Microwave Antigen Retrieval Research Articles

Cüce Hamsterlerin (Phodopus roborovskii) Genital Dokularında Androjen Reseptör Lokalizasyonu

Androgens perform the their effects on target tissues mainly through the androgen receptor (AR). In this study, AR localization was investigated in female and male dwarf hamsters genital tissues. In the present study, 6 female and 6 male animals were used. After anesthesia, the tissues to be worked were quickly removed from the body and fixed in 10% buffered neutral formalin, and embedded in paraffin. Sections were stained after microwave antigen retrieval for immunohistochemistry. Although AR positive immunostaining was observed only in cell nuclei in testis, caput epididymis, prostate gland and vesicle seminalis tissues, it was observed in cytoplasm or nucleus of the cells in the ovarium and oviduct tissues. AR localization in dwarf hamster male and female genital tissues was similar to other species. The presence of AR in genital tissues supports the idea that androgens are essential for these tissues.

Immunohistochemical localization of androgen receptors in female mole rat (Spalax leucodon) tissues

Androgens exert their effects through androgen receptors (AR) in tissues. We investigated the distribution of AR in female mole rat tissues. Tissues were excised, fixed with 10% formalin and embedded in paraffin. Sections were stained after microwave antigen retrieval for immunohistochemistry. Immunostaining of AR immunostaining was detected in the nucleus or cytoplasm of the cells in the cerebral cortex, cerebellum, anterior pituitary, lung, liver, uterus and skin. Granulosa and some thecal cells in the ovary, cardiac muscle cells and adipose cells exhibited a nuclear reaction for AR. In the kidney, labeling of AR was restricted to the cytoplasm of tubule cells. We found that AR could be detected using immunohistochemistry in the nucleus or cytoplasm or both in the presence of androgens.

A microwave antigen retrieval method using two heating steps for enhanced immunostaining on aldehyde-fixed paraffin-embedded tissue sections.

Antigen retrieval is an immunohistochemical procedure that results in better exposure of target antigens in aldehyde-fixed, paraffin-embedded tissue sections to antibodies. However, the commercially recommended or conventional protocols for antigen retrieval do not always succeed in expressing the target antigen. Here, an improved method was developed for antigen retrieval from aldehyde-fixed, paraffin-embedded histological sections. Proliferating cell nuclear antigen (PCNA), tight junction proteins Claudin-2 and Claudin-7, and water channel aquaporins in kidney tissue were selected as test antigens. Typically, PCNA and Claudin-2 and Claudin-7 show negative, weak, or nonspecific immunoreactions with conventional antigen retrieval methods using microwave heating. In the present study, microwave heating was performed twice with an interval of 30min between the two steps to allow the buffer solution to cool. Sodium citrate buffer (10mM sodium citrate, pH 6.0) was used for PCNA, and Tris-EDTA buffer (10mM Tris, 1mM EDTA, pH 9.0) was used for the Claudins. Compared with conventionally prepared tissues, the tissues exhibited both enhanced and specific immunostaining, and well-preserved morphology. In conclusion, the conventional protocol could be supplemented with a second microwave heating step to improve the expression of antigens that do not respond well to the conventional method.

Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride

Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3′-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.

Sclerosing nodular lesions of the gastrointestinal tract containing large numbers of IgG4 plasma cells

Hyalinised fibrous nodules have been encountered within the gastrointestinal tract (GIT) and been labelled as reactive nodular fibrous tumours. Several have a history of abdominal surgery and/or sepsis that acts as a precipitating cause for the fibrosis. Recently, much attention has been focused on IgG4 related fibrosing lesions that are typically associated with a high population of IgG4 positive plasma cells and tissue fibrosis. There may be attendant elevated serum IgG4 levels and associated autoimmune disease. We present four patients with well-circumscribed fibrous nodular lesions occurring in the GIT. Tissue was formalin fixed after microwave antigen retrieval and H&E stains and immunohistochemistry were performed. IgG4/IgG ratios were calculated from the three high power fields containing the densest concentration of positive plasma cells. The patients were two females (45 and 56 years) and two males (47 and 60 years) who presented with gastric (2 cases), caecal and sigmoid flexure involvement. One case had four lesions while the other three cases were solitary nodules. Two patients had coexistent autoimmune disease. All lesions were nodular and composed of paucicellular, hyalinised fibrous tissue associated with chronic inflammation. In all lesions the plasma cell population was strongly IgG4 positive. This paper describes unique, well-circumscribed sclerosing nodules containing IgG4 positive plasma cells within the bowel wall that may cause mucosal polypoid lesions. It is possible that these lesions may be related to the spectrum of IgG4 related sclerosing disease or belong to a separate subset of inflammatory reactive conditions that are rich in IgG4 plasma cells.

