Biomedical subjects
C J Smart
Publications and source records attributed to C J Smart.
Squamous cell carcinoma in a patient on clean intermittent self-catheterization.
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Extramammary Paget's disease of the penis is associated with long-standing transitional cell carcinoma and radiotherapy.
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Multidrug resistance evaluation by confocal microscopy in primary urothelial cancer explant colonies.
Assessing functional multidrug resistance (MDR) status in clinical biopsy material using drug autofluorescence has potential applications to clinical management. The small size of many cystoscopy specimens has led us to develop, as an alternative to flow cytometry, a protocol for studying epirubicin accumulation in adherent colonies of primary bladder cancer cells viewed live and in situ by confocal microscopy. The limitations to quantitation inherent in this technique are compensated for by preservation of cellular organisation and the elimination of non-malignant cells. Biopsy material is disaggregated and explanted into culture-grade petri dishes. After incubation for three to seven days plaques of epithelial cells have developed. Classical patterns of sensitive and resistant drug distribution are observed. Cells of the rolled edges of the colony accumulate more drug than those of the inner epithelial monolayer. Some central areas of larger colonies give the appearance of drug arrested at the intercellular junctions to give a fenestrated pattern. These observations contribute to the understanding of mechanisms in MDR as well as forming the basis for a clinical urological MDR evaluation protocol.
Confocal microscopy of idarubicin localisation in sensitive and multidrug-resistant bladder cancer cell lines.
Idarubicin is a highly lipophilic anthracycline and appears effective against tumours resistant to conventional anthracyclines. Confocal microscopy demonstrates predominantly cytoplasmic idarubicin accumulation. This distribution is unaltered by resistance status or the resistance reversing agent verapamil. Our results contrast with studies on conventional anthracyclines and suggest that nuclear accumulation may not be a prerequisite for anthracycline cytotoxicity.
Determination and reversal of resistance to epirubicin intravesical chemotherapy. A flow cytometric model.
OBJECTIVE: To develop a method of determining the characteristics of epirubicin resistance and to study the reversal of such resistance in the intravesical treatment of superficial bladder cancer, using sensitive and resistant derivatives of a bladder cancer cell line in vitro. MATERIALS AND METHODS: Epirubicin fluorescence and flow cytometry were used to measure the intracellular levels of epirubicin in both sensitive and resistant live cultured bladder tumour cells, with and without different doses of the resistance-reversing agent verapamil. RESULTS: There was a reliable, highly significant and consistent difference in intracellular epirubicin concentration between the resistant and sensitive bladder tumour cells. In addition, it was possible to substantially reverse the features of resistant cell subline with additional verapamil. CONCLUSION: Application of this assay to clinical specimens should allow better targeting of epirubicin intravesical chemotherapy and avoid the premature termination of such treatment in patients whose tumours remain sensitive to this agent. Furthermore, the addition of verapamil to intravesical epirubicin may permit effective treatment of those patients whose tumours have inherent or acquired resistance to epirubicin.
Determination and reversal of resistance to epirubicin intravesical chemotherapy. A confocal imaging study.
OBJECTIVE: To evaluate the use of confocal microscopy in the study of resistance to epirubicin and to determine the effect of temperature, viability and a resistance-reversing agent on the intracellular distribution of this drug in sensitive and resistant derivatives of a superficial bladder cancer cell line. MATERIALS AND METHODS: Viable and non-viable adherent cells were incubated in epirubicin solutions under various conditions. After incubation, the distribution of intracellular epirubicin fluorescence was visualized using confocal microscopy and a x50 water-immersion lens. RESULTS: There was a striking and consistent difference between resistant and sensitive cells in the intracellular distribution of the drug. In addition to having greater overall levels of epirubicin fluorescence, sensitive cells accumulated epirubicin predominantly in the nucleus. Epirubicin fluorescence in resistant cells was cytoplasmic and granular in appearance. When incubated at 0 degrees C, both cell lines showed no nuclear uptake and thus resembled resistant cells at 37 degrees C. However, dead cells rapidly acquired brightly fluorescent nuclei. The resistance-reversing agent verapamil appeared to cause reversion of the resistant to the sensitive phenotype. CONCLUSION: Confocal microscopy allows epirubicin-sensitive and resistant cultured tumour cells to be differentiated reliably and provides information about the mechanisms of action of, and resistance to, epirubicin. Applying this technique to clinical specimens should enable patients who have the resistant phenotype to be detected and the efficacy of intravesical resistance-reversing agents to be evaluated in such cases.
