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E Kay

Publications and source records attributed to E Kay.

At least 37 records · Page 2Linked to original sources
PubMed2002

Telomerase activity detected in oral lichen planus by RNA in situ hybridisation: not a marker for malignant transformation.

BACKGROUND: Oral lichen planus (OLP) is a chronic inflammatory condition. Clinically, it is characterised by the presence of a white lace-like lesion on the buccal mucosa, tongue, and gingivae, with erosions and ulceration. The World Health Organisation considers OLP to be a premalignant condition. AIMS: To investigate expression of the telomerase RNA component (hTR) in OLP compared with normal control buccal mucosa and to assess the possibility of using hTR expression as a marker for malignant transformation in OLP. METHODS: hTR expression was analysed in 40 cases of OLP and 18 normal control buccal mucosa samples using an RNA in situ hybridisation approach. RESULTS: Strong hTR RNA expression was seen in the basal, suprabasal, and to a lesser extent in the upper epithelial layers in 36 of the 40 OLP lesions examined. Infiltrating subepithelial lymphocytes in OLP were also shown to express hTR RNA. Weak hTR RNA expression was seen in seven of the 18 normal control buccal mucosa specimens, with expression confined exclusively to the basal layer of the epithelium and absent in the suprabasal and upper layers. CONCLUSION: The telomerase RNA component hTR is found to be highly expressed in the epithelium of non-dysplastic OLP lesions. It is possible that this high expression is related to the increased cellular proliferation seen in OLP lesions rather than being an indicator of susceptibility to malignancy. Thus, hTR RNA expression may not be a suitable marker for predicting malignant transformation in OLP.

Adult↗
PubMed2001

Immune function, telomerase, and angiogenesis in patients with primary, operable nonsmall cell lung carcinoma: tumor size and lymph node status remain the most important prognostic features.

BACKGROUND: Lung carcinoma usually is advanced at the time of presentation and frequently shows metastatic spread. In recent times, prognostic factors such as c-erbB-2 in patients with breast carcinoma have provided useful information and beneficial therapeutic targets. The objective of this study was to evaluate angiogenesis, immune function, and telomerase expression in patients with nonsmall cell lung carcinoma (NSCLC) to determine their prognostic significance. METHODS: Immunohistochemistry was used to evaluate the expression of human telomerase reverse transcriptase (hTERT; n = 115 patients), interleukin-2r (IL-2r; n = 40 patients), microvessel density (MVD; n = 81 patients), and vascular endothelial growth factor (VEGF; n = 61 patients). Three-year survival follow-up information was available for most patients, and a comprehensive review of clinicopathologic features was carried out. RESULTS: Fifty percent of tumors showed nuclear staining for hTERT, 55% of tumors showed some degree of lymphocyte IL-2r expression, 33% of tumors were recorded with an MVD that was higher than average, and VEGF staining was detected in 85% of tumors. None of the parameters measured had an impact on survival. hTERT expression was correlated with lymph node status. Lymph node status and tumor size were identified as independent prognostic factors. CONCLUSIONS: This study failed to identify a marker of prognosis for patients with NSCLC other than tumor size and lymph node status in this population. Telomerase expression was associated with metastases, raising the possibility that this enzyme is involved in the metastatic process. Tumor cell VEGF expression was identified frequently: This growth factor may have potential as a target for antiangiogenic therapy. Lung carcinoma typically is the result of large numbers of mutations. Further understanding of the biologic implications of these mutations will lead to the development of effective prognostic markers and treatments for patients with NSCLC.

Adult↗
PubMed2001

Tamoxifen inhibits endothelial cell proliferation and attenuates VEGF-mediated angiogenesis and migration in vivo.

