Plasmacytoid acinar adenocarcinoma of the prostate: a newly described variant of prostate cancer.

A plasmacytoid variant of prostatic adenocarcinoma has not been reported to the best of our knowledge. A 54-year-old male presented with recurrent attacks of acute urinary retention. Laboratory findings showed high creatinine and a serum prostate specific antigen of 50.7 µg/L. Magnetic Resonance Imaging showed a locally advanced tumor involving the bladder and extending to the base of prostate with bilateral ureterovesical junction involvement and invasion of the left seminal vesicle and left anterior mesorectal fascia as well as perirectal fat invasion. Diffuse metastases to the abdominopelvic lymph nodes were identified. Bone scintigraphy showed multiple bone metastases. Transrectal ultrasound guided biopsy of the prostate was attempted but the patient could not tolerate the procedure and the procedure was canceled. The patient then underwent transurethral resection of bladder tumor. Microscopic examination showed sheets of malignant cells with prominent plasmacytoid appearance undermining benign urothelium. The tumor cells were positive for PSA, PSAP, NKX 3.1 and Cytokeratin 8/18. The tumor cells were negative for P63, Cytokeratin 34ßE12, Cytokeratin 20, Desmin, CD38, Kappa and Lambda light chains, Chromogranin, Synaptophysin, GATA 3, E-cadherin and CD45. INI1 was retained. Next generation sequencing showed an intermediate tumor mutational burden. Notably, no genomic alterations in the CDH1 gene (encoding for E-cadherin) were present. The patient showed some initial response to antiandrogen therapy with a drop in serum PSA levels following androgen deprivation therapy. However, the patient died 6 months after diagnosis. It is critical to recognize this newly described variant and to distinguish it from plasmacytoid urothelial carcinoma. Recognition of the newly described plasmacytoid variant of adenocarcinoma of the prostate will lead to identification and reporting of more cases and a better understanding of its clinicopathologic features.

Different immunohistochemical localization for TMEM16A and CFTR in acinar and ductal cells of rat major salivary glands and exocrine pancreas.

We investigated the mRNA expression and immunohistochemical localization of Cl- channels, transmembrane member 16A (TMEM16A or anoctamin 1), and cystic fibrosis transmembrane conductance regulator (CFTR) in rat major salivary glands and exocrine pancreas. RT-PCR detected mRNA expression of TMEM16A and CFTR in the extracts of the parotid gland (PG), submandibular gland (SMG), sublingual gland (SLG), and pancreas. Immunoreactivity for TMEM16A was localized in the apical membrane of serous acinar and intercalated ductal cells in the PG and SMG as well as mucous acinar cells in the SLG; however, it was not detected in striated ductal cells of these tissues. Although striated ductal cells in the PG, SMG and SLG, and granular ductal cells in the SMG, were immunoreactive for CFTR in the luminal side, serous, mucous acinar, and intercalated ductal cells were not immunoreactive for CFTR in any of the major salivary glands. In the exocrine pancreas, immunoreactivity for TMEM16A was localized in the apical membrane of acinar cells, while immunoreactivity for CFTR was localized in the luminal side of intercalated ductal cells. These results suggest that different localization of TMEM16A and CFTR immunoreactivities reflects the respective functions of acinar and ductal cells in major salivary glands and exocrine pancreas.

HMGA1 expression levels are elevated in pancreatic intraepithelial neoplasia cells in the Ptf1a-Cre; LSL-KrasG12D transgenic mouse model of pancreatic cancer.

BACKGROUND: Pancreatic cancer is currently the third leading cause of cancer deaths in the United States and it is predicted to become the second by the year 2030. High-mobility group A1 protein (HMGA1) is an oncogenic transcription factor, localised and active in cell nuclei, that is linked to tumour progression in many human cancers, including pancreatic cancer. Overexpression of HMGA1 renders cancer cells resistant to chemotherapy. Although the Ptf1a-Cre; LSL-KrasG12D transgenic mouse is perhaps the most widely utilised animal model for human pancreatic cancer, expression levels of HMGA1 in pancreata from this mouse model have not been characterised. METHODS: Quantitative immunohistochemical analysis was used to determine nuclear HMGA1 levels in pancreatic tissue sections from Ptf1a-Cre; LSL-KrasG12D mice aged 5, 11, and 15 months. The H Score method was used for quantitative analysis. RESULTS: The HMGA1 levels were significantly elevated in pancreatic intraepithelial neoplasia (PanIN) epithelia compared with untransformed acinar tissues or fibroinflammatory stroma. CONCLUSIONS: The PanINs have long been regarded as precancerous precursors to pancreatic adenocarcinoma. Significantly elevated HMGA1 levels observed in the nuclei of PanINs in Ptf1a-Cre; LSL-KrasG12D mice validate this animal model for investigating the role that HMGA1 plays in cancer progression and testing therapeutic approaches targeting HMGA1 in human cancers.

Allograft pancreas: pale acinar nodules.

Microscopic pale-staining acinar nodules were characterized in native pancreas in the 1980s under a variety of names but have been infrequently reported since. We retrospectively studied the frequency and characteristics of pale acinar nodules in allograft pancreas biopsies, as compared to a sampling of native pancreas specimens at our center. Pale acinar nodules were present in 13% (9/69) of allograft biopsies from 22% (7/32) of transplant patients, and 23% (5/22) of native pancreas surgical specimens, although more nodules per pancreas area were present in allograft needle biopsies. Acinar nodules had size of 100 to 700 µm, were periodic acid-Schiff pale, were synaptophysin negative, stained more weakly with keratin CAM 5.2 compared to surrounding parenchyma, and had a low proliferative rate. Ultrastructural evaluation revealed paucity of zymogen granules with dilated cistern-like structures. In our experience, pale acinar nodules have similar features in allograft and native pancreas specimens, yet remain of uncertain etiology and significance.

