Search

Two proteins comprising the ZEB family of zinc finger transcription factors, ZEB1 and ZEB2, execute EMT programs in embryonic development and cancer. By studying regulation of their expression, we describe a novel mechanism that limits ZEB2 protein synthesis. A protein motif located at the border of the SMAD-binding domain of ZEB2 protein induces ribosomal pausing and compromises protein synthesis. The function of this protein motif is dependent on stretches of rare codons, Leu(UUA)-Gly(GGU)-Val(GUA). Incorporation of these triplets in the homologous region of ZEB1 does not affect protein translation. Our data suggest that rare codons have a regulatory role only if they are present within appropriate protein structures. We speculate that pools of transfer RNA available for protein translation impact on the configuration of epithelial mesenchymal transition pathways in tumor cells.

AIM: To determine the optimum form of labelling and the most efficient reporter molecule for non-radioisotopic in situ hybridisation (ISH). METHODS: Nine deoxyoligonucleotides complementary to histone mRNA were synthesised and labelled either enzymatically or during solid-phase synthesis with the reporter molecules digoxigenin, 2,4-dinitrophenyl (DNP), or alkaline phosphatase. Pooled deoxyoligonucleotide cocktails were then used in non-radioisotopic ISH detection of histone mRNA in human tonsil. Hybrid detection was by nitroblue tetrazoleum/5-bromo-4-chloro-3-indolyl phosphate colorimetric development. RESULTS: The use of a spacer in 3' enzymatic labelling and when labelling with alkaline phosphatase significantly increased ISH signal. The 3' and 5' labelling of oligonucleotides with triple DNP groups during solid-phase synthesis produced the strongest signal as determined by the highest cell signal intensity and shortest development time. CONCLUSIONS: 3' and 5' solid-phase labelling with triple DNP groups produced the best labelling for non-isotopic ISH using deoxyoligonucleotide cocktails.

Background Recent studies have identified unique small ribonucleic acids called microRNAs (miRNAs) in colonic tumour tissue and blood that may accurately diagnose the presence of colorectal cancer and help predict disease recurrence. This review explores the potential role of these biomarkers. Methods A literature search identified studies describing miRNAs in colorectal cancers. The outcomes of interest included diagnosis, progression and recurrence of disease, and future therapy. Results Overexpression and silencing of specific miRNAs are associated with the development and progression of colorectal cancer. Such a role in oncogenesis suggest that miRNAs may be important targets for gene therapies. Differential expression of specific miRNAs in tissues and blood offers the prospect of their use in early detection and screening for colorectal cancer. MiRNAs are implicated in metastasis and cytotoxic drug resistance. Their manipulation has potential in both prevention of recurrence and palliation. Conclusion The miRNAs expression profile in tissue and blood has potential for their use in the detection, screening and surveillance of colorectal cancer. Furthermore, miRNAs may be targeted by gene therapy to treat colorectal cancer.

Apoptosis is implicated as a major pathogenic mechanism in chronic hepatitis B and C. Previous studies of the relationship between apoptotic rates and histological necroinflammatory activity have produced conflicting results. Hepatocyte apoptosis was assessed in liver tissue from 32 cases of chronic viral hepatitis, seven cases of hepatocellular carcinoma (HCC) and six cases of steatohepatitis as non-viral disease controls and eight cases of control liver. Apoptotic rates were measured using H&E morphological assessment and immunohistochemical staining with antibodies to activated caspase-3 and M30. Histological necroinflammatory activity of viral hepatitis cases was scored using the Knodell scoring system, and the cases were divided according to their score into group 1 (mean 2.43 +/- 0.48) and group 2 (mean 7.80 +/- 0.49). Apoptotic indices were significantly higher in group 2 than group 1 using H&E (11.53 +/- 2.70 vs. 0 +/- 0, P=0.015) and activated caspase-3 (22.01 +/- 5.27 vs. 1.79 +/- 1.79, P=0.03) methods but were not significantly higher with M30 (3.80 +/- 1.74 vs. 0 +/- 0, P=0.207). Apoptotic scores using an antibody to activated caspase-3 are significantly higher in cases of chronic viral hepatitis with greater histological necroinflammatory scores, supporting a central role for apoptosis in disease pathogenesis. This method offers an alternative to routine histological assessment for measuring disease activity.

Smooth muscle actin (SMA) positive myofibroblasts have been implicated in tumour invasion; however, acquisition of SMA is not limited to peritumorous fibroblasts and other changes in fibroblasts may be more specifically related to the malignant environment. CD34 is a sialomucin expressed by normal breast fibroblasts but lost in invasive carcinomas. The aim of this study was to establish the relation between CD34 and SMA expression in breast fibroblasts and to analyse whether loss of CD34 is specific for invasive disease.Immunohistochemistry for CD34 and SMA was performed on 135 cases including 10 normal, 10 fibroadenomas, 40 infiltrating ductal carcinomas, 55 cases of ductal carcinoma in situ (DCIS), and 20 radial scar/complex sclerosing lesions. The relation between staining pattern and histopathological features was recorded as positive, negative, or reduced.Fibroblasts around all normal duct-lobule units and those showing epithelial hyperplasia were CD34 positive and mainly SMA negative. In fibroadenomas, fibroblasts retained CD34 but acquired SMA expression. In contrast, fibroblasts around invasive carcinoma were CD34 positive and SMA negative. In DCIS, loss of CD34 was significantly more frequent in high grade tumours than in low or intermediate grade ones (p0.001). The acquisition of SMA was seen more frequently than the loss of CD34, particularly in non-high grade DCIS. In all radial scars, fibroblasts were SMA positive but CD34 negative, and a similar pattern was seen in stromal cells in areas of fibrosis following core biopsy.These results show that SMA positive myofibroblasts exhibit variable expression of CD34, indicating that these markers are not coordinately controlled. Loss of CD34 is strongly related to the malignant phenotype, in both invasive and preinvasive disease, but is not entirely specific because radial scar fibroblasts and fibroblasts in reactive fibrosis exhibit a similar phenotype. The functional relevance of altered CD34 expression is unclear but the very focal changes implicate local signalling mechanisms probably of epithelial origin.

