy using carcinoembryonic antigen and epithelial membrane antigen immunocytochemistry.

BACKGROUND AND OBJECTIVES: Traditionally, the diagnosis of meningeal carcinomatosis has been based on clinical suspicion and confirmed by cytologic study of cerebrospinal fluid. However, routine cytologic study may fail to detect malignant cells in a relatively large number of cases. We used immunocytochemistry in an attempt to increase the sensitivity of cytologic detection of malignant neoplasms in cerebrospinal fluid. MATERIALS AND METHODS: Thirty-eight consecutive cerebrospinal fluid specimens from patients with clinically suspected meningeal carcinomatosis were selected for this study. Immunocytochemistry for carcinoembryonic antigen and epithelial membrane antigen were used on the archival Papanicolaou-stained cerebrospinal fluid preparations. RESULTS: Of the 23 specimens from patients with proven meningeal carcinomatosis, 13 were correctly diagnosed using cytomorphologic criteria alone. The diagnosis of malignant neoplasm in 8 cytologically suspicious and 1 cytologically negative specimen was confirmed using immunocytochemistry. All cases that were negative on follow-up were also negative cytologically and immunocytochemically. CONCLUSIONS: We conclude that in using common antibodies, such as carcinoembryonic antigen and epithelial membrane antigen, the sensitivity of the cytologic diagnosis of meningeal carcinomatosis increases, and that previously Papanicolaou-stained preparations are suitable for immunocytochemical studies.

Intestinal-type adenocarcinoma of the gallbladder. A clinicopathologic study of seven cases.

Seven examples of a distinctive morphological variant of well-differentiated adenocarcinoma of the gallbladder with intestinal features are reported. Four tumors were composed predominantly of goblet cells and absorptive columnar cells, two of which had, in addition, a few Paneth cells and neuroendocrine cells. Three neoplasms closely resembled colonic carcinoma, and one of these also contained neuroendocrine cells. Serotonin-immunoreactive cells were demonstrated in three of the seven intestinal-type adenocarcinomas, two of which also had cells that stained for somatostatin pancreatic polypeptide and cholecystokinin. Four adenocarcinomas were associated with cholelithiasis, and three with intestinal metaplasia of the uninvolved mucosa. Despite the well-differentiated character of all neoplasms and the deceptively benign microscopic appearance of two of them, three patients died with extension to the liver and metastasis. Of the two survivors, one had carcinoma in situ and the other had a carcinoma that extended only to the muscle layer of the gallbladder. The various cell phenotypes found in these gallbladder adenocarcinomas can be explained on the basis of intestinal differentiation.

Clear cell variant of thyroid carcinoma.

Three carcinomas composed of a variable proportion of clear cells, oxyphil cells, and cells with combined oxyphil and clear cell features are reported. Cytologically, these tumors were included in the category of oxyphil cell variant of follicular carcinoma. In regard to pattern, two were entirely follicular, and one had follicular and papillary areas. The biologic behavior of these tumors, however, was consistent with that of follicular carcinomas. The clear cell change could be the result of chronic TSH overstimulation. This may explain the variable histological patterns, nuclear features, and biologic behavior associated with these tumors. These tumors bear a striking histologic resemblance to metastatic renal cell carcinoma and clear cell neoplasms from other sites. Immunocytochemical stain for thyroglobulin proved to be a specific and sensitive method for identification of these tumors.

Mucinous cystadenocarcinoma of the pancreas. Morphologic and immunocytochemical observations.

Twenty mucinous cystadenocarcinomas of the pancreas, most of which occurred in the tail of the pancreas in middle-aged women, were examined histologically and by immunohistochemical stains. Thirteen tumors displayed a marked histological heterogeneity and expressed intestinal differentiation as shown by the colonic appearance of the glands both at the light- and electron-microscopic levels. Other intestinal features included varying numbers of goblet cells, argyrophil and argentaffin cells, and even Paneth cells. By immunohistochemistry, endocrine cells were present in 13 of the 20 tumors (65%) and were more numerous in the poorly differentiated than in the well-differentiated epithelial component of the tumors. Serotonin-containing cells were the most common endocrine cells, followed by somatostatin-containing cells and cells that showed immunoreactivity for pancreatic polypeptide and gastrin. However, none of the patients had clinical manifestations of carcinoid, somatostatinoma, or the Zollinger-Ellison syndrome. The findings support the hypothesis that mucinous cystadenocarcinomas of the pancreas arise from an "endodermal stem cell" that differentiates into cells with intestinal phenotypes.