INTRODUCTION: Angiogenesis is fundamental to tumour growth and vascular endothelial growth factor (VEGF) is one of the most potent proangiogenic cytokines known. We have previously demonstrated that tamoxifen reduces serum VEGF in certain cancer patients. We hypothesized that tamoxifen may attenuate the angiogenetic response to VEGF. METHODS: Human dermal microvessel endothelial primary cell cultures (HMEC) were incubated with tamoxifen (1.25-5.0 microg) or vehicle. Cell proliferation was quantified using 5-bromo-2'-deoxyuridine (BrdU) labelling endothelial cell proliferation assay. The effect of oral tamoxifen (20 mg/day) on VEGF-mediated angiogenesis in vivo was assessed using a Matrigel angiogenesis assay in the Sprague-Dawley rat. RESULTS: Tamoxifen (5.0 microg/ml) significantly reduced HMEC proliferation over 24 h when compared with cells treated with vehicle alone. Oral administration of tamoxifen in the rat (20 mg/day) significantly reduced endothelial cell proliferation and migration in response to VEGF. CONCLUSION: Tamoxifen (5.0 microg/ml) reduces proliferation of a VEGF-dependent endothelial cell line in vitro. In vivo, orally administered tamoxifen reduces VEGF-mediated angiogenesis in the rat. These findings indicate that tamoxifen may directly inhibit the effect of VEGF on the endothelial cell, in addition to its previously described effect of reducing serum VEGF levels. This data supports a role for tamoxifen in modulation of the VEGF-dependent angiogenic response to surgical trauma, particularly as an adjuvant therapy for VEGF-dependent tumours.

Administration, Oral↗
PubMed2001

Natural transformation of Pseudomonas fluorescens and Agrobacterium tumefaciens in soil.

Little information is available concerning the occurrence of natural transformation of bacteria in soil, the frequency of such events, and the actual role of this process on bacterial evolution. This is because few bacteria are known to possess the genes required to develop competence and because the tested bacteria are unable to reach this physiological state in situ. In this study we found that two soil bacteria, Agrobacterium tumefaciens and Pseudomonas fluorescens, can undergo transformation in soil microcosms without any specific physical or chemical treatment. Moreover, P. fluorescens produced transformants in both sterile and nonsterile soil microcosms but failed to do so in the various in vitro conditions we tested. A. tumefaciens could be transformed in vitro and in sterile soil samples. These results indicate that the number of transformable bacteria could be higher than previously thought and that these bacteria could find the conditions necessary for uptake of extracellular DNA in soil.

Agrobacterium tumefaciens↗
PubMed2001

Selective inhibition of COX-2 in humans is associated with less gastrointestinal injury: a comparison of nimesulide and naproxen.

BACKGROUND: Selective inhibitors of cyclooxygenase (COX)-2 may provoke less gastric damage and platelet inhibition than conventional non-steroidal anti-inflammatory drugs. AIMS: We compared the biochemical and gastrointestinal effects of nimesulide, a potent and selective COX-2 inhibitor, with naproxen which exhibits no selectivity. SUBJECTS: Thirty six healthy volunteers were randomised to nimesulide 100 mg or naproxen 500 mg twice daily for two weeks in a double blind, crossover study with a washout between treatments. METHODS: Gastrointestinal side effects were assessed by endoscopy, and by estimation of small intestinal absorption-permeability and inflammation. Comparisons were made between variables at the end of each treatment phase. RESULTS: Nimesulide caused significantly less gastric injury using the modified Lanza score (p<0.001) as well as reduced duodenum injury (p=0.039). Nimesulide had lower visual analogue scores (VAS) for haemorrhage and erosive lesions in the stomach (p<0.001) and for mucosal injection in the duodenum (p=0.039). Naproxen increased excretion of calprotectin, a marker of intestinal inflammation (5.5 (1.2) to 12.1 (2.1) mg/l) while nimesulide had no effect (treatment difference p=0.03). Naproxen abolished platelet aggregation to arachidonic acid and suppressed serum thromboxane B(2) (TXB(2)) by 98%, indices of COX-1 activity. In contrast, nimesulide had no significant effect on platelet aggregation, although it reduced serum TXB(2) by 29%. Production of prostaglandin E(2) and prostacyclin by gastric biopsies, also COX-1 dependent, was inhibited by naproxen, but not by nimesulide. COX-2 activity, determined as endotoxin induced prostaglandin E(2) formation in plasma, was markedly suppressed by both treatments. INTERPRETATION: Nimesulide has preferential selectivity for COX-2 over COX-1 in vivo at full therapeutic doses and induces less gastrointestinal damage than that seen with naproxen in the short term.

Adolescent↗
PubMed2001

Role of taurine in preventing acetaminophen-induced hepatic injury in the rat.