In normal breast and ductal carcinoma in situ, myoepithelial cells form an incomplete layer separating the epithelial compartment from the stromal environment. Transition to invasive disease is marked by penetration of the myoepithelial-basement membrane (BM) interface. One mechanism involved in tumour invasion is breakdown of extracellular matrices by matrix metalloproteinases (MMPs). It was hypothesized that myoepithelial cells may modulate tumour invasion by controlling MMP gene expression, both in tumour cells and in peri-ductal fibroblasts. To investigate this, myoepithelial cells from normal breast were purified and characterized and their effect on tumour cell invasive potential was assessed. The effect on MMP gene expression of breast cancer cells cultured alone or in combination with primary normal breast fibroblasts was also analysed using RT-PCR with ELISA quantitation, with zymographic analysis to measure enzyme activity. Normal breast myoepithelial cells significantly reduced invasion by the breast cancer cell lines MCF-7, T47D, MDA-MB 231, and MDA-MB 468 when they were cultured alone or in the presence of a fibroblast population. Reduced invasion was associated with changes in MMP gene expression. In those tumour cells expressing MMP, there was a significant down-regulation of MMP-2 (MDA-MB 468, p

AIM: To investigate vascular endothelial growth factor (VEGF) mRNA expression in glomerular disease in the context of heavy proteinuria. METHODS: Non-radioisotopic in situ hybridisation was performed using a cocktail of 12 deoxyoligonucleotides complementary to VEGF mRNA labelled during solid phase synthesis with 2,4-dinitrophenyl. Archival renal biopsies were studied from cases of minimal change nephropathy, membranous nephropathy, diabetic nephropathy, and controls, matched for age, sex, race, and storage time. Hybrid detection used NBT/BCIP colorimetric development. RESULTS: More VEGF mRNA positive glomerular cells per unit cross sectional diameter were seen in minimal change nephropathy (mean (SEM), 19.35 (1.5)) compared with controls (12.6 (1.73)), p < 0.01. In contrast, fewer were seen in diabetic nephropathy (5.93 (0.97)) compared with controls (9.97 (1.25)), p < 0.03. Analysis of membranous nephropathy (10 (1.62)) showed no difference from controls (10.98 (1.51)), NS. In addition, in minimal change nephropathy there was a significant correlation between 24 hour protein excretion at the time of biopsy and the number of VEGF mRNA cells per glomerulus (r = 0.08, p = 0.01). CONCLUSIONS: Using non-radioisotopic in situ hybridisation, VEGF mRNA is almost exclusively expressed by visceral glomerular epithelial cells. Abnormal numbers of cells are seen in both minimal change and diabetic nephropathy. As VEGF exists in a number of functionally distinct isoforms, further study of qualitative VEGF isoform expression in diagnostic groups is indicated.

the eVacement of foot processes represents a simpliBackground. Nephrin recently has been identified as a fication of architecture which demands massive remodputative adhesion molecule, expressed in the glomer- elling of the podocyte intercellular junction, the slit ulus, in which mutations cause congenital nephrotic diaphragm. syndrome of Finnish type. We sought to determine The molecular composition of the podocyte cell‐mawhether expression of nephrin is altered in human trix adhesion complexes has been largely determined glomeruli in patients with acquired nephrotic in recent years, but the composition of the slit diasyndrome. phragm remains surprisingly obscure. It contains the Methods. We performed PCR amplification of nephrin a-splice variant of ZO-1 [3], a protein which in the cDNA, using cDNA previously prepared from single a+ form is a normal constituent of tight junctions. human glomeruli plucked fresh from the surface of However, ultrastructurally and functionally, the slit human renal biopsies. We had available four cases of diaphragm is not a tight junction. Other tight junction nephrotic syndrome (one membranous, three minimal proteins such as cingulin [4] and occludin [5] are not change) and six normal controls. PCR product quant- present, and no other slit diaphragm-associated molecitation was by gel densitometry, confirmed by enzyme- ule has been identified. linked immunosorbent assay using a specific oligonu- The recent identification and sequencing of nephrin cleotide probe. Results were corrected for reaction has, therefore, provoked considerable interest [6 ]. A eYciency and glomerular cellularity by expression as a mutation in the gene for this protein was found to be ratio to levels of the ‘housekeeping gene’ glyceral- the underlying abnormality in the congenital nephrotic dehyde phosphate dehydrogenase. syndrome of Finnish type, where the glomerular podoResults. Glomerular levels of nephrin mRNA are signi- cytes fail to make normal foot processes or slit diaficantly decreased in cases of minimal change nephrotic phragms. Nephrin mRNA is expressed in normal syndrome. An apparent reduction was also seen in the kidneys, and has been localized by in situ hybridization single case of membranous nephropathy which was to the glomerular podocytes of a single human embryo available for study. [6 ]. Sequence analysis suggests that it is a novel Conclusions. Abnormalities of nephrin expression transmembrane adhesion molecule of the immunoglobappear to be associated with acquired as well as ulin superfamily and, therefore, one might speculate congenital causes of human nephrotic syndrome. that it could be a slit diaphragm component.

AIMS: To visualise directly numerical chromosomal aberrations and polyploidy in both Hodgkin and Reed Sternberg (HRS) cells and background cells from cases of Hodgkin's disease using in situ hybridisation. METHODS: Non-isotopic DNA in situ hybridisation was applied to interphase cell nuclei of Hodgkin's disease within routine paraffin embedded tissue sections. Two a satellite DNA probes, specific for chromosomes 3 and 12, were used to evaluate the feasibility of this approach. Double labelling with immunocytochemical detection of the CD30 antigen was used to identify HRS cells. Cytogenetic normal diploid and triploid placental tissue served as controls. RESULTS: The eight cases of Hodgkin's disease investigated displayed frequent polysomy, while the majority of background cells showed disomy signals. CONCLUSIONS: Numerical chromosomal aberrations were detected in HRS cells from eight cases of Hodgkin's disease by in situ hybridisation. These data show that in Hodgkin's disease HRS cells frequently display polyploidy compared with background cells and are, therefore, probably the only neoplastic component in this disease. Correlations between polysomy and tumour type or grade could not be made from these data owing to the limited number of cases examined and to problems with interpreting data from truncated nuclei.

Angiotropic lymphoma, also known as intravascular lymphomatosis, is characterised by widespread intravascular proliferation of malignant lymphoid cells, usually without evidence of focal disease. A case of a 52 year old man referred for investigation of a two year history of pyrexia of unknown origin, skin rash and multiple organ failure is described. Angiotropic lymphoma was seen in gastric, colonic and skin biopsy specimens, and review of an earlier skin biopsy specimen showed similar morphological features. In contrast to previous cases which showed B or T cell differentiation, immunohistochemical examination was positive for histiocyte markers. Molecular studies showed no evidence of immunoglobulin heavy chain gene or T cell receptor gene rearrangement. The patient responded to combination chemotherapy, comprising cyclophosphamide, doxorubicin, etoposide, and prednisolone. This case highlights the fact that advanced lymphoma may be present without evidence of focal disease and that the diagnosis may be missed easily both clinically and histologically.

