p53 immunostaining is a highly specific and moderately sensitive marker of malignancy in serous fluid cytology.

Several studies have suggested that p53 immunostaining does not occur in benign mesothelium but is common in malignancies involving the serous surfaces, including malignant mesothelioma. As a result, p53 has been advocated as a marker of malignancy in serous fluid cytology. However, the specificity of p53 in this context has been brought into question by some studies that claim to have found immunostaining in benign mesothelium. The aim of our study was to examine p53 immunostaining in a large series of serous fluids to try to resolve the uncertainty. Monoclonal Do-7 antibody was used to immunostain ethanol-fixed cytospin preparations employing an alkaline phosphatase method. Positivity was found in 17 of 35 malignant effusions, including two probable mesotheliomas, but was not found in any of 115 benign effusions. Our study suggests that p53 immunostaining is a highly specific and moderately sensitive marker of malignancy in serous fluids.

Expression of a homologously recombined erythopoietin-SV40 T antigen fusion gene in mouse liver: evidence for erythropoietin production by Ito cells.

We have obtained transgenic mice in which an erythropoietin-SV40 virus T antigen fusion gene is homologously recombined into the native Epo locus. This gene is expressed in a tissue-specific manner closely resembling that of the native Epo gene. Immunohistochemical detection of SV40 T antigen has been used to characterize the hepatic cell populations expressing the transgene. In mice stimulated by anaemia or hypobaric hypoxia, SV40 T antigen was demonstrated in two liver cell populations: a subset of hepatocytes and a nonparenchymal cell type. Immunohistochemical and ultrastructural characterization of these cells by light and electron microscopy showed the nonparenchymal cell type to be the Ito cells, which lie in a persinusoidal position within the space of Disse. We therefore conclude that Ito cells are the nonhepatocytic source of liver Epo production. These cells show many similarities to the Epo-producing fibroblastoid interstitial cells of the kidney.

Identification of the renal erythropoietin-producing cells using transgenic mice.

Regulation of erythropoietin production by the kidneys is central to the control of erythropoiesis. Uncertainty about the identity of the renal cells involved has been a major obstacle to understanding this mechanism. We have used sequence from the mouse erythropoietin locus to direct expression of a marker gene, SV40 T antigen, to these cells in transgenic mice. The transgenic constructs contained an oligonucleotide marker (Epo-M) or SV40 sequence (Epo-TAg) in the 5' untranslated region of the mouse erythropoietin gene, flanked on each side by 9 and 7.5 kb of DNA from the mouse erythropoietin locus. Anemia-inducible expression of Epo-M and Epo-TAg was observed in the kidney. In one of thirteen lines, homologous integration of Epo-TAg into the mouse erythropoietin locus occurred. In transgenic mice bearing Epo-TAg at homologous and heterologous insertion sites, renal expression was restricted to a population of cells in the interstitium of the cortex and outer medulla. Immunohistochemical characterization by light and electron microscopy shows that these are the fibroblast-like type I interstitial cells.

Immunohistochemical properties of the endothelial cells in the human uterus during the menstrual cycle.

Expression of vascular cell adhesion molecule (VCAM) and endothelial leukocyte adhesion molecule (ELAM)-1 on human uterine endothelium was examined by immunohistochemistry. Endomyometrial blocks were obtained from 26 hysterectomy specimens (n = 6, menstrual; n = 6, follicular; n = 14, luteal). VCAM and ELAM-1 expression was compared to that of the classic endothelial markers Factor VIIIRA, CD31, CD34 and CD36, which were found to be present in samples taken from all stages of the cycle. Factor VIIIRA, CD31 and CD34 were expressed on most endothelium whereas CD36 was expressed primarily on that of small vessels. CD36 was also found to be strongly expressed on endometrial epithelium and no changes were apparent during the menstrual cycle. ELAM-1 expression was not detected in any of the tissues examined. VCAM expression varied during the cycle, appearing on endometrial endothelium in the mid- and late luteal phases, stages at which large granular lymphocytes migrate into endometrium. VCAM or a VCAM-like molecule may be involved in migration of large granular lymphocytes into endometrium.