Acetaminophen overdose causes acute liver injury in both humans and animals. This study was designed to investigate the potential role of the conditionally essential amino acid taurine in preventing acetaminophen-induced hepatotoxicity. Male Sprague-Dawley rats were administered acetaminophen (800 mg/kg) intraperitoneally. Taurine (200 mg/kg) was given 12 h before, at the time of, and 1 or 2 h after acetaminophen injection. Acetaminophen treatment increased the plasma levels of aspartate transaminase, alanine aminotransferase, and alkaline phosphatase and caused hepatic DNA fragmentation and hepatocyte necrosis. Taurine administered before, simultaneously with, or 1 h after acetaminophen resulted in significant improvement in hepatic injury as represented by decrease of hepatocellular enzyme release and attenuation of hepatocyte apoptosis and necrosis, and this correlated with taurine-mediated attenuation of hepatic lipid peroxidation. These results indicate that taurine possesses prophylactic and therapeutic effects in acetaminophen-induced hepatic injury.

Acetaminophen↗
PubMed2001

TGF-beta1 is elevated in breast cancer tissue and regulates nitric oxide production from a number of cellular sources during hypoxia re-oxygenation injury.

Cellular response to treatment is dependent on the metabolic preconditioning of individual cells, which is a reflection of environmental conditions. Within solid tumours there are areas of different oxygen tension, which, we hypothesise, may indicate that cells are exposed to conditions that change continually. Other characteristics of the solid-tumour microenvironment include the production of growth factors, one of which is transforming growth factor (TGF)-beta1. As part of this study, we measured TGF-beta1 and found it raised in the serum of breast cancer patients compared with controls (98.24+/-13.25 vs. 48.87+/-12.14 ng/mL; P < 0.05; n = 7), and in the conditioned medium of breast tumour explant tissue compared with matched normal tissue (21.1+/-5.3 vs. 4.7+/-1.2 ng TGF-beta1/gram of tissue; P < 0.05; n = 11). Nitric oxide (NO) is a cytotoxic molecule produced by a large number of cells and thought to have antimetastatic properties. In order to observe the effect of conditions within breast tumours on NO production, we exposed macrophages, endothelial cells and tumour cells to hypoxia re-oxygenation in vitro, both in the presence and absence of TGF-beta1. Hypoxia stimulated increased NO production in both macrophages (normoxia: 0.34+/-0.04 nmol/L nitrite vs. hypoxia: 1.04+/-0.18 nmol/L nitrite; P < 0.05) and endothelial cells (normoxia: 0.02+/-0.01 nmol/L nitrite vs. hypoxia: 0.21+/-0.07 nmol/L nitrite; P < 0.05). NO production in macrophages, endothelial cells and tumour cells was reduced significantly following hypoxia in the presence of TGF-beta1 in a concentration-dependent manner. These results suggest that, within breast tumours, tumour-derived TGF-beta1 in combination with environmental conditions may result in decreased local NO production, which could have implications for tumour growth.

Breast Neoplasms↗
PubMed2000

Expression of CD44 in uterine cervical squamous neoplasia: a predictor of microinvasion?

OBJECTIVE: CD44, an integral membrane glycoprotein, may have an important role in early tumorigenesis, specifically, facilitating early tumor progression. Reports of the expression of CD44 in early uterine cervical squamous carcinogenesis are conflicting. We examined the expression of CD44 in microinvasive carcinoma of the cervix (MIC), as yet unreported, and compared it to that in cervical intraepithelial neoplasia (CIN) 1 and CIN 3 to further elucidate its role in early squamous carcinogenesis. METHODS: Seventeen cases of CIN 1, 24 cases of CIN 3, and 20 cases of MIC were stained with antibodies to CD44s, CD44v5, and CD44v6. Only membranous staining was considered positive. RESULTS: Positive membranous staining (>50% cells) was observed in 97% of cases of CIN 1 using all three antibodies. In CIN 3, positive staining was seen more often with CD44v6 (18/24) and CD44v5 (19/24) than with CD44s (6/24). Expression of CD44v6 was retained more often in MIC (16/20) compared with CD44s (3/20) and CD44v5 (9/20). Those cases of CIN 3 and MIC that failed to meet our criteria for positive staining showed either heterogeneous or absent staining. CONCLUSION: There is a qualitative and quantitative reduction in expression of CD44 in MIC and CIN 3 compared with CIN 1. Down-regulation of CD44 variants may occur later in neoplastic progression than CD44s. This pattern may reflect their important biological function in early progression by cervical cancer cells. Patchy and heterogeneous staining in more advanced lesions limits the usefulness of CD44 and its variants in the assessment of microinvasion.