Aim—To investigate whether clinical features of non-Hodgkin's lymphomas, at the time of first biopsy, correlate with studies of cell proliferation and cell death as well as with the level of bcl-2 expression. Methods—Bcl-2 expression, determined by immunocytochemistry, was compared with cell proliferation, measured using in situ hybridisation for histone mRNA, and cell death by apoptosis, measured using in situ end labelling for DNA cleavage. Results—Histone mRNA staining gave a labelling index of 30% of cells for reactive germinal centres, 5.2-13.5% of cells for low grade non-Hodgkin's lymphoma and 12.1-50.5% of cells for high grade non-Hodgkin's lymphoma. In situ end labelling gave a labelling index of 5.0-10.0% of cells for reactive germinal centres, 1.0-3.7% of cells for low grade non-Hodgkin's lymphoma and 4.7-13.5% of cells for high grade non-Hodgkin's lymphoma. There was a positive correlation between apoptotic index and proliferation index. More cases of low grade than high grade non-Hodgkin's lymphoma expressed bcl-2. There was no correlation between apoptotic index and bcl-2 expression for high grade non-Hodgkin's lymphoma. Conclusions—The molecular mechanisms controlling cell proliferation and death in non-Hodgkin's lymphoma are complex, probably involving a range of genes, including bcl-2. A better understanding of resistance to cell death is needed if the clinical goal of tailoring cancer treatment to individual tumours is to be achieved.

SUMMARYThe origin of mesangial IgA deposits in IgA nephropathy (IgAN) remains obscure. A significant proportion of deposited immunoglobulin is dimeric (J chain-positive). Previous studies of J chain expression within lymphoid tissue in IgAN have utilized antibodies which other investigators have found to be non-specific. To address this problem, we have developed an in situ hybridization (ISH) method for the detection of J chain mRNA within IgA plasma cells. Tonsils from 12 patients with IgAN and 12 controls were studied using (i) non-isotopic ISH for J chain mRNA, and (ii) combined immunofluorescence (IF) and fluorescent ISH. J chain mRNA-positive cells were identified in germinal centres, and within the subepithelial and interfollicular zones. A greater number of J chain mRNA-positive cells were found in the germinal centres of patients (mean 57.7±4.6 cells/105μm2) compared with controls (mean 36.9±3.5 cells/105μm2) (P < 0.001). Combined IF and fluorescent ISH showed a greater proportion of J chain mRNA-positive interfollicular IgA cells in patient tonsils (3.2±3.4%) compared with controls (21±2.3%; P < 0.02). These results indicate a shift towards dimeric IgA production in the tonsils in IgAN. In addition, the finding of excess numbers of J chain-positive IgA-negative cells within germinal centres suggests that an abnormality may be present at the B cell differentiation stage before IgA switching. These results further highlight immune abnormalities within the tonsil as a central feature of abnormal polymeric IgA biology in this common form of glomerulonephritis.

Muhammad Imran Aslam1,2, Maleene Patel1 2, Baljit Singh1,2, John Stuart Jameson2 and James Howard Pringle1 1Department of Cancer Studies and Molecular Medicine, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, 2Department of Colorectal Surgery, University Hospitals of Leicester NHS Trust, Leicester General Hospital, Gwendolen Road, Leicester, United Kingdom

The lineage of Hodgkin and Reed-Sternberg cells is still unclear. Detection of both immunoglobulin light chains in Hodgkin and Reed-Sternberg cells by immunohistochemistry is a well-known phenomenon. However, up to now, in situ hybridization techniques have failed to demonstrate light chain messenger(m) RNA in Hodgkin and Reed-Sternberg cells. In this investigation, we have analysed 26 cases of Hodgkin's disease (nodular lymphocyte predominant Hodgkin's disease, mixed cellularity, and nodular sclerosis type) using digoxigenin-labelled oligonucleotide probes for kappa and lambda light chains by in situ hybridization. In nearly half of the cases of nodular lymphocyte predominant Hodgkin's disease and in one case of mixed cellularity type, mRNA for only one light chain could be clearly demonstrated in the lymphocytic and histiocytic cells, Hodgkin, and Reed-Sternberg cells. These results support the idea that at least some cases of Hodgkin's disease are B-cell neoplasms.

We modified a technique of RNA detection based upon primed in situ labeling of RNA (PRINS) to quantitate poly(A) and histone mRNA in HL60 cell suspensions by flow cytometry. The direct PRINS technique was used to label poly(A) mRNA, based upon the incorporation of fluoresceinated nucleotides into cDNA chains generated by reverse transcription from the site of oligo(dT) primer-specific annealing on the poly(A) template. By this method, poly(A) fluorescent signals of up to 20-fold above background were obtained when primer concentrations were saturating. Nonspecific binding of the fluorochrome-conjugated nucleotide proved to be the greatest source of background fluorescence in cell suspensions. This was considerably reduced by preincubation of the cells in lysine and an excess of cold nucleotides prior to the PRINS reaction. The application of indirectly fluoresceinated antibodies to the FITC moiety of the conjugated nucleotide (indirect PRINS) enhanced detection of a fluorescent signal generated from cells by the direct PRINS technique after stringent histone priming. Cell cycle analysis, based upon DNA histograms obtained after PRINS labeling, showed the histone fluorescence to be primarily in S-phase cells of an exponentially growing population. Confocal laser scanning microscopy revealed good retention of cellular architecture, with cytoplasmic localization of poly(A) and historic mRNA fluorescence and heterogeneous mRNA expression among cells. Our modified PRINS labeling of RNA represents a useful advance in the precise quantitation of RNA species in cell suspension by flow cytometry.

This is a combined study using in situ hybridization and the polymerase chain reaction to investigate the presence of Coxsackie virus RNA in formalin-fixed tissue from cases of childhood myocarditis. Of the ten cases studied, two were positive by both methods. The virus RNA was predominantly located in areas showing an inflammatory cell infiltrate and myofibre necrosis. These findings suggest that direct lytic infection of myocytes by virus is responsible for myocarditis in these cases, rather than an autoimmune process, which has been suggested previously. The findings in one case, where the virus showed a marked sub-endocardial distribution, may have implications for the aetiology of endocardial fibroelastosis by confirming a viral tropism for this location. The techniques used in this study are easily repeatable and can be directly applied to look for viruses in a number of other diseases where a viral aetiology is suspected.