Citraconic anhydride: a new antigen retrieval solution

The introduction of heat-induced antigen retrieval has been a major milestone in diagnostic immunohistochemistry, enabling the application of many antibodies to fixed paraffin-embedded tissue sections. A number of important variables affect the preservation of tissue antigens, among which are analytical variables including the antigen retrieval methodology. Temperature of retrieval, duration of heating, source of heat, pH and nature of retrieval solution are among more important variables pivotal to results. Citrate buffer at pH 6.0 has become widely embraced as the universal fixative but some antibodies remain capricious and yield poor staining results. This study examines the recent suggestion that citraconic anhydride may be a suitable universal retrieval reagent. Immunostaining of 65 commonly employed antibodies following microwave antigen retrieval in 0.05% citraconic anhydride for 10 minutes at 98 degrees C was compared with consecutive tissues sections subjected to antigen retrieval in citrate buffer at pH 6.0 at the same duration and temperature. Thirty-five of the 65 antibodies examined yielded more intense staining following antigen retrieval in citraconic anhydride, including some capricious antibodies such as MyoD1, myogenin, perforin, TIA-1, Tdt, RET and MiTF, confirming the efficacy of this retrieval solution. It is recommended that consideration be given to 0.05% citraconic anhydride as an antigen retrieval solution, particularly for antibodies that fail to work or stain weakly in fixed paraffin-embedded tissue sections.

Protocol modifications influence the result of EGF receptor immunodetection by EGFR pharmDx™ in paraffin-embedded cancer tissues

EGF receptor (EGFR) became a useful target for several recently introduced therapies of various cancer types including colorectal, lung, head and neck cancers and glioblastoma. The successful clinical application of these novel molecularly targeted therapies requires the expression of their target, EGFR, determined by nucleic acid based or immunohistochemical techniques. However, until now, immunohistochemistry has not become a reliable diagnostic approach for this purpose. The golden standard for the determination of EGFR protein expression in paraffin-embedded cancer tissues is the EGFR pharmDxTM kit. Here we show that the recommended protocol may not be optimal for EGFR immunodetection. Microwave antigen retrieval and extended primary antibody incubation time converted four out of eight EGFR-negative tumors into EGFR-positive in a study of 50 lung adenocarcinoma cases. Accordingly, we recommend retesting cases negative for EGFR with EGFR pharmDxTM using protocol modifications optimizing antigen retrieval and the incubation periods.

Combined In Situ Hybridization and Immunohistochemistry for Automated Detection of Cytomegalovirus and p53.

Background: Cytomegalovirus (CMV) infection has been shown to be associated with p53 overexpression in coronary artery restenosis. We investigated the occurance of this association in other forms of CMV infection using an automated in situ hybridization (ISH) technique. Methods and Results: We performed ISH for CMV using digoxigenin-labeled or biotinylated probes followed by avidin-alkaline phosphatase and nitroblue tetrazolium color substrate. Immunohistochemistry (IHC) was then performed using an anti-p53 antibody utilizing streptavidin-immunoperoxidase and 3,3;-diaminobenzidine tetrahydrochloride as a chromogen. Sixteen cases with characteristic cytomegalic inclusions from a variety of body sites were examined. All 16 cases were positive for CMV by ISH. Nine of sixteen expressed nuclear p53. Six of these nine cases showed viral cytopathic effect in the cells with p53 expression. In an illustrative case, double colocalized staining for CMV and p53 protein was demonstrated in individual cytopathic cells. When microwave antigen retrieval was necessary, ISH was performed before IHC, and our standard microwaving time was reduced by two-thirds. Conclusions: The colocalization of p53 protein overexpression with CMV within single cells adds further evidence that this overexpression is a viral-induced phenomenon. The combined ISH and IHC assay can be carried out in a rapid automated mode, increasing the ease of investigating relationships between message and protein expression within single cells in a wide variety of settings.

