Climate deteriorations and Neanderthal demise in interior Iberia.

Time and circumstances for the disappearance of Neanderthals and its relationship with the advent of Modern Humans are not yet sufficiently resolved, especially in case of the Iberian Peninsula. Reconstructing palaeoenvironmental conditions during the last glacial period is crucial to clarifying whether climate deteriorations or competition and contacts with Modern Humans played the pivotal role in driving Neanderthals to extinction. A high-resolution loess record from the Upper Tagus Basin in central Spain demonstrates that the Neanderthal abandonment of inner Iberian territories 42 kyr ago coincided with the evolvement of hostile environmental conditions, while archaeological evidence testifies that this desertion took place regardless of modern humans' activities. According to stratigraphic findings and stable isotope analyses, this period corresponded to the driest environmental conditions of the last glacial apart from an even drier period linked to Heinrich Stadial 3. Our results show that during Marine Isotope Stages (MIS) 4 and 2 climate deteriorations in interior Iberia temporally coincided with northern hemisphere cold periods (Heinrich stadials). Solely during the middle MIS 3, in a period surrounding 42 kyr ago, this relation seems not straightforward, which may demonstrate the complexity of terrestrial climate conditions during glacial periods.

New insights into host-pathogen interactions during Entamoeba histolytica liver infection.

Amoebiasis is the third worldwide disease due to a parasite. The causative agent of this disease, the unicellular eukaryote Entamoeba histolytica, causes dysentery and liver abscesses associated with inflammation and human cell death. During liver invasion, before entering the parenchyma, E. histolytica trophozoites are in contact with liver sinusoidal endothelial cells (LSEC). We present data characterizing human LSEC responses to interaction with E. histolytica and identifying amoebic factors involved in the process of cell death in this cell culture model potentially relevant for early steps of hepatic amoebiasis. E. histolytica interferes with host cell adhesion signalling and leads to diminished adhesion and target cell death. Contact with parasites induces disruption of actin stress fibers and focal adhesion complexes. We conclude that interference with LSEC signalling may result from amoeba-triggered changes in the mechanical forces in the vicinity of cells in contact with parasites, sensed and transmitted by focal adhesion complexes. The study highlights for the first time the potential role in the onset of hepatic amoebiasis of the loss of liver endothelium integrity by disturbance of focal adhesion function and adhesion signalling. Among the amoebic factors required for changed LSEC adherence properties we identified the Gal/GalNAC lectin, cysteine proteases and KERP1.

The folic acid metabolite L-5-methyltetrahydrofolate effectively reduces total serum homocysteine level in orthotopic liver transplant recipients: a double-blind placebo-controlled study.

OBJECTIVE: Hyperhomocysteinemia is a described risk factor of cardiovascular diseases. The aim of this study was the treatment of hyperhomocysteinemia in liver transplant recipients with L-5-methyltetrahydrofolate (L-5-MTHF; 1 mg) vs folic acid (1 mg) vs placebo in a double-blind placebo-controlled study and to compare the relative responsiveness of these patients to L-5-MTHF and folic acid. SUBJECTS/METHODS: Patients were recruited from Hepatology-Transplantation-Unit at Johann Wolfgang Goethe-University, Frankfurt. Sixty patients were included in this study and 12 patients dropped out for different reasons. The patients were treated over 8 weeks with supplemental L-5-MTHF or folic acid or placebo. Serum homocysteine (HCY) was analyzed with high-performance liquid chromatography (HPLC) beside routine lab tests. RESULTS: We observed only a significant decrease of total serum HCY in the L-5-MTHF group during the study period (at week 0: 15+/-7.7 microM; after 8 weeks treatment: 9.41+/-2.6 microM, P<0.001). There was no significant decrease of total serum HCY neither in the folic acid group nor in the placebo group. CONCLUSION: The effects of L-5-MTHF are significantly more potent than folic acid itself. Therefore, lowering serum HCY in liver transplant recipients is effective with L-5-MTHF.

TCDD deregulates contact inhibition in rat liver oval cells via Ah receptor, JunD and cyclin A.