AIM--To analyse the configuration of the antigen receptor genes in Hodgkin's disease. METHODS--DNA extracted from 45 samples of Hodgkin's disease was analysed using Southern blotting and DNA hybridisation, using probes to the joining region of the immunoglobulin heavy chain gene, the constant region of kappa immunoglobulin light chain gene, and the constant region of the beta chain of the T cell receptor gene. RESULTS--A single case of nodular sclerosing disease showed clonal rearrangement of the immunoglobulin heavy and light chain genes, all other samples having germline immunoglobulin genes. The nature of the clonal population in the diseased tissue is uncertain, because the intensity of the rearranged bands did not correlate with the percentage of Reed-Sternberg cells present. The T cell receptor genes were in germline configuration in all the samples. CONCLUSIONS--Antigen receptor gene rearrangement is a rare finding in unselected cases of Hodgkin's disease.

The distribution and expression of parathyroid hormone (PTH) were investigated in normal and abnormal parathyroid tissue. PTH was detected using a monoclonal antibody with specificity for the 44-68 region of the PTH molecule. Prominent reactivity for PTH was seen in normal parathyroid with a granular pattern of staining. Active parathyroid tissue (adenoma and hyperplasia) showed much less reactivity for PTH, although there was prominent reactivity in the normal tissue around adenomas. Comparison of expression of PTH with that of parathormone mRNA showed a reciprocal pattern in normal tissue and, to a less marked extent, in abnormal tissue. Parathyroid carcinoma in particular had coinciding areas of PTH and PTH mRNA expression. Oxyphil cells had little or no PTH expression, except in the associated 'colloid' in some cases. The findings indicate an inverse relationship between storage and cellular synthesis of PTH, this being more marked in physiological than in pathological conditions of the parathyroid.

A simple method for the demonstration of Coxsackie virus RNA by in situ hybridization is described. Oligonucleotides complimentary to conserved sequences of Coxsackie B genome were synthesised and labelled with digoxigenin using commercially available reagents. In addition to detecting all five Coxsackie B strains examined, six strains of Coxsackie A were also demonstrated by these probes. Using one of the oligonucleotides separately it was possible to distinguish Coxsackie A strains from the strains of Coxsackie B virus examined. This study demonstrates the presence of viral RNA in mice tissues showing morphological evidence of damage, confirming the suspected tropisms of these viruses. The method described is directly applicable to the study of the presence of these viruses in human tissue from diseases where a viral aetiology is suspected.

The replication terminus region (31 to 35 min) of the Escherichia coli chromosome contains very few mapped genes (two per min) compared with the remainder of the chromosome, and much of the DNA appears dispensable. In order to determine whether, despite this, the terminus region consists of protein-coding sequences, we cloned 44 kb (1 min) of terminus region DNA (that surrounding trg at 31.4 min) and examined its ability to catalyze protein synthesis in vitro or in minicells. We were able to account for more than half the coding capacity of the cloned DNA with proteins synthesized in these systems, indicating that the sparsity of mapped genes in the terminus region does not result from a lack of identifiable coding sequences. We can therefore conclude that the terminus region is composed mainly of expressable, albeit inessential, protein-encoding genes.

AIMS: The development of a technique for simultaneous in situ hybridisation for native mRNA and conventional immunofluorescence for cytoplasmic antigens in routine pathology specimens. METHODS: Cocktails of synthetic deoxyoligonucleotides coding for immunoglobulin J chain and kappa light chain were 3' end labelled enzymatically with digoxigenin using terminal deoxynucleotidyl transferase. Native mRNA sequences were "unmasked" using proteolytic digestion with proteinase K and hybrid detection was achieved with an alkaline phosphatase labelled anti-digoxigenin antibody. Alkaline phosphatase was visualised with Fast red/naphthol AS-MX phosphate. Fluorescein isothiocyanate (FITC) conjugated anti-isotype antibodies were used simultaneously at the detection stage to identify the isotype production by individual plasma cells in endoscopic duodenal biopsy specimens. RESULTS: The IgA plasma cells of the lamina propria were identified by immunofluorescence and hybrids were detected in the anticipated plasma cell population by Fast red visualisation. The reaction product was visible in bright field or ultraviolet illumination which allowed FITC and Fast red labels to be visualised together under ultraviolet light at 490 nm. Dual labelled cells were clearly visible. Morphology was well preserved throughout. CONCLUSIONS: This technique permits the demonstration of specific mRNA species in cells expressing immunoglobulin. It combines all the advantages of non-radioactive synthetic oligonucleotide probes and conventional immunofluorescence techniques in routine formol-saline fixed and paraffin wax embedded sections with good retention of morphology.

Human insulin and glucagon mRNA were identified in routinely processed pancreatic tissue by non-radioactive in-situ hybridization using digoxigenin-labelled oligonucleotide probes. Cocktails of synthetic oligonucleotides complementary to human insulin and glucagon mRNA were labelled with digoxigenin using terminal deoxynucleotidyl transferase (Tdt). Specific hybrids were detected with alkaline phosphatase-labelled anti-digoxigenin antibody and visualized by BCIP-nitroblue tetrazolium indicator substrate. The results showed highly sensitive and specific staining of islet cells on a range of routinely formalin-fixed and paraffin-embedded tissues. Post-mortem pancreatic tissue from adults and stillborn neonates yielded acceptable signals as long as tissue morphology was well preserved. Preliminary investigations using pancreatic endocrine cell tumours gave clear easily interpretable signals which were comparable to conventional immunostaining. The application of this technique promises to be of value in the investigation of pancreatic disease.

The presence of Epstein-Barr virus (EBV) DNA in biopsy tissues from patients with Hodgkin's disease (HD) was investigated by the polymerase chain reaction (PCR) using primers specific to a sequence within the EBV Bam H1W region. EBV genome was detected in 33 of 57 (58 per cent) cases of HD. Viral DNA was, however, also demonstrated in nine of 24 non-Hodgkin's lymphomas, in three of nine non-neoplastic lymph nodes and in seven of 12 normal peripheral blood samples used as controls. In all cases, the band obtained following PCR was verified using Southern blotting and hybridization with highly specific Bam H1W probes. The results suggest that the technique is sufficiently sensitive to detect EBV in persistent latent infection in B-lymphocytes. Distinction between virus present as a possible aetiological agent of malignancy or as a latent infection is not possible when PCR is used under these conditions. The possible role of EBV as an aetiological agent of HD remains unresolved.

Immunohistologic studies have shown that synthesis of cytoplasmic immunoglobulin (cIg) is a normal function of some follicle centre cells (FCCs). The mechanisms regulating this synthesis of immunoglobulin and its function within the germinal centre are still poorly understood. In this study we applied a recently developed in situ hybridization method for the detection of kappa and lambda light chain mRNA to reactive lymph nodes and tonsils in order to investigate further the immunoglobulin-synthesizing cells of the germinal centre. FCCs containing detectable levels of light chain mRNA corresponded closely to cells containing cIg. The detection of light chain mRNA rather than its immunoglobulin product was found to be an advantage in that problems associated with the detection of extracellular immunoglobulin were eliminated. This was most apparent in germinal centres where the absence of ‘network’ immunoglobulin led to the observations that immunoglobulin-synthesizing FCCs are predominantly small centrocytes and that in a proportion of germinal centres they localize in that part of the light zone closest to the dark zone. This zonal distribution of immunoglobulin-synthesizing FCCs raises the possibility of further functional and micro-environmental subcompartments within the light zone.