Inappropriate expression of blood group antigens on biliary and colonic epithelia in primary sclerosing cholangitis.

The distribution of carbohydrate antigens of the ABO, Lewis, and Kell systems was examined in biliary and colonic epithelial of 11 patients with primary sclerosing cholangitis (PSC) using a panel of 11 monoclonal antibodies. Controls consisted of 27 liver biopsy specimens (11 normal, six alcoholic liver disease, five extrahepatic obstruction, and five primary biliary cirrhosis) and 24 colonic biopsy specimens (six normal, four Crohn's disease, and 14 ulcerative colitis). There was inappropriate staining with anti-A (four of six, 66%) and anti-B (nine of 11, 81%) in biliary epithelium of PSC patients compared with normal and disease controls. Expression of Lewis antigens was increased in patients with cholestatic liver disease. Ninety one per cent of PSC patients showed a similar pattern of inappropriate staining by anti-A and anti-B antibodies in colonic epithelium compared with 33% of normal and 42% of inflammatory bowel disease controls. There is inappropriate expression of A and B carbohydrate antigens in biliary and colonic epithelium in PSC. Whether these oncofetal antigens are implicated in the pathogenesis of this condition is discussed.

Comparison between liver and serum concentrations of mannan binding protein.

AIMS: To investigate staining patterns for mannan binding protein (MBP) by immunocytochemistry in liver biopsy specimens from patients with various hepatic disorders; to measure the serum MBP concentration in the patients at the time of biopsy; and to compare these to define further the role of MBP in disease. METHODS: Fifty seven consecutive patients with a variety of types of liver disease were studied. Fresh liver biopsy specimens were immunostained with anti-MBP and graded for intensity of staining. Serum MBP concentrations were measured on samples obtained on the day of biopsy, as were a full range of liver blood tests. RESULTS: MBP was only detectable in liver biopsy specimens from patients with morphological evidence of liver disease. MBP was most prominent in the livers of patients with severe alcoholic liver disease; livers harbouring metastases or showing biliary disease had moderate concentrations. Patients with liver disease were more likely to have raised serum MBP concentrations, but there was no correlation between these values and those found in the biopsy specimens. There was also no significant correlation between either of these concentrations and liver blood test abnormalities. CONCLUSIONS: Patients with liver disease tend to have raised MBP concentrations in both the liver and serum, but the exact relation between the two is as yet undefined.

Intramural platelet deposition in cerebral vasculopathy of systemic lupus erythematosus.

AIMS: To test the hypothesis that fragments of platelet thrombi and vascular endothelium are incorporated into the walls of small cerebral vessels in systemic lupus erythematosus (SLE). METHODS: Six varied necropsy cases of central nervous system (CNS) SLE and 15 controls were studied. The controls were selected to represent a wide range of diseases in which the cerebral circulation is compromised. Tissue sections were stained by standard histochemical and immunocytochemical methods, the latter using antibodies to platelet membrane glycoprotein IIIa (CD61), and vascular endothelium (CD31). RESULTS: In four of six cases of CNS SLE characterised by small vessel hyalinization and thickening, fragments of platelet membrane were found in the walls of small cortical and meningeal vessels. Similar findings were not evident in two other SLE cases that were characterised by relatively short clinical histories and an acute vasculitis. One control case of severe polyarteritis nodosa showed platelet fragment deposition in arteries larger than the vessels so affected in SLE. CONCLUSIONS: Previous studies have suggested that neuropsychiatric symptoms in SLE may be related to repeated episodes of vasculitis in small cerebral vessels that are triggered by antiphospholipid antibodies. Concurrent thrombus formation might facilitate the incorporation of platelet fragments into small vessel walls. This process contributes to the thickening and irregularity of small vessels, a major feature of longstanding cases of CNS SLE.

p53 expression in colorectal adenomas.