The aryl hydrocarbon receptor (AhR) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Activation of the AhR by TCDD leads to its dimerization with aryl hydrocarbon nuclear translocator (ARNT) and transcriptional activation of several phase I and II metabolizing enzymes. However, this classical signalling pathway so far failed to explain the pleiotropic hazardous effects of TCDD, such as developmental toxicity and tumour promotion. Thus, there is an urgent need to define genetic programmes orchestrated by AhR to unravel its role in physiology and toxicology. Here we show that TCDD treatment of rat liver oval cells leads to induction of the transcription factor JunD, resulting in transcriptional upregulation of the proto-oncogene cyclin A which finally triggers a release from contact inhibition. Ectopic expression of cyclin A in confluent cultures overcomes G(1) arrest, indicating that increased cyclin A levels are indeed sufficient to bypass contact inhibition. Functional interference with AhR-, but not with ARNT, abolished TCDD-induced increase in JunD and cyclin A and prevented loss of contact inhibition. In summary, we have discovered a novel AhR-dependent and probably ARNT-independent signalling pathway involving JunD and cyclin A, which mediates TCDD-induced deregulation of cell cycle control.

Polymorphisms in the methylenetetrahydrofolate reductase gene are determinant for vascular complications after liver transplantation.

OBJECTIVE: The aim of this study was to evaluate the role of the C677T-MTHFR (methylenetetrahydrofolate reductase)-polymorphism (CC, CT and TT) for vascular complications in liver transplant recipients. DESIGN: Retrospective study. SETTING: Hepatology-Transplantation-Unit, Johann Wolfgang Goethe-University, Frankfurt am Main. SUBJECTS: 48 liver transplant recipients were included, no dropouts. METHODS: MTHFR polymorphism was detected by PCR amplification and digestion with Hinfl restriction enzyme. Vascular complications after liver transplantation were detected from the patients' records. The total serum homocysteine (HCY) was analyzed with high-pressure liquid chromatography. RESULTS: In the wild-type group (CC), the HCY levels were slightly high (14.0+/-1 micro M). Among the patients with the CT polymorphism, the HCY values were elevated (22.5+/-3 micro M). In the homozygous TT group, there was a significant increase (31.2+/-6 micro M, P<0.01) of the HCY values. The percentage of vascular complications was higher in the heterozygous CT (47%) and homozygous TT (62.5%) group compared with wild-type CC (21%). Patients with a homozygous TT genotype of the MTHFR polymorphism with a vascular complication had a highly significant elevated HCY level compared to the other genotype groups, both with and without any vascular complications (P<0.001). Recipients with an elevated HCY and the TT polymorphism have a higher probability of developing a vascular complication after transplantation (odds ratio: 4.3 and 11.0; 95% confidence interval: 1.15, 12.25 and 1.41, 85.24). CONCLUSIONS: The C677T polymorphism in the MTHFR gene and subsequent elevation of the total serum HCY is significantly associated with an increased incidence of vascular complications in liver transplant recipients.

Long-term therapy of acute chronic liver failure to successful transplantation with an extracorporeal liver support system.

We report the case of a 38-year-old Caucasian male who was admitted because of end-stage liver failure due to primary sclerosing cholangitis. Because of the rapidly progressive severe hepatic encephalopathy and development of hepatorenal syndrome type I, the patient was immediately upgraded to a high priority status on the liver transplantation waiting list (T2 status according to Eurotransplant criteria). Intermittent therapy with an extracorporeal liver support system (Prometheus) was initiated in order to bridge the time period until the expected transplantation date. Under therapy with the extracorporeal liver support system, total serum bilirubin decreased significantly from 33 to 15 mg/dL after 8 sessions. Simultaneously the encephalopathy resolved gradually within 3 weeks (10 sessions) from initially grade 3 to grade 1. Extracorporeal detoxification therapy was continued for 51 days (23 sessions) until the patient underwent his successful liver transplantation in good general clinical condition. Prometheus, a new liver support system, seemed to sufficiently replace hepatic detoxification on a long-term basis in this patient with end-stage liver failure in order to bridge the time period until liver transplantation.

Does the liver ever age? Results of liver transplantation with donors above 80 years of age.