An in situ hybridization technique has been developed for the detection of immunoglobulin light chain mRNA in routine pathology specimens. The method detects kappa or lambda constant region sequences using a cocktail of synthetic oligonucleotide probes labelled with biotin or fluorescein 5-isothiocyanate (FITC) reporter molecules. The probes were labelled at flanking sites chemically by primary amine directed acylation and by 'homopolymer tailing' with terminal deoxynucleotidyl transferase using non-radioactive nucleotide analogues. The mRNA was unmasked in the formalin-fixed tissue sections by digestion with varying concentrations of proteinase K, and the hybrids were demonstrated using alkaline phosphatase with either a streptavidin/biotin based four-stage system or an anti-FITC antibody based detection system. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results confirm that the method is specific for kappa or lambda mRNA and show that specific mRNAs can be detected in routine formalin-fixed sections using non-radioactive techniques with retention of good morphology. The method reliably detects light chain mRNA in cells expressing secretory immunoglobulin. The protocol can also be applied to tissue rich in endogenous biotin by using hapten-labelled probes.

BACKGROUND Cervical epithelium is known to contain receptors for Epstein-Barr virus (EBV) and is a recognized site of viral shedding. Recent cases of nasopharyngeal carcinoma have been associated with cervical carcinoma, leading to speculation of a possible etiologic link between EBV and cervical carcinoma. METHODS This study investigates the presence of EBV in 24 cases of invasive cervical carcinoma (9 adenocarcinomas and 15 squamous cell carcinomas) and 10 cases of cervical intraepithelial neoplasia (4 adenocarcinomas in situ and 6 squamous: 3 CIN II, 3 CIN III), using in situ hybridization. RESULTS In one case, EBV was demonstrated in infiltrating lymphocytes adjacent to tumor epithelium, but in no cases did the malignant epithelium show the presence of EBV. CONCLUSIONS These results suggest that EBV does not play a role in the etiology of cervical carcinoma, although further studies are needed.

Background Chronic allograft nephropathy is characterized by an excessive accumulation of extracellular matrix proteins leading to glomerular and interstitial fibrosis. The aim of this study was to determine the effects of two different immunosuppressive agents (cyclosporin and tacrolimus) on the expression of the genes controlling extracellular matrix deposition in renal transplant glomeruli. Methods Fifty-one renal transplant recipients were randomized to receive immunosuppression with either microemulsion cyclosporin or tacrolimus. Isolated glomeruli were plucked from protocol transplant biopsies performed 1 week, 3 months and 6 months after transplantation. Expression of the genes for collagen IVα2, collagen III, matrix metalloproteinase 2, tissue inhibitor of metalloproteinases (TIMP) 1 and TIMP-2, tenascin and transforming growth factor (TGF) β1 was studied by quantitative reverse transcriptase–polymerase chain reaction. Results The expression of messenger RNA (mRNA) for collagen III and TIMP-1 was significantly higher in patients receiving cyclosporin therapy than in those having tacrolimus (P < 0·01); this finding was accounted for by differences in the biopsy material at 1 week. A significant difference in collagen III, TIMP-1 and TIMP-2 mRNA expression was also detected between patients depending on the source of renal donor (cadaveric or living). There were no significant differences in the level of glomerular TGF-β1. Conclusion The data provide new in vivo evidence that tacrolimus may exert a less fibrogenic influence on transplant glomeruli than cyclosporin.

Remodelling of the extracellular matrix requires tight control not only of matrix synthesis, but also of matrix degradation. Control of matrix degradation is achieved mainly through the matrix metalloproteinase (MMP) enzymes. In the glomerulus, MMP-2 and MMP-9 are believed to be particularly important, as they have activity against type IV collagen. This study has demonstrated by immuno-electron microscopy that most of the immunoreactivity for MMP-2 in the normal glomerulus is located within the glomerular basement membranes and mesangial matrix. mRNA for MMP-2 is also detectable in normal glomeruli, but the other main gelatinase, MMP-9, could not be localized by immuno-electron microscopy. In the normal glomerulus, it seemed likely that MMP-2 is present in an inactive form. To confirm this, in situ zymography was carried out using frozen sections of normal kidney. Baseline activity of normal kidney was relatively weak, but this was dramatically increased by chemical activation of metalloproteinases. The results imply that MMP-2, in an inactive form, is a normal constituent of the extracellular matrix and glomerular basement membranes. Activation would presumably render the matrix 'self-degrading'; membrane-bound MMPs (MT-MMPs) seem particularly likely to be involved in leukocyte penetration of basement membranes in inflammation.

Although B-cell diffuse large-cell lymphoma (DLCL) can respond to chemotherapy and radiotherapy, a large number of patients are still resistant to treatment. Caspase-3 is an enzyme crucial to the apoptotic process and may be important in the clinical outcome of these patients. The pattern of caspase-3 expression was studied in 54 cases of DLCL using immunohistochemistry and quantitative reverse-transcription PCR. Tumor cells displayed both a diffuse cytosolic and a punctate cytosolic staining for caspase-3. Kaplan-Meier survival curves indicated that tumor cells with a diffuse cytosolic expression of caspase-3 correlated with a poor prognosis (P0.0004). In addition, a punctate cellular localization was associated with complete response to treatment (P = 0.011). Cases with a small percentage of lymphoma cells expressing caspase-3 also tended to show poor survival (P0.09). Levels of caspase-3 mRNA were not significant (P0.17), although a weak trend was observed similar to the immunohistochemical analysis. The pattern of expression of caspase-3 was also assessed with respect to terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) positivity in both reactive lymph nodes and B-cell DLCL cases. Our results suggest that TUNEL-positive cells are not caspase-3-positive and that there is no correlation between DLCL cases with a high degree of DNA fragmentation and caspase-3 immunostaining. Furthermore, a survival curve indicated that a high TUNEL positivity was associated with a poor survival probability (P0.02) and a poor response to treatment (P = 0.04). These results confirm the dynamic nature of caspase-3 expression in DLCL and suggests that the pattern of expression of the enzyme has prognostic significance.