Adult↗
PubMed2000

A simple and rapid technique for the detection of Epstein-Barr virus DNA in HIV-associated oral hairy leukoplakia biopsies.

A method of generating nucleic acid probes by polymerase chain reaction (PCR) for the detection of Epstein-Barr virus (EBV)-DNA by in situ hybridization in oral hairy leukoplakia (OHL) lesions is described. This method has the advantage over older methods of being cheaper, quicker and retaining sensitivity and specificity. Purified PCR products of Epstein-Barr virus DNA of 110 bp and 328 bp were labelled with biotin by nick translation or random primer labelling and were compared in in situ hybridization experiments with probes prepared by incorporation of biotin-labelled nucleotides in the PCR reaction mixture, with EBV viral DNA as a template. These probes were applied to 18 OHL tongue biopsies known to be positive for EBV-DNA, using a commercially available biotin-labelled BamHI "V" fragment EBV-DNA probe. To determine the specificity of the probes, we applied them to 20 normal tongue tissue samples and to 12 biopsies taken from keratotic tongue lesions from patients without risk factors for HIV infection and known to be negative for EBV-DNA. Clear positive signals for EBV-DNA were detected in all 18 cases of OHL biopsies using the amplimer of 328 bp labelled by PCR and random primer labelling. However, nick translation labelling was less efficient and sensitive. All control specimens were negative for EBV-DNA.

AIDS-Related Opportunistic Infections↗
PubMed2000

Diaphragmatic dysfunction secondary to experimental lower torso ischaemia-reperfusion injury is attenuated by thermal preconditioning.

BACKGROUND: Preconditioning describes the process whereby tissue exposure to a subcritical stress confers protection from subsequent injuries. This study assessed diaphragmatic muscle function after lower torso ischaemia-reperfusion (IR) and the role of thermal preconditioning in attenuation of this injury. METHODS: Sprague-Dawley rats were randomized into three groups (24 per group): a control group, an IR group that had aortic cross-clamping for 1 h followed by reperfusion, and a third group that received thermal preconditioning 18 h before IR. Diaphragmatic function was assessed at 24 h, 48 h and 7 days. RESULTS: IR resulted in significant diaphragmatic twitch and tetanic dysfunction compared with control muscle. Thermal preconditioning significantly attenuated this injury (P < 0.05). Mean(s.e.m.) muscle twitch and tetanic forces in the IR group were 204.9(17.2) and 282.7(19.2) g respectively at 24 h. Corresponding twitch and tetanic forces in preconditioned muscle were 270.4(25.1) and 552.0(35.2) g. CONCLUSION: This study demonstrated that systemic IR injury produced a respiratory muscle mechanical dysfunction that was attenuated by thermal preconditioning, at 24 h, 48 h and 7 days. Preconditioning may have a role in clinical practice, particularly before elective surgery.

Animals↗
PubMed2000

Taurine protects against early and late skeletal muscle dysfunction secondary to ischaemia reperfusion injury.

OBJECTIVE: To assess the efficacy of the cytoprotective B-amino acid taurine in prevention of skeletal muscle dysfunction secondary to ischaemia-reperfusion (IR) injury. DESIGN: Randomised controlled animal study. SETTING: Biomedical research laboratory, teaching hospital, Republic of Ireland. ANIMALS: 96 Sprague Dawley rats. INTERVENTIONS: Rats were randomised into three groups (n = 24/group): control (sham); ischaemia-reperfusion (untreated); and ischaemia-reperfusion + taurine. A further 24 rats were given taurine alone. The rat cremaster skeletal muscle model of four hours of ischaemia followed by reperfusion was used. Taurine 4%wt/vol was given in the animals' water throughout the experiment, beginning 48 hours before the ischaemia was initiated. OUTCOME MEASURES: 8 rats were killed from each group and muscle contractile function was assessed using electrical field stimulation in a tissue bath at 24 hrs, 48 hrs and 7 days. RESULTS: Ischaemia followed by 24 hours, 48 hours or 7 days of reperfusion resulted in significant reduction in both muscle twitch and tetanic contractile function (p < 0.05). This was reversed by taurine, which resulted in significant preservation of twitch and tetanic contractility at all time points including one week of reperfusion (p < 0.05). There was no difference in muscle function between muscle treated with taurine after ischaemia-reperfusion and control muscle, with the same operation but without ischaemia, from 48 hours onwards. CONCLUSIONS: These data show that pharmaceutical use of the endogenous amino acid taurine, unlike many-other agents, protects electrophysiological function in skeletal muscle against both early and late ischaemia-reperfusion injury.