INTRODUCTION: Facing an increasing shortage of donor organs, donor criteria become more extended and so-called marginal organs are accepted for transplantation. For liver donation donor age above 70 years is accepted as a risk factor concerning primary dysfunction or nonfunction. Therefore, the aim of this study was to compare the early outcome of grafts older versus younger than 80 years of age. PATIENTS AND METHOD: Between August 2002 and February 2004, 40 adult liver transplants were performed using triple immunosuppression with tacrolimus, MMF, and low-dose corticosteroids. Recipients with HCC received low-dose rapamycin after postoperative day 14. The outcome of grafts from donors under 80 years of age (n=35) was compared with those from donors 80 years old or more (n=5). For statistical analysis Mann-Whitney-U-Test and Fisher's Exact Test were used with P < .05 considered statistically significant. RESULTS: The average donor age of our population was 54.4 +/- 17.3 years with five donors older than 80 years (80-83 years). These donors all had additional risk factors. The recipients of the latter grafts suffered from HCC and liver cirrhosis Child A (n=2) or from viral hepatitis (n=3). One recipient had advanced cirrhosis with severe complications. The outcomes of both groups were comparable concerning intraoperative and postoperative courses. All recipients of old liver grafts left the hospital with stable graft function. CONCLUSION: Liver grafts over 80 years can be transplanted with good results, especially if given to recipients with malignancy and otherwise stable liver function.

Sonographic characterisation of hepatocellular carcinoma at time of diagnosis.

BACKGROUND: Hepatocellular carcinoma (HCC) is a malignant liver tumour with a high prevalence world-wide. For screening procedures conventional transabdominal B-mode ultrasound and AFP determination are commonly used. We investigated 100 consecutive patients with histologically proven hepatocellular carcinoma in order to evaluate sonographic characteristics in unselected patients and to compare native and contrast-enhanced ultrasonographic techniques. PATIENTS AND METHODS: We investigated 100 consecutive patients with hepatocellular carcinoma at time of diagnosis with respect to echogenicity, patterns of vascularity, and portal/hepatic vein thrombosis. In addition to B-mode and native power Doppler sonography, contrast-enhanced power Doppler sonography with SHU 508A was used in 65 patients. RESULTS: The ultrasound appearance with conventional B-mode of hepatocellular carcinoma was hypoechoic in 48 % of the cases, isoechoic in 9 %, hyperechoic in 19 %, and in 25 % a mixture between hyper- and hypoechoic appearance was found compared to the surrounding liver tissue. Contrast-enhanced power Doppler sonography with SHU 508A changed the pattern of tumour vascularity in 27 % of patients into hypervascular, mainly in small lesions. DISCUSSION: At the time of diagnosis, the most commonly observed finding in hepatocellular carcinoma is that they appear hypervascular, independent of their size. The use of ultrasound contrast media should be considered to achieve characterisation of liver nodules in cirrhotic livers because they can improve the evaluation of tumour vascularity. Hypovascular HCC are found in about 10 % even after the administration of a contrast agent.

13C-methacetin breath test as liver function test in patients with chronic hepatitis C virus infection.

BACKGROUND: The 13C-methacetin breath test enables the quantitative evaluation of the cytochrome P450-dependent liver function. AIM: To find out whether this breath test is sensitive in noncirrhotic patients also with chronic hepatitis C in early stages of fibrosis. METHODS: Sixty-one healthy controls and 81 patients with chronic hepatitis C underwent a 13C-methacetin breath test. In all patients, a liver biopsy was performed. The liver histology was classified according to the histology activity index-Knodell score. RESULTS: Delta over baseline values of the patients at 15 min significantly differed from controls (19.2 +/- 9.2 per thousand vs. 24.1 +/- 5.7 per thousand; P < 0.003). The cumulative recovery after 30 min in patients was 11.4 +/- 4.8% and in healthy controls 13.8 +/- 2.8% (P < 0.002). However, patients with early fibrosis (histology activity index IVB) did not differ in delta over baseline values of the patients at 15 min (23.2 +/- 7.9 per thousand vs. 22.6 +/- 7.2 per thousand; P = 0.61) or cumulative recovery (13.6 +/- 3.7% vs. 13.2 +/- 3.8%; P = 0.45) from patients with more advanced fibrosis (histology activity index IVC). Patients with clinically nonsymptomatic cirrhosis (histology activity index IVD; Child A) metabolized 13C-methacetin to a significantly lesser extent (delta over baseline values of the patients at 15 min: 8.3 +/- 4.9 per thousand; P < 0.005 and cumulative recovery after 30 min: 5.6 +/- 3.2%; P < 0.003). The 13C-methacetin breath test identified cirrhotic patients with 95.0% sensitivity and 96.7% specificity. CONCLUSION: The non-invasive 13C-methacetin breath test reliably distinguishes between early cirrhotic (Child A) and noncirrhotic patients, but fails to detect early stages of fibrosis in patients with chronic hepatitis C.