Primary pure teratoma of the testis.
PURPOSE: Despite its histologically benign appearance, primary pure teratoma of the testis is believed to have metastatic potential and behave similarly to other nonseminomatous germ cell tumors. We present our experience with the natural history and management of pure teratoma. MATERIALS AND METHODS: We reviewed the histological findings and clinical history of 15 patients with primary pure teratoma who were treated during a 15-year period, accounting for 4.2% of all nonseminomatous germ cell tumors treated during the same period. Fourteen patients were available for followup and are included in this report. RESULTS: In 8 patients the tumor was composed entirely of mature teratoma and in 6 immature elements were also present, although this finding was not associated with an increased frequency of metastatic disease. Carcinoma in situ was found adjacent to the tumor in 12 cases. Of 10 patients with stage I disease at presentation who were entered on a surveillance program only 2 have had relapse. The remaining 4 patients had metastatic disease at presentation and, thus, metastatic disease occurred in a total of 6 of the 14 patients (43%) with a median followup of 46 months (range 5 to 197). Metastatic disease was confined to the retroperitoneum in all 6 patients and only 2 patients had elevated serum marker levels. Five patients were treated with primary chemotherapy followed by resection of a residual mass and in all cases teratoma was identified in the resected mass. One patient underwent surgical excision of a retroperitoneal mass, which contained teratoma and yolk sac tumor, followed by chemotherapy. All patients are alive without evidence of progressive disease. CONCLUSIONS: In patients with primary pure teratoma of the testis metastatic disease may develop and the metastases may contain other subtypes of nonseminomatous germ cell tumors in addition to teratoma. There is probably a reduced frequency of relapse, which should be considered when advising patients with stage I disease, but otherwise management should be the same as for other testicular nonseminomatous germ cell tumors and the prognosis should be excellent.
Ruptured kidney--an unusual presentation of renal malakoplakia.
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Orchiectomy after chemotherapy in patients with metastatic testicular cancer. Is it indicated?
BACKGROUND: A small proportion of patients with testicular germ cell tumors present with widely metastatic disease and are treated initially with chemotherapy. Little is known about the efficacy of systemic chemotherapy in eradicating the primary testicular germ cell cancer; however, there is concern that the testis may act as a sanctuary site for germ cell cancer in these patients, and orchiectomy, is, therefore, recommended after chemotherapy. METHODS: The results from a clinical and pathologic review of 24 patients who underwent delayed orchiectomy after chemotherapy are presented. The testicular pathologic findings are correlated with those in extragonadal masses and also with a blinded review of postchemotherapy testicular ultrasound scans. RESULTS: The most common testicular pathological finding was a dense fibrous scar that was found in all patients. Three patients had persistent testicular germ cell cancer, six had mature teratoma, and one had carcinoma in situ. There was a strong concordance between the major testicular pathologic findings and those in the resected extragonadal masses. All three patients with persistent testicular germ cell cancer subsequently had disease progression in the extragonadal sites. Testicular ultrasound examination did not distinguish accurately between residual tumor or scar in the testis. CONCLUSION: Persistence of the primary testicular germ cell cancer is most likely due to the same heterogeneous response to chemotherapy observed in different metastatic sites. Because current imaging techniques cannot identify accurately those patients with residual testicular germ cell cancer or related testicular abnormalities that may predispose to subsequent relapse, orchiectomy after chemotherapy remains appropriate.
Nuclear restoration of cytoplasmic male sterility in sunflower is associated with the tissue-specific regulation of a novel mitochondrial gene.