Frequency and pattern of p53 gene mutation in a cohort of Spanish women with node-negative breast cancer.

Ethnic, racial and regional differences in the frequency and pattern of p53 gene mutations have been well documented. Some of these differences have been shown to have an impact on the survival of patients with breast cancer. In this study we explored the frequency and pattern of p53 abnormality in a cohort of Spanish women with node-negative breast cancer using PCR, subcloning and DNA sequencing of archival tumors. One hundred and seventy-eight cases of breast cancer diagnosed between 1981 and 1986 at the University of Oviedo Hospital in Oviedo, Spain were subjects of this study. Sequence analysis of exons 5 through 8 of p53 was performed on subcloned PCR-amplified DNA, extracted from formalin-fixed, paraffin-embedded tumors. Appropriate positive, negative, PCR, and polymerase controls were utilized and evaluated. Duplicate samples of the genomic DNA were re-evaluated on all cases showing more than one mutation. One hundred and five out of 178 breast cases (59%) carried one or more p53 gene mutations. Mutations were distributed randomly from codon 128 to 305. There were 123 (88%) transition, 10 (7%) transversion, 5 (3.5%) splice junction mutations, and 2 (1.5%) deletions. Eighty-three cases (61.5%) had missense mutation, 45 (33.5%) silent, 5 (3.5%) nonsense and 2 (1.5%) frameshifts. Eighty (75%) of 120 transitions were G:C to A:T, 11 (25%) of which occurred at CpG sites. Sixteen mutations were in novel codons not reported in breast cancers previously. Codons with the highest frequency of mutations in this group were 278, 273, 213 and 227. We also detected 27 tumors with more than one mutation within a single exon or in different exons in the same patient. These findings suggest that the frequency and pattern of p53 mutations in this group of Spanish women with breast cancer is different than those reported in the United States and Northern Europe.

Thyroglobulin in carcinoma of the thyroid: an immunohistochemical study.

To determine the value of thyroglobulin as an immunohistochemical marker for thyroid neoplasms, we studied 42 primary thyroid carcinomas, 38 metastatic carcinomas, and four sarcomas involving the thyroid gland. All follicular and papillary carcinomas, regardless of their morphologic variation, stained positively for thyroglobulin, whereas the medullary carcinomas, metastatic tumors, and sarcomas showed negative staining reactions. The only small-cell variant of follicular carcinoma and ten of 14 spindle and giant cell carcinomas showed the lowest thyroglobulin reactivity. It is concluded that immunohistochemical demonstration of thyroglobulin is a sensitive and specific method of identifying thyroid carcinomas of follicular cell origin.

C-cell hyperplasia in thyroid tissue adjacent to follicular cell tumors.

An immunohistochemical study was conducted on the number and distribution of C-cells in the nonneoplastic thyroid tissue adjacent to tumors of follicular cell origin. It consisted of 49 cases, of which 25 were papillary carcinomas, 22 were follicular adenomas, and 2 were follicular carcinomas. Twenty normal adult thyroids from the Broward's Medical Examiner's morgue served as controls. In 17 of the 49 cases (34.6%), there was a statistically significant increase in the number of C-cells in the normal-appearing thyroid tissue adjacent to follicular cell tumors, with at least 50 C-cells in one low power field, while only one of 20 normal thyroids had a similar number of cells. (P = .02; chi 2 = 5.05). In two tumor cases there were more than 100 C-cells in several low power fields with formation of small C-cell nodules similar to those described in the type II Multiple Endocrine Neoplasia Syndrome (MEN). It was concluded that the nonneoplastic thyroid tissue adjacent to 34.6% of tumors with follicular cell phenotypes contains significantly more C-cells than those present in normal adult thyroids. The possible pathogenesis and clinical significance of these findings are discussed.

Intestinal metaplasia of the gallbladder: a morphologic and immunocytochemical study.