[Consensus-recommendations for sirolimus in liver transplantation]. / Konsensusempfehlung zum Einsatz von Sirolimus in der Lebertransplantation.

Sirolimus is an m-TOR inhibitor without renal side effects and potentially protects against the development of malignancy. Due to a higher incidence of complications in two trials and an official warning in the drug information, the use of Sirolimus in liver transplantation is limited. The participants of this consensus meeting had to analyse and evaluate the literature with respect to the potential role of Sirolimus in liver transplantation. This consensus statement follows the scheme normally employed for the presentation of guidelines including the grading of evidence (1a-5) and the extent of recommendation (A-C). Moreover, the consensus included the experience of the authors with respect to the handling of Sirolimus after liver transplantation.

Open reduction and screw fixation of mallet fractures.

Twelve patients with mallet fractures treated by open reduction and internal fixation with small screws were reviewed at an average of 31 months after surgery. The indication for surgery was a fracture involving more than one-third of the distal phalanx articular surface or with subluxation of the distal interphalangeal joint. Loss of reduction occurred in one patient and in another one screw loosened slightly without loss of reduction. There were no nail deformities, infections, or secondary procedures. The mean range of motion was from 6 degrees (range, 0-30 degrees ) (extensor lag) to 70 degrees (range, 60-90 degrees ) flexion. Ten patients had no evidence of degenerative changes, one had minor joint space narrowing and one had significant deformity. Open reduction and screw fixation with small screws can lead to satisfactory outcome in appropriate patients.

Human peritoneal macrophages in culture: a model for studying inflammatory disorders in vitro.

In this study we characterized a model of human peritoneal macrophages maintained in culture for up to 48 h that can be used to study different functions of this cell population in vitro. The cells remained viable and functionally active over time, with well-preserved phagocytic properties. They expressed a macrophage marker, CD14. Once in culture, human peritoneal macrophages secreted C1q and nitric oxide in a pattern described in murine, guinea pig, and rat peritoneal macrophages. The described model can be used to study physiology and pathophysiology of peritoneal macrophages in vitro, offering all the advantages of the use of a human cell population.

Increased serum bone sialoprotein concentrations in patients with Crohn's disease.

INTRODUCTION: Impaired calcium homeostasis and/or the administration of corticosteroids are considered to be among the factors contributing to the pathogenesis of osteopenia in patients with inflammatory bowel disease. There is an increasing evidence suggesting that certain pro-inflammatory cytokines may also directly influence the bone metabolism in these patients. Routine measurement of bone mass and loss usually include dual energy X-ray absorptiometry as well as urinary and serum assessment of collagen crosslinks. More recent studies include likewise the detection of bone sialoprotein into a specific diagnostics of bone turnover. PATIENTS AND METHODS: We investigated 47 patients with inflammatory bowel disease (Crohn's disease N = 41, ulcerative colitis N = 6) and 17 healthy volunteers to assess and compare serum levels of bone sialoprotein and other routine parameters of bone turnover. Bone sialoprotein levels were measured by using a recently described radioimmunoassay. RESULTS: In comparison to the control group, bone sialoprotein and urinary crosslinks were significantly increased only in patients with Crohn's disease, while other markers of bone turnover (e. g. alkaline phosphatase, carboxylterminal propeptide of typ I procollagen, urinary deoxypyridinoline, vitamin D, phosphate and calcium) did not differ significantly between the patients' groups. CONCLUSION: According to these data, increased serum bone sialoprotein concentrations seem to be an additional valuable and sensitive marker of bone resorption in patients with Crohn's disease.

Nodular regenerative hyperplasia of the liver: a rare differential diagnosis of cholestasis with response to ursodeoxycholic acid.