Neuroendocrine secretory protein-55 (NESP-55) expression discriminates pancreatic endocrine tumors and pheochromocytomas from gastrointestinal and pulmonary carcinoids.

Neuroendocrine secretory protein-55 (NESP-55), the latest addition to the chromogranin family, is a product of a genomically imprinted gene transcribed exclusively from the maternal allele. Initial studies have shown it to have a less widespread distribution than that of chromogranin A in normal tissues. It has also been suggested that NESP-55 may be a marker of neuroendocrine tumors differentiating toward the adrenal chromaffin and pancreatic islet cells. Metastatic gastrointestinal and pulmonary carcinoids may occasionally be difficult to distinguish from pancreatic endocrine tumors (PETs) and pheochromocytomas on morphologic grounds alone. We studied neuroendocrine tumors from these sites to see if NESP-55 expression could reliably discriminate pulmonary and gastrointestinal carcinoids from neuroendocrine tumors arising in the pancreas or the adrenal medulla. Sixty-three neuroendocrine tumors positive for one or more immunohistochemical marker of neuroendocrine differentiation (chromogranin A, chromogranin B, synaptophysin, secretogranin II, neuron-specific enolase) were selected for the study and consisted of 34 typical carcinoids (15 pulmonary, 11 ileal, 4 gastric, and 4 rectal), 19 PETs, and 10 pheochromocytomas (4 sporadic, 3 MEN-2, 2 neurofibromatosis type 1, and 1 VHL). All cases were stained for NESP-55 after microwave antigen retrieval using a rabbit polyclonal antibody at a dilution of 1:1000. Sections of normal adrenal medulla were used as positive controls for NESP-55 staining. Negative controls consisted of omission of primary antibody and replacement with normal rabbit serum at an equivalent concentration. NESP-55 immunoreactivity was seen as brown finely granular cytoplasmic staining with prominent perinuclear accentuation. All gastric and ileal carcinoids studied were completely negative for NESP-55. One of four rectal and 1 of 15 pulmonary carcinoids showed focal positivity for it in less than 5% of tumor cells. In contrast, all 10 pheochromocytomas and 14 of 19 PETs showed strong immunohistochemical staining in a variable proportion of tumor cells. Diffuse positivity (>75% of tumor cells) was seen in 6 of 14 PETs and 8 of 10 pheochromocytomas. Our results indicate that, in contrast to the other granins, NESP-55 reactivity is restricted to endocrine tumors of the pancreas and the adrenal medulla. Immunohistochemical expression of NESP-55 may thus be useful in assigning a pancreatic or adrenal origin to metastatic endocrine tumors of unknown origin.

Immunohistochemical Detection ofAspergillusSpecies in Pediatric Tissue Samples

Definitive diagnosis of invasive aspergillosis often requires tissue samples for histologic evidence of fungal infection and culture confirmation of Aspergillus species. However, the culture frequently fails to isolate Aspergillus species. Alternative approaches to confirm Aspergillus infection use polymerase chain reaction, in situ hybridization, and immunohistochemical analysis on paraffin-embedded sections. These approaches are well characterized in animals and adult patients but not pediatric patients. We studied the immunoreactivity of a commercially available monoclonal antibody, Mab-WF-AF-1 (DAKO, Carpinteria, CA), on paraffin-embedded sections from 16 pediatric cases with invasive aspergillosis, of which 12 were proven by culture. Optimal immunoreactivity required microwave antigen retrieval using high pH; 5 other antigen retrieval approaches were unsuccessful. With optimization, the monoclonal antibody was strongly immunoreactive in all cases with staining of the Aspergillus cell wall, septa, and cytoplasm. Background was minimal with no cross-reactivity to Candida albicans. These findings demonstrate the usefulness of the Mab-WF-AF-1 antibody in pediatric tissues suspected of invasive aspergillosis.

Immunohistochemical detection of Aspergillus species in pediatric tissue samples.