We have previously shown that cytoplasmic male sterility in sunflower is associated with the insertion into the mitochondrial DNA of a novel open reading frame (ORF) located 3' to the atpA gene. Here, we show that in mitochondria from the sterile line, this novel ORF (ORF522) is cotranscribed with atpA. We have identified the product of the ORF522 as being a 15 kDa protein previously observed in sterile plant mitochondria by in organello translation. Both Western blot analysis and in organello translation assays show reduced levels of the 15 kDa polypeptide upon restoration of fertility. Interestingly, this reduction is tissue specific since it is only observed in the male florets from restored hybrid plants. These results suggest that the 15 kDa novel polypeptide is probably responsible for the CMS phenotype. Northern blot analysis using RNA from both seedlings and male florets shows a flower-specific reduction in the level of the ORF522 transcript in the restored hybrid line. The reduction is not due to a reduced transcription rate as demonstrated by 'run-on' experiments using mitochondria isolated from male florets. This suggests that the product of the nuclear restorer gene acts at the post-transcriptional level to destabilize the novel mitochondrial transcript in a tissue-specific manner and restore male fertility.
Cell-specific regulation of gene expression in mitochondria during anther development in sunflower.
Mitochondrial gene expression was characterized during meiosis in sunflower anthers. In situ hybridization experiments showed that there was a marked accumulation of four mitochondrial gene transcripts (atpA, atp9, cob, and rrn26) in young meiotic cells. This pattern of transcript accumulation was only detected for mitochondrial genes and not for transcripts of two nuclear genes (atpB and ANT) encoding mitochondrial proteins or another nuclear gene transcript (25S rRNA). Immunolocalization studies showed that the pattern of accumulation of the protein product of the atpA gene, the F1-ATP synthase alpha subunit, reflects that of the transcript. The expression of the novel mitochondrial orf522, which is associated with the cytoplasmic male-sterile (CMS) phenotype, was also studied by in situ hybridization. The orf522 transcripts were reduced in abundance in meiotic cells in the presence of fertility restorer genes. These results suggest that mitochondrial gene expression is regulated in a cell-specific fashion in developing anthers and that the restorer gene(s) may act cell specifically.
Painful shrinking testis and Pagetoid spread of germ cell neoplasia in the rete testis.
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Flexible cystoscopy in men: is topical anaesthesia with lignocaine gel worthwhile?
OBJECTIVE: The value of 2% lignocaine gel as a topical anaesthetic for flexible cystoscopy in men was tested in a prospective, randomized, double-blind, placebo controlled trial. PATIENTS AND METHODS: A total of 138 patients were entered into the study. Group A patients (n = 75) received 10 ml of 2% lignocaine gel intraurethrally whilst 10 ml of plain lubricating gel was instilled in the remaining 63 patients (Group B). Patient discomfort was recorded by means of a four-point descriptive pain scale (completed by both surgeon and patient) and a 100 mm non-graphic rating visual analogue scale (patient only). RESULTS: The results showed no significant difference between the two treatments irrespective of the method of recording. CONCLUSION: This study shows that 2% lignocaine gel offers no advantage over plain lubricating gel in providing analgesia for flexible cystoscopy. In this situation good lubrication may be a more important factor than topical anaesthesia.
Specific circulating anti-gliadin IgG-class antibody does not mediate intestinal enteropathy in gliadin-fed mice.
The effects of specific circulating IgG antibody on the uptake of dietary antigen and in the generation of intestinal enteropathy have been investigated in Balb/c mice bred on a gluten-free diet. A monoclonal IgG1 antibody (GD3) was prepared against gliadin. After adoptive transfer into mice, this antibody was capable of mediating a type III hypersensitivity response in vivo to footpad challenge with gliadin. The titres of circulating GD3, as estimated in vitro by ELISA, correlated well with the degree of inflammation at sites of type III responses in vivo. Following footpad challenge with gliadin, titres of circulating GD3 antibody were reduced. GD3 antibody was tested for its ability to mediate inflammatory responses in vivo in the intestinal mucosa of mice fed with gliadin. Circulating GD3 antibody was removed selectively and specifically by dietary gliadin, compared to feeding with bovine serum albumin or maintenance on a gliadin-free diet only. However, we were unable to demonstrate any pathological changes in the intestine as a result of possible local antigen-antibody complex formation or deposition. Using radio-iodinated gliadin as a trace marker, no significant retention of gliadin in the intestinal mucosa was found in mice pre-injected with GD3 antibody. These data suggest that circulating IgG antibody has little effect on dietary antigen uptake in the gut, and alone is insufficient to mediate an enteropathy.
Regulation of intestinal immunoglobulin production in response to dietary ovalbumin.
