20-Hydroxyecdysone-upregulated proteases involved in Bombyx larval fat body destruction.

During insect larval-pupal metamorphosis, the obsolete larval organs and tissues undergo histolysis and programmed cell death to recycle cellular materials. It has been demonstrated that some cathepsins are essential for histolysis in larval tissues, but the process of tissue destruction is not well documented. Fat body, the homologous organ to mammalian liver and adipose tissue, goes through a distinct destruction process during larval-pupal transition. Herein, we found that most of the Bombyx proteases - including Bombyx cathepsin B (BmCatB) (BmCatLL-2), Bombyx cathepsin D (BmCatD), Bombyx cathepsin L like-1 (BmCatLL-1) and -2(BmCatLL-2), Bombyx fibroinase (BmBcp), Bombyx matrix metalloprotease (BmMmp), Bombyx A disintegrin and metalloproteinase with thrombospondin motifs 1 (BmAdamTS-1), Bombyx A disintegrin and metalloproteinase with thrombospondin motifs like (BmAdamTS L) and Bombyx cysteine protease inhibitor (Bmbcpi)- were expressed highly in fat body during feeding and metamorphosis, with a peak occurring during the nonfeeding moulting or prepupal stage, as well as being responsive to 20-hydroxyecdysone (20E). The aforementioned protease genes expression was upregulated by injection of 20E into the feeding larvae, while blocking 20E signalling transduction led to downregulation. Western blotting and immunofluorescent staining of BmCatB and BmBcp confirmed the coincident variation of their messenger RNA (mRNA) and protein level during the development and after the treatments. Moreover, BmCatB, BmBcp, BmMmp and BmAdamTS-1 RNA interference all led to blockage of larval fat body destruction. Taken together, we conclude that 20E regulates larval fat body destruction by upregulating related protease gene expression and protein levels during larval-pupal transition.

Hepatocellular carcinoma and non-Hodgkin lymphoma in a patient with chronic hepatitis C and cirrhosis.

Hepatitis C virus (HCV) is a hepatotropic virus, but its genome and replicative intermediates also have been detected in peripheral blood mononuclear cells in patients with chronic hepatitis C. Chronic HCV infection may lead to hepatocellular carcinoma and, in a small percentage of cases, to B-cell non-Hodgkin lymphoma. To our knowledge, coexistence of these 2 tumors has not been reported previously. We describe a case of chronic hepatitis C and cirrhosis with 2 small hepatocellular carcinomas and incidental non-Hodgkin lymphoma of a hilar lymph node found during liver transplantation. Although the mechanisms of HCV oncogenesis in hepatocellular carcinoma and in lymphoma are unclear, the presence of these 2 tumors in a single patient are in agreement with the tropism of HCV and its role in oncogenesis.

Primary hepatic marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue type in a patient with primary biliary cirrhosis.

Primary lymphoma of the liver is rare. Recently, marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (MALT) type have been described in the liver. Most of these cases occurred without known underlying liver disease, while others were seen in patients with chronic hepatitis. A case of primary hepatic MALT lymphoma in a patient with primary biliary cirrhosis was reported recently. Some authors have proposed that chronic persistent immunogenic stimulation causes development of acquired MALT and subsequently MALT lymphoma, based on the observation of MALT lymphoma in association with infectious agents, such as Helicobacter pylori and hepatitis C virus, and autoimmune diseases, such as Hashimoto thyroiditis and Sjögren syndrome. Primary biliary cirrhosis is a chronic, progressive, cholestatic liver disease characterized by destruction of intrahepatic small to medium-sized bile ducts; this disease is mediated by a cytotoxic T-cell reaction. The prolonged immune activation in primary biliary cirrhosis may play a role in the lymphomagenesis of hepatic MALT lymphoma. We describe another case of primary hepatic MALT lymphoma, which was found incidentally in a patient with end-stage primary biliary cirrhosis. This case further supports the role of immunogenic stimulation in the pathogenesis of this particular low-grade B-cell lymphoma.

Detection of hepatitis C virus RNA sequences in cholangiocarcinomas in Chinese and American patients.