Nodular regenerative hyperplasia of the liver (NRHL) is an uncommon non-malignant finding typically associated with haematological or auto-immune disease. The main clinical symptom is portal hypertension in the absence of underlying liver cirrhosis. The pathogenesis of NRHL remains unknown. We report a case of NRHL with cholestasis and progression to liver insufficiency without any underlying disease and no association with systemic disease or drug intake. Cholestasis and liver function tests improved significantly during treatment with ursodeoxycholic acid (750 mg per day). Based on this case, it may be concluded that treatment with ursodeoxycholic acid might be beneficial in patients with NRHL and progression to liver insufficiency.

2,3,7,8-tetrachlorodibenzo-p-dioxin-dependent release from contact inhibition in WB-F344 cells: involvement of cyclin A.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent tumor promoter ever tested in rodents. Although it is known that most of TCDD actions are mediated by binding to the aryl hydrocarbon receptor (AhR), the mechanisms leading to tumor promotion still remain to be elucidated. Loss of contact inhibition is one characteristic hallmark in tumorigenesis. In rat liver epithelial WB-F344 cells, TCDD induces a release from contact inhibition, which is manifested by a twofold increase in cell number when TCDD (1 nM for 48 h) is added to confluent cells in the presence of serum, but not when given to exponentially growing or subconfluent, serum-deprived WB-F344 cells. Loss of G1 arrest was also shown by flow cytometric analysis. We demonstrate that TCDD treatment significantly increases cyclin D2 and cyclin A protein levels and show by immunofluorescence that these proteins accumulate in the nucleus. Although TCDD treatment leads to a strong increase in cyclin D2/cdk4 and cyclin A/cdk2 complex formation, we could only detect an elevation of cyclin A/cdk2 activity. In accordance with a lack of elevated cdk4 activity, no decrease in the amount of hypophosphorylated retinoblastoma protein could be shown after TCDD treatment. The importance of increased cyclin A/cdk2 activity for TCDD-dependent release from contact inhibition was shown by the fact that the cdk2/cdc2-specific inhibitor olomoucine (25 microM) abolished TCDD response. These data indicate cyclin A-dependent loss of G1 arrest after TCDD treatment mainly downstream of the retinoblastoma protein.

Regulation of alpha1-proteinase inhibitor release by proinflammatory cytokines in human intestinal epithelial cells.

alpha1-Proteinase inhibitor (alpha1-PI) is the main serine proteinase inhibitor in human plasma. Apart from its synthesis in the liver, this anti-inflammatory protein is also synthesized by and excreted from human intestinal epithelial cells. Antiinflammatory actions of alpha1-PI are thought to be of relevance in the pathogenesis of inflammatory bowel disease. To investigate the role of macrophage-derived cytokines on alpha1-PI secretion from intestinal epithelial cells, we cultured Caco-2 cells until differentiation (14 days in culture) on permeable filter supports. Monolayers of differentiated Caco-2 cells were then co-cultured with human peritoneal macrophages, grown on plastic in the basolateral chamber. Under these conditions, alpha1-PI secretion from Caco-2 cells was enhanced by 45%, probably by a direct action of macrophage-derived cytokines on Caco-2 cells. To extend this observation further, we treated differentiated Caco-2 cells with macrophage-derived proinflammatory cytokines (IL-1beta, IL-8, TNF-alpha), as well as with lymphocyte-derived cytokines IL-2, IL-6 and IFN-gamma. As early as after 24h treatment, IL-2 and IL-8 induced a significant and dose-dependent increase of alpha-1-PI secretion into cell culture medium; this effect was completely reversed after immunoneutralization by the antibodies against IL-2 and IL-8 alpha1-PI secretion was only slightly decreased after treatment with IFN-gamma, while IL-1beta, IL-6 and TNF-alpha had no effect. alpha1-PI secretion correlated well with the expression of this protein in differentiated Caco-2 cells after cytokine treatment, as confirmed by Western blot. Our data imply that, in vitro, alpha1-PI secretion in enterocyte-like Caco-2 cells is up-regulated by IL-2 and IL-8. Our results suggest that both lymphocyte- and macrophage-derived cytokines regulate secretion of the anti-inflammatory protein alpha1-PI in intestinal epithelial cells.

Effects of deoxycholate on human colon cancer cells: apoptosis or proliferation.