Animals↗
PubMed2000

Potential dissemination of antibiotic resistance genes from transgenic plants to microorganisms.

Evidence that genes were transferred during evolution from plants to bacteria was obtained from nucleotide and protein sequence analyses. However, the extent of such transfers among phylogenetically distant organisms is limited by various factors, including those related to complexity of the environment and those endogenous to the bacteria, designed to prevent a drift of the genome integrity. The goal of this article is to give an overview of the potentials and limits of natural interkingdom gene transfers, with a particular focus on prokaryote originating sequences fitting the nuclear genome of transgenic plants.

Drug Resistance, Microbial↗
PubMed2000

Predicting population dental disease experience at a small area level using Census and health service data.

BACKGROUND: Information on the dental disease patterns of child populations is required at a small area level. At present, this can be provided only by expensive whole population surveys. The aim of this study was to evaluate the ability of Census data combined with health service information to provide estimates of population dental disease experience at the small area level. METHOD: Clinical dental data were collected from a large cross-sectional survey of 5-year-old children. A preliminary series of bivariate linear regression analyses were undertaken at ward level with the mean number of decayed, missing or filled teeth per child (dmft) as the dependent variable, and the Census and health service and lifestyle variables suspected of having a strong relationship with dmft as independent variables. This was followed by fitting a multiple linear regression model using a stepwise procedure to include independent variables that explain most of the variability in the dependent variable dmft. RESULTS: All deprivation indicators derived from the Census showed a highly significant (p<0.001) bivariate linear relationship with ward dmft. The Jarman deprivation score gave the highest R2 value (0.45), but the Townsend index (R2=0.43) and the single Census variable 'percentage of households with no car' (R2 = 0.42) gave very similar results. The health and lifestyle indicators also showed highly significant (p<0.001) linear relationships with dmft. The R2 values were generally much lower than the deprivation-related Census variables, with the exception of the percentage of residents who smoked (R2 = 0.42). None of the health or lifestyle variables was included in the final dental disadvantage model. This model explained 51 per cent of the variability of ward dmft. CONCLUSIONS: The results demonstrate the strong relationship between dental decay and deprivation, and all of the commonly used measures of deprivation exhibited a similar performance. For this population of young children health and health services shelf data did not improve on the ability of deprivation-related Census variables to predict population dental caries experience at a small area level.

Child, Preschool↗
PubMed2000

Immunohistochemical measurement of tumor vascular endothelial growth factor in breast cancer. A more reliable predictor of tumor stage than microvessel density or serum vascular endothelial growth factor.

Microvessel density counting is commonly proposed as a method of assessing angiogenesis. However, results have been difficult to reproduce because of many methodological inconsistencies. Vascular endothelial growth factor (VEGF), an angiogenic regulator, is also a poor prognostic indicator in breast cancer, correlating in many studies with microvessel density. In this study, VEGF and microvessel density counting were examined as methods of assessing angiogenesis in breast cancer and correlated with tumor stage. A representative tumor section was stained with anti-CD34 and anti-VEGF. Microvessel density was evaluated using the manual "hot-spot" procedure and a semiquantitative image analysis system. Serum VEGF levels were available from an additional nine patients. Results were analyzed using Kendall's tau correlation. Tumor stage correlated with tumor VEGF, but not with microvessel "hot-spot" or vessel counts. There was no correlation between scores obtained from the manual or semiquantitative methods. Serum VEGF did not correlate with either tumor VEGF or tumor stage. The prognostic importance of VEGF in invasive breast cancer is associated with tumor stage. Measurement of tumor VEGF, as an indicator of angiogenesis, is more reliable prognostically than measurement of microvessel density or serum VEGF. Routine measurement of microvessel density in breast cancer is less reliable.

Adult↗
PubMed1999

The role of endotoxin/lipopolysaccharide in surgically induced tumour growth in a murine model of metastatic disease.