Definitive diagnosis of invasive aspergillosis often requires tissue samples for histologic evidence of fungal infection and culture confirmation of Aspergillus species. However, the culture frequently fails to isolate Aspergillus species. Alternative approaches to confirm Aspergillus infection use polymerase chain reaction, in situ hybridization, and immunohistochemical analysis on paraffin-embedded sections. These approaches are well characterized in animals and adult patients but not pediatric patients. We studied the immunoreactivity of a commercially available monoclonal antibody, Mab-WF-AF-1 (DAKO, Carpinteria, CA), on paraffin-embedded sections from 16 pediatric cases with invasive aspergillosis, of which 12 were proven by culture. Optimal immunoreactivity required microwave antigen retrieval using high pH; 5 other antigen retrieval approaches were unsuccessful. With optimization, the monoclonal antibody was strongly immunoreactive in all cases with staining of the Aspergillus cell wall, septa, and cytoplasm. Background was minimal with no cross-reactivity to Candida albicans. These findings demonstrate the usefulness of the Mab-WF-AF-1 antibody in pediatric tissues suspected of invasive aspergillosis.

Characterization of membrane rafts isolated from rat sertoli cell cultures: caveolin and flotillin-1 content.

Membrane rafts from Sertoli cell cultures were isolated as detergent-insoluble glycosphingolipid-enriched (DIG) fractions on the basis of their enriched content of glycosphingolipids and cholesterol and the resulting insolubility in 1% Triton X-100 and their low buoyant density. Because lipid rafts have been implicated in numerous cell functions, including cell signaling and sites for actin/membrane attachment, studies were initiated to characterize Sertoli cell rafts. This study reports the distribution of the raft structural proteins, caveolin and flotillin-1, implicated in raft microdomain organization. Methods employed included the immunoblotting of cell lysates and detergent-insoluble glycosphingolipid-enriched (DIG) fractions, the immunofluorescent microscopy of peritubular myoid cell (PMC) cultures and cryostat-sectioned testis, and the immunohistochemical staining of paraffin-embedded sections following microwave antigen retrieval techniques. Sertoli cells and Sertoli DIG fractions were found to lack the common raft-associated protein, caveolin, a marker protein for caveolae, but they are enriched in the 48-kd protein, flotillin-1, a protein also implicated in raft formation, cell signaling, and cell motility. Since the primary cell contaminant of Sertoli cell cultures is the PMC, these cells, along with spermatogenic cell fraction (SPGC), were also examined for caveolin and flotillin-1 content. The PMCs contained significant concentrations of both caveolin and flotillin-1. PMCs in culture exhibited a punctate caveolin staining pattern at the cell surface characteristic of a caveolar location. These data support the idea that the pinocytotic vesicles observed in PMCs are caveolae. PMCs also show a perinuclear location for caveolin characteristic of a Golgi location. Cryostat sections of rat testis showed a marked concentration of caveolin in the PMCs. The PMC location of caveolin was also confirmed by the immunohistochemical staining of sections from paraffin-embedded rat testis following microwave antigen retrieval techniques. Similar experiments showed a more ubiquitous, stage-specific distribution of flotillin-1 among testicular cell types.

Growth factors and receptors in juvenile nasopharyngeal angiofibroma and nasal polyps: an immunohistochemical study.

Juvenile nasopharyngeal angiofibroma is a rare nasopharyngeal tumor that occurs exclusively in adolescent boys. It is a histologically benign but locally persistent growth of stromal and vascular tissue. Although male hormones and some growth factors, such as transforming growth factor beta1 (TGF-beta1), insulin-like growth factor II (IGF-II), and, lately, the proto-oncogene beta-catenin, have been implicated in the histogenesis of the tumor, the biologic signaling pathways that drive this peculiar fibrovascular proliferation are still nuclear. To evaluate immunoexpressions of beta-catenin, c-Kit, p130Cas, TGF-beta3, bone morphogenic protein 4, nerve growth factor (NGF), and the IGF receptor (IGF-1R) in a series of juvenile nasopharyngeal angiofibromas and to compare to that of a group of nasal polyps. A standard immunohistochemical technique was used on paraffin sections of 12 sporadic juvenile nasopharyngeal angiofibromas and 15 nasal polyps with microwave or steam antigen retrieval. Immunoreactivity was analyzed semiquantitatively in stromal cells and endothelial cells of each case. The expressions of beta-catenin (nuclear), c-Kit (cytoplasmic), and NGF (cytoplasmic) were higher and more frequent in stromal cells of juvenile nasopharyngeal angiofibromas than those of nasal polyps. Both juvenile nasopharyngeal angiofibromas and nasal polyps showed similarly frequent and strong immunoreactivity for p130Cas and TGF-beta3 and weak immunoreactivity for bone morphogenic protein 4 in both stromal cells and endothelial cells. No IGF-1R immunoreactivity was detected in any case of either group. Our results support the role of beta-catenin in juvenile nasopharyngeal angiofibromas and suggest a potential involvement of c-Kit and NGF signaling pathways in the juvenile nasopharyngeal angiofibromas. Although the biologic significance of c-Kit in juvenile nasopharyngeal angiofibromas has yet to be defined, the finding of frequent and high c-Kit expression might have therapeutic importance for patients with juvenile nasopharyngeal angiofibromas.