The uterus and the placenta synthesize insulin-like growth factors (IGFs) and insulin-like binding proteins (IGFBPs). These growth factors are implicated in processes of proliferation and differentiation that occur in the uterus. To determine the patterns of expression of IGFs during rat pregnancy we used in situ hybridization with digoxigenin labeled probes on uterus from day 7 to day 16 of pregnancy. In early gestation days (7-8) both IGF mRNAs showed similar tissue distribution with relative abundance in the stroma and circular muscle layer. On days 11 and 12 expression for IGF-I mRNA was found in the mesometrial decidua and metrial gland and in the ectoplacental cone while clear expression of IGF-II mRNA could only be found in the latter. On days 13 and 14, expression for IGF-I mRNA could be detected in the mesometrial decidua and metrial gland but no expression was observed for IGF-II mRNA. A gradient of IGF-I mRNA expression could be observed in the placenta on day 16, with the trophoblastic cells of the basal zone expressing the signal with stronger intensity than in the labyrinthine zone. For IGF-II mRNA the highest expression was associated with the labyrinthine zone. Endovascular trophoblast was positive for both mRNAs. The spatial and temporal patterns of expression suggests a role for IGFs in the process of decidualization as well as in the establishment, growth and differentiation of the various trophoblast cells of the placenta.

Background: Membranous nephropathy (MN) is a ‘non-proliferative’ glomerulonephritis. However, visceral glomerular epithelial cell (vGEC) proliferating cell nuclear antigen staining and increased glomerular histone mRNA in passive Heymann nephritis (PHN), suggest that vGECs may enter the cell cycle and undergo DNA synthesis. We used in situ hybridisation for histone mRNA, an S-phase specific marker, to investigate this possibility and identify the cellular origin of histone mRNA in PHN and MN. Methods: PHN was induced in 16 Sprague-Dawley rats. There were 8 saline/serum controls. 12 animals were sacrificed on days 5 and 10. Renal biopsies from 10 proteinuric cases with MN and matched controls were studied. Results: Day-5 Heymann animals demonstrated more S-phase cells/glomerulus than controls (0.53 ± 0.09 vs. 0.195 ± 0.045; p < 0.01). Glomerular S-phase cells were also increased in patients compared to controls (0.24 ± 0.07 vs. 0.04 ± 0.018; p < 0.03). In both experimental and human MN, the peripheral location and morphology of glomerular histone mRNA-positive cells was typical of vGECs. Conclusion: The results in PHN indicate that vGECs recently injured with antibody and complement enter into the cell cycle and undergo DNA synthesis. The S-phase vGECs in MN may indicate the persistence of immune injury. Whether or not this process leads to cell replication is open to question.

The use of reverse transcription (RT) PCR for relative quantitation of gene transcripts relies on the reproducibility of the individual RT, PCR and product measurement steps. Semi-competitive RT-PCR (RT-cPCR) uses an internal competitor template in the PCR step to improve quantitation. We have surveyed the reproducibility of RT, PCR, RT-cPCR and measurement, amplifying the glyceraldehyde-3-phosphate dehydrogenase “housekeeping” gene from isolated renal glomeruli. We used an enzyme-linked immunosorbent assay (ELISA) to quantify PCR products. We also report our PCR-based method for constructing a competitor DNA identifiable independently of the native product. Our results show that the entire RT-PCR and ELISA process had a standard deviation (SD) of less than 10% (n = 10). This compared to an SD of less than 13% (n = 10) in PCR and ELISA. The SD for ELISA alone was less than 11% (n = 10). RT-cPCR quantitation gave an SD of approximately 15% (n = 10). These results support the use of standard RT-PCR for the relative quantitation of mRNA. RT-cPCR is also suited to relative quantitation, but it is also independent of the amplification saturation curve and permits the identification of differences in cellularity between samples.

The patterns of expression of mRNA encoding decidualization-associated protein (DAP) in the rat uterus from early, mid-, and late stages of pregnancy were examined by in situ hybridization to provide an insight into the function of DAP during pregnancy. Parallel studies were performed on rat uteri during the oestrous cycle and oil-induced deciduomata. DAP transcripts were absent in virgin uteri and during the day of implantation (day 5), low in early gestation tissues (days 8, 9) and high during midgestation (days 10-14). Expression of mRNA encoding DAP was first detected on day 8 of pregnancy in decidual cells of the lateral decidual glycogen wing areas. On day 9, mRNA encoding DAP was found in the same area as on day 8 as well as in the outer layer of cells in the antimesometrial decidua adjacent to the myometrium termed the fibrinoid capsula. On days 11 and 12, expression appeared in the decidual cells of the mesometrial decidua, and was particularly strong around the ectoplacental cone and blood vessels that were invaded by the trophoblast. By day 13 and throughout the remainder of pregnancy, expression occurred in the mesometrial decidua and metrial gland. No signal was observed in granulated metrial gland cells. mRNA encoding DAP was also found in the smooth circular muscle coat adjacent to the mesometrial decidua. A similar pattern and cellular localization of expression of mRNA encoding DAP expression was detected during the development of the deciduomata, artificially induced in the absence of a conceptus. The spatial and temporal patterns of expression correlated with a possible role for DAP in mediating fetal-maternal interactions and the establishment of the placental structure.

A method has been developed by which single human glomeruli may be plucked from fresh renal biopsies under direct vision, followed by separation of mRNA using oligo-dT-linked paramagnetic beads. The mRNA was amplified by reverse transcription and polymerase chain reaction (RT-PCR). Primers for a variety of human and rat proteins have been developed. The quantity of the amplified cDNA was measured by an enzyme-linked immuno-sorbent assay (ELISA), where biotinylated forward strands of DNA were captured, probed with a fluorescein-conjugated DNA oligomer, and then assayed with an enzyme-linked anti-fluorescein antibody. The cDNA-linked beads are reported to be stable and can be reused with different primer sets, thus forming a "bank' of samples from cases with defined glomerular disorders, which can be used to address new questions as they arise. Using rat glomeruli, a method has been devised which permits at least partial separation of epithelial cell mRNA from mesangial and endothelial cell mRNA.

AIM: To investigate the possible role of the systemic IgA immune system in the pathogenesis of IgA nephropathy METHODS: J chain mRNA expression in the IgA cells of the bone marrow was studied. Bone marrow trephine biopsy specimens from seven patients with IgA nephropathy and seven matched controls were examined by (1) non-isotopic in situ hybridisation (ISH) and (2) combined immunofluorescence and non-isotopic ISH to identify the plasma cell type. Serum polymeric IgA was also determined using standard high pressure liquid chromatography and sandwich enzyme linked immunosorbent assay. RESULTS: Non-isotopic ISH revealed a similar number of J chain mRNA positive cells/unit length in biopsy specimens from patients (16.5 +/- 2.7 cells/mm) and controls (17.7 +/- 2.4 cells/mm). Combined immunofluorescence and ISH revealed a greater proportion of J chain mRNA positive IgA cells in patients (7.6 +/- 1.45%) compared with controls (3 +/- 0.8%). Serum polymeric IgA was similar in both patients (91 +/- 22 mg/l) and controls (77 +/- 24 mg/l). CONCLUSION: These data suggest that excess production of dimeric IgA occurs in the bone marrow in IgA nephropathy.