Surgical removal of a primary tumour is often followed by rapid growth of previously dormant metastases. Endotoxin or lipopolysaccharide, a cell wall constituent of Gram-negative bacteria, is ubiquitously present in air and may be introduced during surgery. BALB/c mice received a tail vein injection of 10(5) 4T1 mouse mammary carcinoma cells. Two weeks later, animals were subjected to surgical trauma or an intraperitoneal injection of endotoxin (10 microg per animal). Five days later, animals which underwent open surgery, laparoscopy with air sufflation or received an endotoxin injection displayed increased lung metastasis compared to anaesthetic controls. These increases in metastatic tumour growth were reflected in increased tumour cell proliferation and decreased apoptosis within lung metastases. Circulating levels of the angiogenic cytokine, vascular endothelial growth factor (VEGF), were also elevated in these groups and correlated with increased plasma levels of endotoxin. Endotoxin treatment for 18 h (>10 ng ml(-1)) directly up-regulated VEGF production by the 4T1 tumour cells in vitro. Metastatic tumour growth in mice undergoing carbon dioxide laparoscopy, where air is excluded, was similar to anaesthetic controls. These data indicate that endotoxin introduced during surgery is associated with the enhanced growth of metastases following surgical trauma, by altering the critical balances governing cellular growth and angiogenesis.

Animals↗
PubMed1999

Phenotyping of immune cell infiltrates in breast and colorectal tumours.

White cell infiltration of solid tumors is an important prognostic indicator in malignant disease. Although macrophage infiltration is associated with good outcome in colorectal cancer, a high macrophage content is associated with poor prognosis in breast cancer. Suppressor macrophages prevent T cell activation in normal tissues such as mucosal linings exposed to continuous antigenic challenge. Interleukin 10 (IL-10), an immunosuppressive cytokine, inhibits macrophage co-stimulation of T cells. Suppressor macrophage numbers, T cell numbers and T cell activation status were assessed in cell suspensions obtained from fresh specimens of breast and colorectal tumours and matched normal tissues. IL-10 production by both malignant and matched normal tissue was also assessed. This study identified elevated numbers of suppressor macrophages in breast tumors compared to matched normal breast tissue. Colorectal tumors did not contain significant numbers of these cells. Although T cell numbers are increased in breast tumors, these cells do not appear to be fully activated, as assessed by major histocompatibility complex class II and Interleukin 2 receptor expression. In contrast, T cells in colorectal tumors exhibit greater expression levels of these markers. Breast tumors produce significantly higher levels of IL-10 than normal breast tissue whereas IL-10 levels in colorectal tumors are similar to normal colon tissue. Our findings of high suppressor macrophage numbers, high levels of IL-10 and poorly activated T cells in breast tumors compared to low suppressor macrophage numbers, low IL-10 and fully activated T cells in colorectal tumors may explain why high macrophage content is associated with poor prognosis in breast cancer and good prognosis in colorectal malignancy.

Adult↗
PubMed1999

Malignant lymphoma of the cervix. An unusual presentation and a rare disease.

Malignant lymphomas arising in the uterus are uncommon and are more commonly seen in the cervix than the corpus. Involvement of the cervix as part of a systemic lymphoma is more common than primary lymphoma, but the cervix as the site of presentation is unusual. We report two cases of malignant lymphoma of the cervix. The first patient, a 52-year-old woman, was referred to colposcopy following persistent low grade dyskaryosis on cervical cytology. At colposcopy a Lletz biopsy was performed and a diagnosis of CIN 1 and focal CIN 2 was made. In addition the subepithelial zone revealed a non-Hodgkin's (NHL) B-cell follicular lymphoma. The patient was subsequently staged as NHL Stage 3E. The second patient, a 35-year-old woman, was referred to the gynaecology department with a history of abnormal vaginal bleeding and two abnormal smears. Subsequent cervical biopsy revealed a high grade, large cell, malignant lymphoma, diffuse, B-cell. The patient was staged as Stage IE. Primary lymphoma of the uterine cervix as illustrated in the second case is very unusual. One case had negative cytology and one case had abnormal cells of uncertain origin. This highlights the difficulty of diagnosing cervical lymphoma, a rare but treatable malignancy, on cytology and suggests that cervical biopsy is needed for the confirmation of the diagnosis.

Adult↗