OBJECTIVE: To investigate the role of hepatitis C virus (HCV) in the malignant transformation of bile duct cells. Tissues from 6 Chinese patients and 6 American patients with cholangiocarcinoma were studied. METHODS: RNA was extracted from the selected tumor areas of formalin-fixed, paraffin embedded sections, followed by reverse transcription double polymerase chain reaction (RT-PCR) and Southern blotting. RESULTS: Positive and negative strand HCV RNA sequences were detected in seven out of twelve patients with cholangiocarcinoma. A high positive rate was found in Chinese patients (83%) as compared to US patients (33%). CONCLUSION: Our finding suggests HCV may play a role in the malignant transformation of bile duct cells.

Hepatic epithelioid hemangioendothelioma: resection or transplantation, which and when?

Hepatic epithelioid hemangioendothelioma (HEHE) is a rare tumor with an unpredictable course and prognosis. The aim of this study is to describe our experience with liver resection, as well as transplantation, in the treatment of this tumor. We retrospectively analyzed the clinical features, pathological findings, and postoperative results in a series of 11 patients presenting between 1990 and 1998. Five patients (45%) presented with abdominal pain, 3 patients (27%) with jaundice and ascites, and the rest were asymptomatic. Computed tomography or magnetic resonance imaging showed localized lesions in 2 patients (18%) and multifocal disease in the others. Seven patients (64%) had extrahepatic lesions, detected either by preoperative imaging or discovered at exploration. Two resections of apparently localized lesions were followed by rapid and aggressive recurrence. Five patients were treated with transplantation, including 1 patient who had previously undergone resection. Of these 5 patients, 2 patients are currently free of detectable disease, 1 patient who had severe ascites and jaundice is now asymptomatic with stable extrahepatic lesions, and 2 patients (including 1 who had previously undergone a resection) died of tumor recurrence. One patient with advanced tumor died while waiting for transplantation. The remaining 4 patients are free of symptoms and have stable hepatic and extrahepatic disease. HEHE is nearly always multifocal, and our results with resection were dismal. Because of the unpredictable nature of the tumor, the indications for transplantation in patients without liver-related symptoms should be carefully evaluated. Nevertheless, extrahepatic disease should not be an absolute contraindication for liver transplantation in patients with severe liver dysfunction.

A 42-year-old woman with liver masses and long-term use of oral contraceptives.

A 42-year-old woman with a history of 25-year oral contraceptive use presented with abdominal pain and was found to have two exophytic liver masses. She had no known prior liver diseases, and her serum liver enzyme and AFP levels were normal. One of the masses was a hepatocellular adenoma and the other was a pigmented hepatocellular carcinoma. The exophytic appearance of both lesions was unusual. This case, once more, demonstrated the risk of hepatocellular adenomas to undergo malignant transformation. The reason for the brown pigment deposition in the hepatocellular carcinoma was not clear. The prognosis was expected to be excellent following complete surgical resection.

De novo hepatocellular carcinoma in a hepatic allograft with recurrent hepatitis C cirrhosis.

We report a case of de novo hepatocellular carcinoma (HCC) in a patient with recurrent hepatitis C (HCV) and cirrhosis 7 years after orthotopic liver transplantation (OLT). This is a previously unreported observation in the natural history of posttransplantantion HCV infection and reiterates the strong oncogenic potential of HCV.

Aggressive surgical treatment of intrahepatic cholangiocarcinoma: predictors of outcomes.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer and constitutes 10% of primary liver malignancies. Surgery is the optimal therapy; the majority of the patients will require extensive resections that are associated with significant morbidity. METHODS: We retrospectively studied the records of 26 patients who underwent exploratory laparotomy for intrahepatic cholangiocarcinoma between June 1991 and December 1997 at the Mount Sinai Hospital. Patients with perihilar (Klatskin) tumors were excluded. All patients were considered resectable based on CT or MRI findings. Patients with positive margins or nodal invasion received adjuvant chemotherapy and radiation. RESULTS: Sixteen patients underwent 18 resections; in 10 patients the tumors were unresectable at laparotomy and only biopsy was performed. The mean age (62 versus 53 years) was significantly higher, and the mean total bilirubin level (0.71 versus 6.17 mg/dL) was significantly lower in the resected group (p=0.031 and 0.017, respectively). No patient with a total bilirubin over 1.2 mg/dL was found to be resectable. Median actuarial survivals were 42.9+/-8.9 months for resectable and 6.7+/-3.6 months for unresectable patients (p=0.005). Positive margins were associated with significantly shorter disease-free survival. But resected patients with positive margins survived significantly longer than those who were unresectable. Tumor size, presence of satellite nodules, and degree of tumor necrosis on histologic examination were significant predictors of outcomes. Survival among patients receiving adjuvant therapy was not significantly altered. CONCLUSIONS: We conclude that an aggressive surgical approach is warranted in patients with ICC because resection offers the only hope for longterm survival. Our findings emphasize the importance of achieving tumor-free margins. Noncurative resection offers a survival advantage over no resection. Histologic examination of resected specimens can help select patients with poor prognoses.