To assess the expression of p53 in premalignant lesions, we examined by immunohistochemistry benign colorectal adenomas (n = 72, measuring more than 6 mm and less than 95 mm in diameter) from patients without (group I, n = 23) or with (group II, n = 49) concurrent sporadic colorectal carcinomas. Using a panel of three monoclonal antibodies (PAb 240, PAb 421, PAb 1801) and two polyclonal antibodies (CM1, C19) immunohistological staining was demonstrated in 26% of the cases (19 of 72 adenomas, 7 of 23 from group I and 12 of 49 from group II). In the majority of the cases, p53 positive foci in the adenomas occurred in the most dysplastic areas, although focal positivity was detected in glands that were histologically normal. Expression of p53 protein was also detected in 21 of 30 (70%) colorectal carcinomas of group II. In two cases focal positive staining was observed in the polyps but not in the concurrent carcinomas. Non-neoplastic colonic mucosa and stromal lymphoid cells were negative in all cases examined. Over-expression of p53 in neoplastic tissues detected by immunocytochemistry is generally believed to correlate with the presence of mutation in the gene. This may not be an absolute rule, because in some hemopoietic malignancies, there is evidence that p53 protein may be detectable in the absence of an underlying mutation. These findings therefore represent the highest incidence in colorectal adenomas of abnormalities in the p53 protein expression, probably largely due to underlying mutations. This study also suggests that immunocytochemical demonstration of p53 protein may be a suitable method for the routine detection of subpopulations of cells which, by clonal expansion, could acquire a growth advantage within an adenoma during the neoplastic process.

An immunohistologic study of endothelialization of uteroplacental vessels in human pregnancy--evidence that endothelium is focally disrupted by trophoblast in preeclampsia.

OBJECTIVE: Our objective was to study the endothelial status of the luminal lining of uteroplacental vessels in the human placental bed in normal and abnormal pregnancy in the third trimester. STUDY DESIGN: Six placental basal plates from uncomplicated pregnancies and five from pregnancies complicated by preeclampsia (n = 3), preeclampsia and a small-for-gestational-age infant (n = 1), and diabetes mellitus (n = 1) were accessioned from the archives because of documentation of their containing uteroplacental vessels. Five placental bed biopsy specimens with intraluminal endovascular trophoblast in the third trimester were also studied. Sections were subjected to immunohistochemical analysis with monoclonal and polyclonal antibodies labeling endothelium and trophoblast. RESULTS: In third-trimester normal uncomplicated pregnancies the uteroplacental arteries and veins were completely endothelialized with no disruption of the endothelium. In third-trimester abnormal pregnancies the uteroplacental veins were also completely endothelialized. However, intraluminal endovascular trophoblast was seen within the uteroplacental arteries in eight of the 10 complicated pregnancies; this finding was associated with disruption of the endothelium. CONCLUSION: In preeclampsia there is an aberrant wave of endovascular trophoblast migration in the third trimester, resulting in focal disruption of the endothelium. This may be responsible for the endothelial cell dysfunction thought to be of pathogenetic importance in preeclampsia.

Co-expression of markers for hepatitis delta and hepatitis B viruses in human liver.

Delta hepatitis (HDV) infection can only occur in the presence of hepatitis B (HBV) infection, as HDV requires a coat of HBV surface antigen (HBsAg) for assembly of complete virus. A number of studies have examined the variation of HBV markers in serum and liver during establishment of HDV infection, but none has systematically examined the relationship between the two viruses in individual hepatocytes. Liver biopsies from five patients with HDV/HBV infection were stained for HBsAg, HBV core antigen (HBcAg) and hepatitis D (delta) antigen (HDAg). Double immunostaining was performed with a combination of indirect immunoperoxidase and alkaline phosphatase/antialkaline phosphatase techniques. HDV and HBV antigens were expressed in all five liver biopsies. Co-localization of HBsAg was seen in up to 39% of HDAg positive cells, and HBcAg in up to 8% of HDAg positive cells. HBcAg was detectable in approximately 9% of HBsAg positive cells, and HBsAg in approximately 12% of HBcAg positive cells. HDV can replicate without HBV but ultimately requires HBV to produce complete virus and subsequently infect other cells. In this study the majority of HDV positive cells did not appear to contain HBV markers. This might suggest delta virus replication without assembly, or possibly sequential production/assembly of the virus.

Immunocytochemical staining of serous effusions with the monoclonal antibody Ber-EP4.