Sjogren's syndrome (SS) is an autoimmune disease characterised by a generalised lymphoproliferation. Patients have an increased risk of developing lymphomas which are usually of the type associated with mucosa-associated lymphoid tissue (MALT). Histological examination of the minor salivary glands of the lower lip is a common and useful diagnostic test for SS but has not been able to provide information with regard to potential malignant change. In this study, a sensitive in situ hybridisation technique for the detection of kappa and lambda immunoglobulin light-chain mRNA was applied to labial salivary glands of 14 patients with SS. 7 cases showed light chain restriction, in 5 cases this was kappa(kappa:lambda ratio > 8.0) and in 2 it was lambda(kappa:lambda ratio < 0.6). Of these 7, 5 developed lymphomas--4 were low grade lymphomas of MALT type and the fifth patient died of disseminated lymphoma. The finding of light chain restriction in lip minor salivary glands is strong evidence of a monoclonal population of B-cells at this site. It is concluded that in patients with SS who develop lymphomas, dissemination of malignant cells may result in detectable disease in the minor salivary glands. Determination of kappa:lambda ratios in labial minor salivary glands may thus provide important prognostic information.

The role of coxsackie virus infection in the pathogenesis of idiopathic inflammatory muscle disease (IIMD) has been investigated by many workers with conflicting results. This study uses in situ hybridization, with digoxigenin-labelled oligonucleotide probes complementary to the coxsackie B virus genome, to investigate the presence of virus RNA in muscle biopsies from 26 patients with IIMD. In the five cases of inclusion body myositis studied, there was focal probe-binding to nuclei and cytoplasm, and in nine cases probe-binding to mast cells was seen. In both of these instances probe-binding was non-specific and not due to hybridization. None of the cases showed the presence of coxsackie virus RNA within muscle and it is concluded that lytic infection of myocytes by coxsackie virus does not occur in IIMD.

Expression of J chain mRNA in duodenal IgA plasma cells in IgA nephropathy. Glomerular IgA in IgA nephropathy (IgAN) is at least in part polymeric, and is thought to derive from the mucosal IgA system in view of the association between mucosal infection and haematuria in this condition. To investigate this hypothesis, an in situ hybridization (ISH) technique was developed for the detection of J chain mRNA, the expression of which has been correlated with the secretion of high level polymeric immunoglobulin (pIg). Endoscopic duodenal biopsies from ten patients and matched controls were examined by: (i) two color immunofluorescence (IF); (ii) ISH; and (iii) combined ISH and IF, to permit simultaneous identification of plasma cell type. IF revealed a reduction in the percentage of IgA plasma cells (P < 0.02) and increased absolute numbers of IgG cells (P < 0.02) in patient biopsies. ISH demonstrated fewer J chain mRNA expressing plasma cells (P < 0.005) with lower signal intensity (P < 0.002) in patients' biopsies compared with controls. Combined ISH and IF confirmed a reduction in J chain mRNA-positive IgA plasma cells in the patient biopsies (P < 0.02). The reduction in J chain mRNA expression in duodenal IgA plasma cells in IgAN argues against the gastrointestinal lamina propria as the source of glomerular pIgA.

AIM--To detect clonal rearrangements of the immunoglobulin (Ig) heavy chain gene in Hodgkin's disease tissue using the polymerase chain reaction (PCR). METHODS--DNA extracted from 36 samples of Hodgkin's disease was analysed using PCR and primers from conserved sequences in the variable (VH) and joining (JH) regions. RESULTS--Clonal rearrangement was detected only in one case. Evidence of clonal immunoglobulin gene rearrangement had been detected previously in this case using conventional Southern blot analysis. CONCLUSIONS--The sensitivity of the two techniques is equivalent and clonal Ig heavy chain gene rearrangements are rare in Hodgkin's disease.

Cervical epithelium is known to contain receptors for Epstein-Barr virus (EBV) and is a recognized site of viral shedding. Recent cases of nasopharyngeal carcinoma have been associated with cervical carcinoma, leading to speculation of a possible etiologic link between EBV and cervical carcinoma.This study investigates the presence of EBV in 24 cases of invasive cervical carcinoma (9 adenocarcinomas and 15 squamous cell carcinomas) and 10 cases of cervical intraepithelial neoplasia (4 adenocarcinomas in situ and 6 squamous: 3 CIN II, 3 CIN III), using in situ hybridization.In one case, EBV was demonstrated in infiltrating lymphocytes adjacent to tumor epithelium, but in no cases did the malignant epithelium show the presence of EBV.These results suggest that EBV does not play a role in the etiology of cervical carcinoma, although further studies are needed.

AIMS: To see if the relative expressions of proto-oncogenes that are increased in acute myeloid leukaemia are raised in patients with myelodysplastic syndromes (MDS), and to see if they increase with progression to leukaemia. To note if there is a correlation between morphology, karyotype, and these proto-oncogene expressions and if any one proto-oncogene can predict prognosis. METHOD: Bone marrow from 130 patients was analysed at six monthly intervals over two years for relative mRNA expression of seven oncogenes, karyotype, and morphology. The technique used slot blot hybridisation and densitometric analysis. The results were compared with 14 surgical controls and 30 people with vitamin deficiency anaemia. RESULTS: Six of seven oncogenes showed increased expression which progressed with time, but did not correlate with morphological or karyotypic changes. Expression of four of the seven oncogenes was increased in megaloblastic and iron deficiency anaemia. C-mos showed differences among the five morphological subgroups; it correlated with abnormal location (p = 0.025) and seemed to influence prognosis. CONCLUSION: Increased proto-oncogenes reflect the overall marrow perturbation in MDS. C-mos may reflect persistence of monocyte pathway which confirms marrow stability.

The distribution and expression of preproparathyroid hormone (PTH) mRNA were investigated in parathyroid tissue from 57 parathyroidectomy specimens. PTH mRNA was detected by in situ hybridization using digoxigenin-labelled oligonucleotide probes. Cell morphology was seen to correlate with PTH mRNA expression. Strong expression of PTH mRNA was confined to cells which on haematoxylin and eosin staining had large vesicular nuclei. These included both vacuolated and non-vacuolated cells. Chief cells with small dark nuclei and scanty cytoplasm had little or no expression. In both adenoma and chief cell hyperplasia, the striking difference from normal was the greatly increased proportion of cells expressing PTH mRNA. In adenomas, the rim of uninvolved parathyroid tissue showed PTH mRNA expression similar to that of normal parathyroid. In hyperplasia, there was frequently concordance of staining within individual nodules. The findings establish morphological criteria for activity of parathyroid tissue and support current concepts of the different pathogenesis of hyperplasia and adenoma. The expression of PTH mRNA in oxyphil change and parathyroid carcinoma was also investigated.