Cytogenetic analyses of a murine carcinoma cell line and six metastatic derivatives with different degrees of radioresistability.

We reported that a murine carcinoma (DEN3) an its six pulmonary metastases (M2, M4C, M4D, M4E, M4F, and M6) exhibited different degrees of radioresistability (In Vitro Cell. Dev. Biol.26:222-228; 1990). While the M2, M4C, M4E, and M4F cultured cells survived up to 2.5 Gy, the cells of DEN3 and M6 tolerated up to 5.0 Gy, and the M4D cells could withstand up to 10.0 Gy of X-irradiation. In the present investigation, the cytogenetic features of these cell lines were examined: (a) to determine the degree of cytogenetic heterogeneity among these cell lines, and (b) to investigate whether any association between the cytogenetic anomaly and the degree of radioresistability could be established. Heterogeneous cytogenetic aberrations were detected in all of the above lines. Karyotype analysis of the M4D and M6 cell lines displayed both numerical and structural abnormalities. The gain and loss of chromosomal copies were observed. Structural aberrations, such as translocation and deletion appeared in both cell lines. However, correlation between the cytogenetic abnormality and the degree of radioresistability was not demonstrated except for a dramatic reduction in one or more copies of the X-chromosome that occurred in 86% and 93% of the M6 and M4D cells, respectively. The results suggest heterogeneous cytogenetic aberrations among these cell lines and a possible association between the loss of X-chromosome and radioresistability of these tumor cells.

Localization of angiotensin II type 1 receptor subtype mRNA in rat kidney.

The physiological effects of angiotensin II (ANG II) on the kidney are mediated primarily by the ANG II type 1 (AT1) receptor. Two highly similar AT1 receptor subtypes have been identified in the rat by molecular cloning techniques, namely AT1A and AT1B. The intrarenal localization of the AT1A and AT1B receptor subtypes has not been studied by hybridization methods with subtype-specific receptor probes. Using radiolabeled probes from the 3' noncoding region of the AT1A and AT1B cDNAs, we localized AT1 mRNA in rat kidney by in situ hybridization. Specificity of the 3' noncoding region probes was tested by Northern blot and solution hybridization methods. AT1A mRNA levels were highest in the liver, kidney, and adrenal. In contrast, AT1B mRNA levels were highest in the adrenal and pituitary and low in kidney. Autoradiographic localization of 125I-[Sar1,Ile8]ANG II binding indicated that the highest levels of AT1 receptors were found in glomeruli and vascular elements. In situ hybridization with a nonselective AT1 receptor riboprobe indicated that the highest levels of AT1 mRNA were in the outer medullary vasa recta and cortical glomeruli with additional diffuse labeling of the cortex and outer medulla, consistent with labeling of tubular elements. In contrast, in situ hybridization with the AT1 subtype selective probes revealed that AT1A receptor mRNA was primarily localized to the vasa recta and diffusely to the outer stripe of the outer medulla and the renal cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of intracerebroventricular captopril on angiotensin-converting enzyme and angiotensinogen mRNA levels in rat brain.