The morphologic spectrum of intestinal metaplasia was studied in 49 gallbladders that had been excised because of cholelithiasis. Based on the absence or presence of endocrine cells, the cases of intestinal metaplasia were arbitrarily divided into two groups. The gallbladders from the first group (26 cases) contained isolated or small clusters of mature goblet cells, while those from the second group (23 cases), in addition to the goblet cells, contained argyrophil and argentaffin cells and, less frequently, Paneth cells and gland-like structures similar to colonic crypts. Pseudopyloric glands and superficial gastric-type epithelium were present in both groups. Argyrophil cells outnumbered argentaffin cells by a ratio of 4 to 1. By immunocytochemical methods serotonin-containing cells were found to be the most common endocrine cells. Other endocrine cells showed immunoreactivity for somatostatin, cholecystokinin, gastrin, and pancreatic polypeptide. The presence of gut endocrine cells and Paneth cells in the pseudopyloric glands suggests that these glands are also an integral component of intestinal metaplasia of the gallbladder. The findings support the hypothesis that cholelithiasis induces the appearance of a stem endodermal cell that, in turn, may differentiate into cells with mature intestinal or gastric phenotypes.

Cathepsin D in host stromal cells, but not in tumor cells, is associated with aggressive behavior in node-negative breast cancer.

One hundred fifty-four axillary lymph node-negative invasive ductal carcinomas of the breast were immunohistochemically evaluated for the expression of cathepsin D. Formalin-fixed paraffin sections of each tumor were stained using a polyclonal antibody raised against recombinant procathepsin D. Cathepsin D content of tumor cells and host histiocytes and fibroblasts within or immediately at the invasive border of tumors were assessed separately and correlated with histomorphology, estrogen-receptor content, and patients' survival data. Positive cathepsin D staining of tumor cells was associated with a lower nuclear grade and well-differentiated histology, whereas moderate to strong staining of host cells correlated with larger tumor size, higher nuclear grade, poorly differentiated histomorphology, and lack of estrogen-receptor (ER) protein. No statistically significant correlation was found between cathepsin D in tumor cells and survival. There was, however, a statistically significant correlation between moderate to strong cathepsin D staining of host cells and shorter disease-free and overall survivals. Expression of cathepsin D by host cells, however, did not have an independent influence on survival. The authors conclude that cathepsin D in stromal cells, but not in tumor cells, is associated with aggressive behavior in node-negative invasive ductal carcinomas of breast. Furthermore, determination of cathepsin D in cytosolic extracts of tumors is of no practical value because it may represent cathepsin D content of tumor cells, intratumoral host cells, or both.

Malignant angioendotheliomatosis--a true lymphoma: a case of intravascular malignant lymphomatosis studied by southern blot hybridization analysis.

Malignant angioendotheliomatosis is a rare, systemic, usually fatal disease characterized by a massive proliferation of large, bizarre-looking mononuclear cells within small and medium-sized blood vessels. The histogenesis of the neoplastic cells has been the subject of long-standing controversy since the disease's initial description. Early investigators concluded that the entity represented a neoplasm of endothelial cells, but recently others have suggested that it is of lymphoid origin. We studied a case of malignant angioendotheliomatosis by Southern blot hybridization analysis which showed clonal rearrangements of the immunoglobulin heavy-chain gene strongly suggesting a B-lymphocyte origin. Our results provide additional evidence that malignant angioendotheliomatosis is an intravascular malignant lymphomatosis.

Hemangioblastomas: histogenesis of the stromal cell studied by immunocytochemistry.

Twenty-one cases of hemangioblastoma from the cerebellum, spinal cord and retina were studied using the unlabeled antibody peroxidase-antiperoxidase technique with antibodies directed against glial fibrillary acidic protein (GFAP) and factor VIII related antigen (VIIIR:Ag). In 19 of 21 cases studied with anti-GFAP, astrocytes were identified peripherally, and in 13 cases they were found centrally within the tumor. In no instance did stromal cells react positively for GFAP. Sixteen cases with anti-VIIIR:Ag antibody were examined, and in all cases many stromal cells showed positive staining. It is concluded that the stromal cells were of endothelial origin. The occasional stromal cells that other investigators have identified as reacting positively for GFAP may represent stromal cells capable of ingesting extracellular GFAP derived from reactive astrocytes within the tumor, or they may be lipidized astrocytes.

Orbital leiomyosarcoma.

A 36-year-old man had a six-month history of painless swelling in the medial orbit. Surgical exploration of this mass revealed a leiomyosarcoma. The tumor was entirely removed, as was all local surrounding tissue.

Special report. Immunocytochemistry in diagnostic cytology: a 12-year perspective.

In recent years immunocytochemistry has become an important addition to diagnostic cytology. Its routine application in cytology, however, has not yet reached the practical levels it has achieved in diagnostic histopathology. This review examines the values and limitations of immunocytochemistry in diagnostic cytology and addresses some of the most common technical and analytical factors that can affect the outcome of the procedure.