The effects of oral administration of ovalbumin (OVA) on intestinal immunoglobulin production was examined. Balb/c mice, bred and reared on an OVA-free diet, received either one dose or 14 consecutive daily intragastric doses of 25 mg OVA/dose. Single dose administration of OVA resulted in significant suppression of total immunoglobulins in the intestinal mucosa, particularly of the IgA isotype, although a very low titre anti-OVA IgG class antibody response was induced. After multiple peroral immunisations, there was more intestinal anti-OVA antibody induction and less suppression of total immunoglobulins. However, all the anti-OVA antibody was of the IgG isotype. In vitro production of mucosal immunoglobulins was not significantly reduced over 5 days, compared with controls, in either single or multiple administration groups, suggestive of a loss of T suppressor cell function in culture. Prior adoptive transfer of splenic and lymph node cells from mice preimmunised with OVA was capable of abrogating the local suppression of immunoglobulin production in vivo. Although adoptive transfer of bovine serum albumin-sensitised cells could also overcome some of the local suppression, complete restoration of normal immunoglobulin levels was not achieved. These data suggest that single oral administration of a novel dietary antigen induces a transient, non-specific suppression of intestinal immunoglobulin production, which can be overcome by antigen-specific T cells.
Expression of leukocyte adhesion molecules by mucosal mononuclear phagocytes in inflammatory bowel disease.
Leukocyte adhesion molecules are important in cell-cell interactions of the immune system. Lymphocyte function-associated antigen 1 (cluster designation 11a) mediates interactions between T cells and mononuclear phagocytes through its ligand, the intercellular adhesion molecule 1 (CD54), whereas complement receptors 3 (CD 11b) and 4 (CD11c) are involved in complement-mediated phagocytosis. Expression of CD11 molecules and intercellular adhesion molecule 1 was studied in colonic biopsy specimens from 20 patients with inflammatory bowel disease and 10 normal controls. In normal colon, few mononuclear phagocytes expressed lymphocyte function-associated antigen 1 and intercellular adhesion molecule 1 at high densities. The major adhesion molecule was CD11c. Thus, the largest population of normal colonic mononuclear phagocytes was represented by quiescent, resident macrophages with likely phagocytic function. In inflammatory bowel disease, mononuclear phagocytes showed only a slight increase in CD11a expression and no significant change in expression of CD11b and CD11c. By contrast, the percentage of mononuclear phagocytes expressing intercellular adhesion molecule 1 was increased from 6.9% +/- 3.9% in controls to 69.2% +/- 12.8% in ulcerative colitis (P less than 0.001) and to 45.7% +/- 22.8% in Crohn's disease (P less than 0.01), showing a close relationship with histological activity. The increased expression of intercellular adhesion molecule 1 in inflammatory bowel disease indicates a state of immunological activation induced by local release of inflammatory cytokines. Such induction of intercellular adhesion molecule 1 on mononuclear phagocytes may be important in the maintenance of chronic inflammation by facilitating interactions with T cells and T-cell antigen recognition.
Expression of the LFA-1 beta 2 integrin (CD11a/CD18) and ICAM-1 (CD54) in normal and coeliac small bowel mucosa.
The leucocyte adhesion molecules (beta 2 integrins) comprise CD11 alpha-chains and a common beta-chain (CD18). CD11a (leucocyte function-associated antigen 1, LFA-1) is expressed by most T cells, and is involved in antigen presentation by macrophages via its counter-receptor, intercellular adhesion molecule (ICAM-1, CD54). By criteria of double-label immunofluorescence of cryostat tissue sections, virtually all lamina propria T cells of the normal small bowel were found to express LFA-1 strongly. By contrast, only 30-60% of intra-epithelial lymphocytes (IEL) expressed detectable LFA-1, most of which were LFA-1 weak and CD18-. ICAM-1 was expressed strongly only by vascular endothelium. In coeliac disease, there was a modest increase of diffuse ICAM-1 expression in the lamina propria, mainly in the subepithelial zone, where ICAM-1+ macrophages were occasionally seen. There was also a slight overall increase in CD11a expression by IEL, seen predominantly in surface epithelium and mainly by the CD4+ minority subset, but not by CD4-CD8- (TcR gamma delta +) cells. These data suggest that the LFA-1/ICAM-1-dependent antigen presentation pathway is of minor importance to IEL in the normal small bowel, and does not assume a major role in coeliac disease.