The effects of intracerebroventricular (i.c.v.) infusion of the angiotensin-converting enzyme (ACE) inhibitor captopril on angiotensin-induced drinking, brain ACE activity, and ACE and angiotensinogen (A-ogen) mRNA levels were examined. I.c.v. infusion of captopril at a rate of 1 microgram/microliter per h for 7 days resulted in a 60% reduction in brain ACE activity and an 80% reduction in the drinking response to i.c.v. angiotensin I. Quantitative solution hybridization experiments indicated that brain ACE mRNA levels were decreased by 40%, whereas brain A-ogen mRNA levels were unchanged. These results suggest that ACE and A-ogen mRNA levels are regulated differently in the brain than in the peripheral renin-angiotensin system.

Stimulation of angiotensinogen mRNA levels in rat pituitary by estradiol.

Angiotensin II (ANG II) is a putative paracrine hormone in the anterior pituitary. Angiotensinogen mRNA, however, is not detectable by Northern blot hybridization, suggesting that ANG II may not be synthesized within the pituitary. An alternative explanation may be that angiotensinogen gene activity is low under normal conditions, with angiotensinogen mRNA being below the level of detection. Utilizing a sensitive solution hybridization method, we sought to determine whether angiotensinogen mRNA could be detected in pituitaries from normal male rats or ovariectomized (OVX) rats treated with estradiol (E2) for 4 days. Very low levels of angiotensinogen mRNA were detected from male or OVX rat pituitaries, but E2 treatment resulted in a marked dose-dependent increase in pituitary angiotensinogen mRNA levels. Levels of angiotensinogen within the pituitary were not significantly different after the E2 treatment. Angiotensinogen mRNA levels in liver and brain were much higher than in the pituitary but were not altered significantly by the chronic E2 treatment. These results are consistent with the local synthesis of angiotensinogen in the pituitary and further suggest that pituitary angiotensinogen gene transcription is regulated by estrogen.

Characterization of an angiotensin type-1 receptor partial cDNA from rat kidney: evidence for a novel AT1B receptor subtype.

We sought to determine if multiple forms of mRNA for the angiotensin type-1 (AT1) receptor could be detected in rat kidney using the polymerase chain reaction (PCR) procedure. Amplification of rat kidney cDNA with oligonucleotide primers derived from the second and sixth transmembrane domains of the rat AT1 receptor yielded a single cDNA fragment 528bp in size. Sequence analysis indicated, however, that the cDNA fragment was a mixture of two highly similar gene products: the first cDNA was identical to the previously cloned AT1 receptor (termed here AT1A) whereas the second cDNA (termed here AT1B) was 92% identical at the nucleotide level and 96% identical at the amino acid level. Nucleotide substitutions were dispersed throughout the cDNA and 80% (33 of 41) were conservative. Significant levels of AT1A and AT1B mRNA were detected by PCR amplification of kidney poly(A)+ RNA and restriction enzyme analysis. These results indicate that at least two distinct AT1 receptor genes are expressed in rat kidney.

Characterization of a dopamine receptor (DA2K) in the kidney inner medulla.

Dopamine (DA) produces a natriuretic/diuretic response in the kidney by mechanisms that are still not well understood. There is some indication that DA2 receptors may be involved in mediating the effects of DA, but little is known regarding the nature of this receptor in the kidney. Autoradiographic localization of [3H]spiperone, a DA2 antagonist, indicated that high-density binding was restricted to inner medullary collecting ducts (IMCDs). [3H]Spiperone binding was saturable, high affinity (Kd, 17.2 +/- 1.65 nM), and high density (Bmax, 935 +/- 83 fmol per mg of protein). The photosensitive spiperone analogue N-(p-azido-m-[125I]iodophenethyl)spiperone labeled similar sized proteins of Mr = 120,000 in membranes prepared from the kidney inner medulla, striatum, and pituitary. However, the rank-order competition profile for the [3H]spiperone binding in the kidney inner medulla differed from the DA2 receptor in striatum and pituitary and, furthermore, RNA (Northern) blot analyses of kidney inner medullary RNA with brain DA2 receptor oligonucleotide probes were negative. Functionally, DA stimulated prostaglandin E2 production by IMCD cells, an effect that could be blocked by the DA2 antagonist domperidone. These results indicate that the kidney inner medulla expresses a functional DA receptor that may represent a newly identified DA receptor subtype (here designated DA2K). Moreover, these results suggest that the kidney inner medulla may be a significant site at which DA, either directly or indirectly, influences water and electrolyte excretion.