Cytospin preparations were made from 102 serous effusions for immunocytochemical staining using a panel of monoclonal antibodies including a new monoclonal antibody Ber-EP4. On cytological examination, 32 fluids were reported to contain tumour cells consistent with metastatic adenocarcinoma; 66 contained benign cells only and three were reported to contain cells suspicious of malignancy. One effusion contained tumour cells consistent with malignant mesothelioma. Positive staining of the tumour cells with Ber-EP4 was observed in the 32 effusions (100%) which contained adenocarcinoma cells. No staining of the mesothelial cells in these 32 specimens was observed. Carcinoembryonic antigen, epithelial membrane antigen Ca2 and CD15 staining of tumour cells was noted in 53%, 50%, 50% and 9% of these cases, respectively. None of the mesothelial cells in the benign effusions stained with Ber-EP4. Nor did the malignant mesothelial cells in the only case of malignant mesothelioma. These findings suggest that Ber-EP4 is a valuable addition to antibodies available for the differential diagnosis of mesothelial cells and adenocarcinoma cells in serous effusions.

The L1 antigen and squamous metaplasia in the bladder.

The L1 antigen was investigated as a marker of squamous differentiation in urothelium using a monoclonal antibody Mac387, and the results were compared with the expression of high molecular weight cytokeratins. L1 antigen was consistently demonstrated in all instances of partial and complete squamous metaplasia and in squamous carcinomas. In contrast, pure transitional cell carcinomas (except one with minor focal staining), adenocarcinomas and normal and hyperplastic urothelium did not label. In a few squamous carcinomas in situ, the pattern of labelling obtained with Mac387 was different from that seen in invasive squamous carcinomas and metaplasias. Compared with high molecular weight cytokeratins, the expression of L1 was more intense in areas of squamous differentiation. L1 expression, as identified by antibody Mac387, may therefore serve as a useful marker of squamous differentiation in urothelial lesions.

Mannan-binding protein in human liver.

Mannan-binding protein (MBP) is a Ca(2+)-dependent lectin which was first described in 1978 in rabbit liver, and subsequently in serum and liver tissue from humans and a range of animal species. MBP structurally resembles C1q, and may act both as a focus for complement activation on the surface of microorganisms and as an opsonin in its own right. Low serum levels of serum MBP have been described in a group of children known to suffer from severe recurrent infections. MBP has also been reported to behave as an acute phase reactant. This preliminary study has investigated the localisation of MBP in human tissues using material obtained both at post mortem and from diagnostic liver biopsies. Using the IgG fraction of rabbit anti-human MBP, immunoperoxidase staining showed no evidence of significant MBP in a wide range of normal tissues, including liver taken both at post mortem and needle biopsy. However, there was a significant degree of staining for MBP in liver biopsies showing a variety of different pathologies, in particular severely damaged alcoholic livers, and those harbouring metastatic tumour. Moderate degrees of staining were also seen in liver biopsies from patients suffering from chronic biliary disease. It is uncertain whether this localisation of MBP in abnormal liver is an acute phase response, or represents a more fundamental link with liver disease. This question could be the focus for future studies.

Immunophenotyping of Nigerian cases of non-Hodgkin's lymphomas on paraffin sections.

One hundred cases of routinely fixed and processed non-Hodgkin's lymphoma from Nigeria were immunostained with a small panel of monoclonal antibodies against B-, T- and macrophage antigens. The aims of the study were to assess the suitability of stored material from a country like Nigeria for immunohistochemical examination and the ability of the antibody panel to evaluate the distribution of B- and T-cell neoplasms. Eighty-seven of the 100 cases gave interpretable immunostaining, with 75 being B-cell and 12 T-cell neoplasms. Eighty-seven of the 100 cases gave interpretable immunostaining, with 75 being B-cell and 12 T-cell neoplasms. There were no tumours of macrophage lineage. Four cases gave satisfactory staining of reactive lymphoid cells but no reactivity with malignant cells and thus were not phenotyped. The remaining nine cases gave no staining of neoplastic or reactive cells, suggesting that they were unsuitable for immunohistochemical study, presumably because of inappropriate fixation and handling. We concluded that a panel of three monoclonal antibodies is suitable for routine immunostaining of conventionally fixed and processed blocks in Third World countries and will give diagnostically useful information in approximately 95% of cases.