BACKGROUND: Deoxycholic acid has long been attributed as a tumour promoter in the colon. It exerts its growth-related actions in a phorbol ester-like manner, by stimulating protein kinase C. The aim of this study was to investigate the effect of deoxycholic acid on proliferation and apoptosis in the colon, by exposing colon cancer cells to it in increasing concentrations. METHODS: Human colon cancer cells (Caco-2 and HT-29) were treated with deoxycholate or its two structural isomers, 3-beta-12-alpha-dihydroxy-5-beta-cholan-24-oic acid and 3-alpha-12-beta-dihydroxy-5-beta-cholan-24-oic acid. Proliferation was evaluated by cell counting, and apoptosis by estimating percentage cell survival and assessment of nuclear morphology. RESULTS: Within the concentration range of up to 20 microM, deoxycholate stimulated growth of both human colon cancer cell lines. Its growth-promoting effect was abolished after inhibition of protein kinase C. At concentrations above 100 microM, deoxycholate induced apoptosis in both cell lines. Epimers of deoxycholate were significantly less potent in stimulating growth. CONCLUSION: Low-dose deoxycholate stimulates colon cancer cell proliferation while > 100 micromol L(-1) of this secondary bile acid induces apoptosis in colon cancer cells. Deoxycholate might promote the likelihood of malignant transformation by increasing epithelial cell turnover in the colon.

Transmural drainage of cystic peripancreatic lesions with a new large-channel echo endoscope.

BACKGROUND AND STUDY AIMS: The availability of a new large-channel echo endoscope led us to develop a new needle-stent device for endoscopic puncture and drainage of pancreatogenic cystic lesions. The purpose of this study was to examine whether endoscopic ultrasound (EUS)-guided one-step 10-F puncture and drainage with the new equipment could be feasible and successful. The use of the technique and the short-term outcome in our first four patients are described and discussed. PATIENTS AND METHODS: Cystic lesions were drained using the new technique in four patients. All the patients had symptomatic peripancreatic lesions, one with intrahepatic and one with intrasplenic extension. Punctures were carried out using a new echo endoscope with a 3.7 mm working channel and an Albarran lever. The 10-F transmural stents were placed over a 1 mm stainless steel needle and a 6-F Teflon catheter using a special assembly designed for controlled one-step placement and stent release. RESULTS: Puncture and drainage were technically successful in two patients. In one patient, the 10-F component failed to pass the cystic wall. Drainage was successful in the same session using a 7-F one-step device. In one patient, no stent was placed, but the 1 mm needle was used for diagnostic tissue sampling during the procedure because of the suspicious cyst morphology. Surgical resection revealed a ganglioneuroma. CONCLUSIONS: The new echo endoscope allows endoscopic interventions for peripancreatic cystic lesions under excellent endoscopic and EUS control. The Albarran lever was reliable and helpful for optimal direction of the needle. The new echo endoscope combined with special needle-stent devices was effective and safe for diagnosis and therapy in the first four cystic lesions. Cystic tumors must always be considered as a differential diagnosis in patients with apparently pancreatitis-induced cystic lesions.

[Diagnostic imaging in liver cirrhosis]. / Bildgebende Diagnostik der Leberzirrhose.

For the diagnosis of liver cirrhosis ultrasound, computed tomography, magnetic resonance imaging, and angiography are recommended as imaging modalities. Ultrasound of the liver is used as a screening imaging tool in cases of patients suspicious for diffuse liver disease and is helpful in the term of follow-up examinations. Computed tomography is mainly performed to clarify the presence of liver disease detected by ultrasound. In this context, accurate examination of the vascular structures of the liver as well as extrahepatic situation, is of the essence. Diagnosis of diffuse liver disease and characterization of morphologic changes is improved using contrast-enhanced MR imaging with liver specific contrast media. Combined magnetic resonance imaging can provide comprehensive evaluation of cirrhosis. An improved detection rate and characterization of regenerating nodules can be achieved compared to imaging modalities such as ultrasound and computed tomography. MR imaging can be performed in a one-stop-technique using unenhanced and liver-specific-contrast-enhanced sequence protocols to evaluate the liver parenchyma itself, MR cholangiography to verify the bile duct system, and MR angiography to specify the vascular situation. This technique is the optimal protocol for diagnostic imaging in patients suffering from liver cirrhosis and the method of choice to reach the final diagnosis.