Tyramide signal amplification enhances the detectable distribution of connexin-43 positive gap junctions across the ventricular wall of the rabbit heart.

Previous mapping studies examinig the distribution and pattern of staining for connexin-43 expression (the major ventricular gap junction protein) across the ventricular wall have yielded variable findings. The aim of this study was to determine if variations in the distribution of connexin-43 were due to histochemical detection problems, i.e. cross-linking of antigenic sites as a consequence of aldehyde fixation and/or due to low levels of protein expression within the epicardial or endocardial regions of the heart. Immunoperoxidase staining of connexin-43 using the ABC method was carried out in crosssections of rabbit hearts at the level of the papillary muscle. The following treatments were examined: the antibody (Ab) only, Ab with 1/2 Tyramide Signal Amplification (TSA) or full TSA; antibody with microwave antigen retrieval (AR); Ab + 1/2 TSA + AR and finally Ab + TSA + AR. Under light microscopy and using computerized image analysis the percentages of ventricular cross-sectional transmural staining for the different treatment groups were calculated: Ab amounted to only 55%; Ab + 1/2 TSA 63%; Ab + TSA 78%; Ab + AR 72%; Ab + AR + 1/2 TSA 72% and Ab + AR + TSA 88%. The percentages of transumural connexin-43 staining in both TSA + Ab and Ab + TSA + AR groups when compared to Ab only were significantly greater p < 0.01. The antigenic cross-linking due to aldehyde fixation and low levels expression of connexin-43 are contributing factors that influence the immunohistochemical detection of connexin-43 in the mammalian heart. Methodological enhancement for the detection of connexin-43 in this study was derived primarily from amplification of low background levels of connexin-43 being expressed using the TSA protocol. This is supported by the significant differences encountered when TSA was utilized in the protocol and compared with antibody treatment only.

Immunohistochemical Detection of Melanoma-specific Antigens in Spontaneous Canine Melanoma

Twenty-five formalin-fixed, paraffin wax-embedded canine melanomas were examined immunohistochemically by an immunoperoxidase method to assess their reactivity with three human melanoma-specific monoclonal antibodies (HMB-45, MEL-1, NK1/C3). HMB-45 and MEL-1 reacted with 22/25 (88%) and 18/25 (72%) of canine melanomas, respectively, but only after microwave antigen retrieval and pretreatment with potassium permanganate and oxalic acid (KMnO 4/OA). Positive reactivity to HMB-45 and MEL-1 was as follows: oral melanomas, 13/16 and 9/16, respectively; cutaneous melanomas, 8/8 and 8/8; melanoma of the digit, 1/1 and 1/1; all pigmented melanomas, 16/18 and 14/18; all amelanotic tumours, 6/7 and 4/7. HMB-45 immunolabelling was characterized by a diffuse granular cytoplasmic pattern within the tumour cells, and MEL-1 labelling by a cytoplasmic pattern with sporadic nuclear localization. In most tumours the labelling was homogeneous, but some showed a multifocal distribution. Generally, a higher percentage of canine melanomas was labelled by HMB-45 than by MEL-1. NK1/C3 failed to label any of seven melanomas tested, regardless of KMnO 4/OA-pretreatment. Of 16 non-melanocytic tumours (specificity controls), 15 showed no signficant reactivity with HMB-45, the exception being one of three plasmacytomas. Immunolabelling by MEL-1 on non-melanocytic tumours was not pursued, due to the poor sensitivity of this antibody as compared with that of HMB-45.