JC70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections.

A new monoclonal antibody, JC70, raised against a membrane preparation from a spleen affected by hairy cell leukaemia, recognises a membrane bound glycoprotein identical with that of the CD31 group of monoclonal antibodies. The antibody stains a fixation resistant epitope on endothelial cells in benign and malignant conditions in a wide variety of paraffin wax embedded tissue. JC70 stained malignant endothelial cells in 10 angiosarcomas with more consistency than monoclonal or polyclonal antibodies to factor VIII related antigen (FVIII-Rag). In four cases of Kaposi's sarcoma the antibody stained malignant endothelial cells but not spindle cells. It is concluded that antibody JC70 is of value for studying benign and malignant human vascular disorders in routinely processed tissue.

Megakaryocytes in myelodysplasia: an immunohistochemical study on bone marrow trephines.

Megakaryocytes in 63 bone marrow trephine biopsies were examined for their staining characteristics, location and size distribution using the monoclonal antibody Y2/51 directed against platelet glycoprotein IIIa (CD61). Megakaryocytes in normal bone marrow were evenly distributed and demonstrated homogeneous staining with Y2/51. In addition, there was little variation in their size or shape. In contrast, myelodysplastic and myeloproliferative bone marrow trephines showed considerable dysmegakaryopoiesis demonstrated by heterogeneity of staining, an altered architectural distribution with a predominantly paratrabecular location and considerable variation in size and shape. Furthermore, in myelodysplasia 25% of the CD61 positive cells were micromegakaryocytes as opposed to less than 10% in normal or reactive marrows. Such dysmegakaryopoiesis is believed to be a clinically important feature of myelodysplasia, although until now it has only been possible to assess it subjectively. The availability of the monoclonal antibody Y2/51 provides a rapid and reproducible means of studying megakaryocyte size, shape and distribution in routine trephine specimens and may help to overcome some of the diagnostic problems currently associated with myelodysplasia and other intrinsic bone marrow neoplasias.

Angioleiomyomas: an immunohistological study.

In a series of nine angioleiomyomas, nerves were identified immunohistologically within the capsule and interstitium only in those that were painful. We believe that it is stretching of capsular nerves that underlies most of the patients symptoms.

Combined non-isotopic in situ hybridisation and immunohistochemistry on routine paraffin wax embedded tissue: identification of cell type infected by human parvovirus and demonstration of cytomegalovirus DNA and antigen in renal infection.

A method for combined immunohistochemistry using alkaline phosphatase antialkaline phosphatase (APAAP) and in situ hybridisation using biotinylated probes was developed. The method requires no change to either technique and no additional procedures between them. The procedure was able to show the cell types involved in parvovirus infection of the fetus. The efficiency of immunohistochemistry and in situ hybridisation for the detection of cytomegalovirus in kidney were also compared: occasional cells contained cytomegalovirus DNA but not antigen. The method is rapid, straightforward, and has wide applications in the study of viral infections, genes, and gene products in tissue sections because it permits the combined demonstration of antigen and nucleic acid on the same section in routine clinical material.

Peripheral blood and portal tract lymphocyte populations in primary sclerosing cholangitis.

The relative distribution of lymphocyte subpopulations in the blood and liver of patients with primary sclerosing cholangitis (PSC) and related diseases has been studied using immunoenzyme techniques. The peripheral blood CD4/CD8 T lymphocyte ratio was significantly higher in active ulcerative colitis (UC) and in PSC with inactive UC than in inactive UC alone. In contrast, no relationship with disease activity was seen in Crohn's disease. The portal tract t lymphocyte count per high power field (mean +/- S.D.) was higher in pre-cirrhotic PSC (173 +/- 105) and primary biliary cirrhosis (PBC: 210 +/- 110) than in histologically normal liver (42 +/- 27). However, the overall portal tract CD4/CD8 ratio was similar in PSC (1.49), PBC (1.89) and normal controls (1.63). The results are consistent with immunological involvement in the pathogenesis of PSC, but argue against the hypothesis that changes in the peripheral blood T cell subsets are due to sequestration at the site of tissue inflammation.