Summary Twenty-five patients with squamous cell carcinoma (SCC) of the anus have presented over an 8 year period; 18 were female. Six of 9 patients aged under 50 years were female. Five of these women had been treated for a previous cervical malignancy (2 invasive) and 4 practised anal intercourse; human papillomavirus (HPV) type 16 DNAs were isolated from their arcival anal/cervical paraffin sections. Signals were confined to the nuclei of the invasive anal SCC cells and the transformation zone of the cervix. HPV 6, 11 and 18 DNAs were not identified. Young women with cervical intraepithelial (CIN) III or invasive cervical SCC found in association with HPV infection are at risk of developing anal SCC (P

In situ hybridization techniques were used to detect immunoglobulin light chain messenger RNA (mRNA) in 28 formalin-fixed, paraffin-embedded samples of Hodgkin's disease. Cocktails of biotinylated oligonucleotide probes specific for the constant regions of kappa and lambda light chain mRNA were used. None of the Reed-Sternberg cells or their variants in any of the cases studied showed positive staining with either probe, in contrast to normal plasma cells which showed strong staining in the same sections. It was concluded, therefore, that the cytoplasmic immunoglobulin frequently detected within these cells by immunocytochemistry is present not as a result of synthesis, but as a result of some other mechanism.

The presence of Epstein-Barr virus (EBV) DNA in formalin-fixed, paraffin-embedded samples of Hodgkin's disease (HD) was investigated by Southern blot hybridization using a specific EBV Bam H1W fragment probe. DNA from 16 samples of HD in children and young adults was compared with that from ten samples in adults older than 45 years. A further eight cases of DNA extracted from fresh samples of HD were also studied, in order to compare the results obtained with fresh and fixed tissue samples. Hybridization was demonstrated in 15 of the 34 cases of HD studied in contrast to 3 or the 34 control specimens of non-Hodgkin's lymphoma, non-neoplastic lymph nodes, and carcinomas. No differences between the two age groups compared were found. The results of this study suggest that there is a definite association between EBV and HD, although the exact nature of this association remains to be established. It has also been shown that DNA of sufficient quality for the detection of EBV DNA can be extracted from formalin-fixed, paraffin-embedded material, and that comparable results can be obtained using DNA extracted from fresh and fixed tissue.

Epidemiological and serological evidence has suggested a role for Epstein-Barr virus infection in the aetiology of multiple sclerosis. Epstein-Barr virus-specific RNA was looked for in the brains of 10 patients with multiple sclerosis by in situ hybridisation. A total of 21 plaques was examined. In all of these preservation of RNA was shown by hybridisation of control probes to mitochondrial rRNA but no signal was detected with the Epstein-Barr virus probes. It is unlikely that persistent or latent Epstein-Barr virus infection of the CNS occurs in multiple sclerosis, although present findings do not exclude a role for Epstein-Barr virus in the initiation of this disorder.

To elucidate the synthetic activity in relation to the morphology of the epithelial cells of the small and large intestine, in situ hybridization with a poly-deoxyribothymidine (poly d(T)) probe was applied to paraffin sections of formalin-fixed blocks. This effectively displays poly-adenylated RNA and, by implication, messenger RNA (mRNA). By minimizing proteinase K pretreatment, the relative concentrations of cellular mRNA were visualized. This revealed minimal mRNA in crypt columnar cells, and maximal mRNA in proliferating cells and in cells showing terminal differentiation. The latter include surface epithelial cells, endocrine cells, Paneth cells, and maturing, but not mature, goblet cells. The goblet cells showing positive hybridization can be regarded as active cells and show characteristic morphology. Such cells are particularly evident in untreated coeliac disease, remitting ulcerative colitis, and transitional mucosa. The proliferating cells showing increased hybridization include normal mitotically active crypt epithelium, regenerating epithelium in ulcerative colitis, adenomatous epithelium, and adenocarcinomatous epithelium. The similarity of hybridization between metaplastic polyp epithelium and surface colonocytes is consistent with the concept that metaplastic polyps are formed of cells showing premature terminal differentiation.

SUMMARY: Extracellular polysaccharide (EP) and lipopolysaccharide (LPS) were isolated from three strains of Pseudomonas fluorescens (a wild-type and two mutants) and the adsorption isotherms (relationship between amount of polymer adsorbed and bulk liquid concentration of polymer in solution) of these polymers to hydrophobic, tissue culture treated and sulphonated polystyrene surfaces were measured. The adsorption properties of the polymers were then related to the ability of the three bacterial strains to attach to the polystyrene surfaces in an attempt to elucidate the attachment mechanisms. A Langmuir adsorption isotherm equation was applied to the data, and the mathematical constants thus derived indicated if and at what concentration each surface became polymer-saturated and whether multilayer adsorption occurred. EP isolated from a crenated mutant (strain with the greatest attachment ability) adsorbed at higher concentrations than EP from wild-type and mucoid strains, and the isotherm indicated multilayer adsorption. EP from the mucoid strain (strain with little attachment ability) showed comparatively little adsorption. The isotherm of wild-type LPS was very similar to that of EP from the mucoid strain. Polymer adsorption to the three surface types was different and was generally consistent with the different degrees of bacterial attachment to the surfaces.

The DnaA protein of Escherichia coli, essential for initiation at oriC, binds at a defined sequence which occurs at the chromosomal origin, near plasmid replication origins and in the promoters of the dnaA and mioC genes. This sequence also occurs at many other sites on the E. coli chromosome including three sites within the essential cell division genes ftsQ and A. Using an fts-lac fusion phage, lambda JFL100, we show here that fts gene expression responds both to reduced and increased intracellular levels of DnaA protein in a manner consistent with the hypothesis that DnaA protein regulates fts gene expression. Experiments using dnaC and dnaB-ts strains, however, suggest that DnaA control of fts transcription may be indirect, at least in part, with fts responding to the rate of initiation at oriC as well as to changes in DnaA protein level per se. It differs in this respect from dnaA gene expression which is unaffected when initiation of replication is inhibited by DnaB or DnaC inactivation. Strains integratively suppressed with pKN500 behave anomalously; neither fts nor dnaA transcription is significantly increased when DnaA is inactivated in these strains.