The role of p53 and Ki67 in Paget's disease of the vulva and the breast.

Paget's disease of the vulva (PDV) and Paget's disease of the breast (PDB) are uncommon diseases, accounting for approximately 1% of all vulval neoplasms and 0.5-4% of all breast cancers, respectively. In 10-30% of vulval cases an invasive adenocarcinoma is present. In such cases the disease is often aggressive and recurrence rate is high. This is in contrast to PDB where the general consensus is that almost all cases are associated with an in situ or invasive ductal carcinoma. Our aim was to examine the presence of the tumor suppressor protein p53 and the proliferation marker Ki67 in PDV and PDB and correlate any differences in the expression of these two proteins with the presence of an underlying carcinoma. Immunohistochemistry was performed on 52 archival cases of PDV, which included 10 with associated invasive adenocarcinoma of the vulva, and on 37 archival cases of PDB, including 26 with available associated ductal carcinoma in situ (DCIS) or invasive carcinoma of the breast. All cases were formalin-fixed and paraffin wax-embedded. Monoclonal antibodies were used with microwave antigen retrieval. Streptavidin-biotin-horseradish peroxidase and 3,3'-diaminobenzidine detection methods were employed to visualize antibody binding and staining. A section was scored positive for p53 if more than 10% of cell nuclei were stained brown and Ki67 was expressed as a percentage of positive cells to the nearest 5% of cells showing nuclear positivity (Ki67 staining index). p53 was expressed in 15 of 52 (29%) PDV cases and 5 of 37 (13%) cases of PDB. Four of the ten cases (40%) of PDV associated with invasive disease expressed p53 compared with 11 of 42 (26%) cases without invasive disease. The mean Ki67 staining index for PDV associated with invasion was 19%, and for that without invasion, 16%. In the breast cases, the mean staining index was 11%. Our data suggest that p53 may have a role to play in PDV progression, and may be a late event in some cases, especially those associated with invasive disease. Ki67 has no apparent prognostic role in PDV as there was no significant difference between those cases associated with and those without invasive disease. Neither p53 nor Ki67 appears to have a prognostic role to play in PDB.

Patterns of immunoglobulin staining in paraffin-embedded malignant lymphomas.

The demonstration of immunoglobulin light chain restriction in paraffin-embedded B cell lymphomas is a capricious and difficult procedure that has been abandoned by many diagnostic laboratories. Using a combination of microwave antigen retrieval performed in a pressure cooker and proteolytic digestion with trypsin, we were able to demonstrate immunoglobulin light chain restriction in 66 B cell lymphomas comprising 25 follicular lymphomas, 29 diffuse large cell lymphomas, 6 small lymphocytic lymphomas, 2 mantle cell lymphomas, 1 nodal marginal zone lymphoma, 1 Burkitt lymphoma, 1 hairy cell leukemia, and 1 plasmacytoma. There was concordance of results in 13 cases in which flow cytometry immunoglobulin analyzes were performed. Perinuclear staining was demonstrated in all cases with dot-like staining of the Golgi present in 23 cases. Perinuclear staining occurred in combination with cytoplasmic staining in 12 cases and membrane staining in 8 cases with 12 lymphomas showing more than two patterns of staining.

Human immunodeficiency virus infection, Bcl-2, p53 protein, and Ki-67 analysis in ocular surface squamous neoplasia.

To determine the age and human immunodeficiency virus (HIV) status of patients with ocular surface squamous neoplasia (OSSN), and to analyze tumor proliferation, Bcl-2, and p53 oncoprotein expression in OSSN. Only patients with histologically proved neoplasia were included in this study. The HIV status was obtained only with informed consent. Monoclonal antibodies to p53 and Bcl-2 protein were used after microwave antigen retrieval to enhance immunohistochemical staining of the sections. Proliferation was assessed by means of Ki-67 antigen expression. Positive staining in each specimen was expressed as a percentage and graded accordingly. Forty-one eyes in 40 black patients with a mean age of 37 years were found to have OSSN. Of the 41 lesions, 35 represented in situ or invasive carcinoma. The remaining 6 had mild or moderately dysplastic lesions. Seventeen patients agreed to an HIV test and, of these, 12 (70.6%) were HIV positive. All 12 were younger than 50 years, and 11 had either carcinoma in situ or invasive lesions. Twenty-two of 40 lesions expressed significant (greater than 50% of neoplastic cells) p53 positivity, while Bcl-2 expression was detected in 10. Ki-67 expression was low, even in the HIV-positive lesions. At our institution, OSSN occurs in young patients, many of whom are HIV positive. Expression of p53 is a common finding, whereas Bcl-2 immunoexpression occurs in the minority of cases. Ki-67 analysis showed that OSSN is a slow-growing tumor, even in the presence of HIV infection.

MIB-1 expression in cervical Papanicolaou tests correlates with dysplasia in subsequent cervical biopsies.

Ki-67 nuclear antigen is present in proliferating cells. MIB-1 antibody, raised to the recombinant part of the Ki-67 antigen, is a widely used biologic marker to assess cell proliferation. Ki-67 expression is normally observed in parabasal and basal cells in the cervix. With increasing severity of dysplasia, MIB-1 labeling is seen in cells of the superficial layers of cervical epithelium, which are exfoliated. The purpose of this study was to determine the sensitivity and specificity of presence of MIB-1-positive cells in Papanicolaou tests for predicting cervical neoplasia, condyloma, or both, on follow up. Using microwave antigen retrieval method, 49 air-dried cervical smears in two-smear cases were evaluated with immunostaining with MIB-1 monoclonal antibody. Presence of MIB-1 positivity was arbitrarily set at > or = 4 MIB-1 immunoreactive cells in each smear. The degree of positive staining was correlated with the cytologic diagnoses, subsequent colposcopy-directed biopsies, endocervical curettage, and/or cytologic follow ups. Follow-up findings correlated with cytology in 33 cases (67%), with MIB-1 positivity in 35 cases (71%). Three cases with positive follow ups were missed by cytology but detected by MIB-1 staining, and three cases were missed by MIB-1 but detected by cytology. Both cytology and MIB-1 staining failed to detect a subsequent cervical lesion in two cases, and in six cases each, an abnormal finding was not substantiated on follow ups. MIB-1 immunostaining is a powerful technique for evaluating gynecologic smears and is as equally sensitive and specific as cervical cytology. It is able to identify cervical disease overlooked by cytologic screening; therefore, it may serve as an adjunct and complimentary tool to cervical cytology.

Effect of bone morphogenetic protein 2 (BMP2) on oestradiol and inhibin A production by sheep granulosa cells, and localization of BMP receptors in the ovary by immunohistochemistry

The bone morphogenetic proteins (BMPs) have been implicated in the paracrine regulation of ovarian follicular development. In this study, we investigated the expression of the BMP receptors (BMPRs) in sheep ovaries by immunohistochemistry and the effect of BMP2, a natural ligand for these receptors, on granulosa cells cultured in vitro. Ovaries from cyclic ewes were fixed, embedded in paraffin wax and cut into sections. The sections were rehydrated, submitted to microwave antigen retrieval and treated with polyclonal antibodies against BMPR1A, BMPR1B and BMPR2. Strong immunostaining for all three receptors was observed in the granulosa cell layer of follicles from the primary to late antral stages of development. Staining was also present in the oocyte, corpus luteum, ovarian surface epithelium and, to a lesser extent, the theca layer of antral follicles. For functional studies, granulosa cells were obtained from immature follicles 1-3 mm in diameter. The cells were cultured for 6 days in serum-free medium containing 1 ng oFSH-20 ml(-1) in the presence of 0, 3, 10 or 30 ng ml(-1) human recombinant BMP2. The medium was replaced every 2 days and oestradiol and inhibin A concentrations were measured in the spent medium. In the absence of BMP2, oestradiol and inhibin A production increased as the granulosa cells differentiated in vitro. The addition of the highest dose of BMP2 enhanced oestradiol production (P < 0.05) without affecting the proliferation of the cells. It is concluded that BMP receptors are present in sheep ovaries and that BMPs may have a role in the differentiation of granulosa cells by enhancing the action of FSH.