MUC1/SEC and MUC1/Y overexpression is associated with inflammation in Sjögren's syndrome.

OBJECTIVES: To evaluate the expression and localization of MUC1/SEC and MUC1/Y isoforms in labial salivary glands (LSG) from Sjögren's syndrome patients (SS patients), as well as their in vitro expression induced by cytokines. SUBJECTS AND METHODS: Labial salivary gland from 27 primary SS patients and 22 non-SS sicca subjects were studied. Relative MUC1/SEC and MUC1/Y mRNA levels were determined by qPCR and protein levels by Western blotting. Induction of mucin mRNAs was assayed in vitro. Immunohistochemistry was used for localization. RESULTS: Relative MUC1/SEC and MUC1/Y mRNA and protein levels were significantly higher in LSG from SS patients. These mRNAs were induced by cytokines. MUC1/SEC and MUC1/Y were detected in acini apical region of control LSGs, and significant cytoplasmic accumulation was observed in acini of SS patients. MUC1/Y localized in acinar nuclei and cytoplasm of inflammatory cells of LSG from SS patients. A strong positive correlation was observed between cellular MUC1/SEC levels and glandular function determined by scintigraphy. CONCLUSIONS: We show for the first time that MUC1/SEC and MUC1/Y are expressed in LSG of both SS patients and non-SS sicca subjects. The observed overexpression and aberrant localization of MUC1/SEC and MUC1/Y and their induction by pro-inflammatory cytokines may favor the perpetuation of the inflammatory environment that disrupts the salivary glandular homeostasis in SS patients.

[Laser scanning confocal microscopic imaging for Ca2 + oscillations of pancreatic acinar cells in mice].

OBJECTIVE: To establish a simple but effective method of laser scanning confocal microscopic imaging for Ca2+ oscillations of pancreatic acinar cells in adult mice. METHODS: Pancreatic acinar cells from adult Kunming mice were isolated acutely with collagenase, and then loaded with fluo-4-AM, a Ca2+ indicator. A laser scanning confocal microscope armed with 488 nm laser was employed to record the dynamic fluorescent signals in-time and synchronously while acetylcholine (ACh) was added in the pancreatic acinar cells. RESULTS: (1) The classic pancreatic acinar cell Ca2+ oscillations were induced by a certain concentration of ACh (100 nmol/L) successfully and steadily, which could be blocked by atropine completely. (2) Plasmic Ca2+ oscillations from different parts of one acinar cell were usually with different amplitudes and almost the same frequencies. But both of amplitudes and frequencies were different among different cells. (3) The acinar cell Ca2+ oscillations were induced by ACh in a concentration-dependent manner. CONCLUSION: The laser scanning confocal microscopic imaging for adult mouse pancreatic acinar cell Ca2+ oscillations was established successfully. The features of being easy to use, direct to see lively, high efficiency and good flexibility make it a popular tool for researchers to choose.

Autophagy in pancreatic acinar cells in caerulein-treated mice: immunolocalization of related proteins and their potential as markers of pancreatitis.

Drug-induced pancreatitis (DIP) is an underdiagnosed condition that lacks sensitive and specific biomarkers. To better understand the mechanisms of DIP and to identify potential tissue biomarkers, we studied experimental pancreatitis induced in male C57BL/6 mice by intraperitoneal injection of caerulein (10 or 50 µg/kg) at 1-hr intervals for a total of 7 injections. Pancreata from caerulein-treated mice exhibited consistent acinar cell autophagy and apoptosis with infrequent necrosis. Kinetic assays for serum amylase and lipase also showed a dose-dependent increase. Terminal deoxynucleotidyl transferase-mediated biotin-dNTP nick labeling (TUNEL) detected dose-dependent acinar cell apoptosis. By light microscopy, autophagy was characterized by the formation of autophagosomes and autolysosomes (ALs) within the cytoplasm of acinar cells. Immunohistochemical studies with specific antibodies for proteins related to autophagy and pancreatic stress were conducted to evaluate these proteins as potential biomarkers of pancreatitis. Western blots were used to confirm immunohistochemical results using pancreatic lysates from control and treated animals. Autophagy was identified as a contributing process in caerulein-induced pancreatitis and proteins previously associated with autophagy were upregulated following caerulein treatment. Autophagosomes and ALs were found to be a common pathway, in which cathepsins, lysosome-associated membrane protein 2, vacuole membrane protein 1, microtubule-associated protein 1 light chain 3 (LC3), autophagy-related protein 9, Beclin1, and pancreatitis-associated proteins were simultaneously involved in response to caerulein stimulus. Regenerating islet-derived 3 gamma (Reg3γ), a pancreatic acute response protein, was dose-dependently induced in caerulein-treated mice and colocalized with the autophagosomal marker, LC3. This finding supports Reg3γ as a candidate biomarker for pancreatic injury.

Whole-exome sequencing of pancreatic neoplasms with acinar differentiation.

Pancreatic carcinomas with acinar differentiation, including acinar cell carcinoma, pancreatoblastoma and carcinomas with mixed differentiation, are distinct pancreatic neoplasms with poor prognosis. Although recent whole-exome sequencing analyses have defined the somatic mutations that characterize the other major neoplasms of the pancreas, the molecular alterations underlying pancreatic carcinomas with acinar differentiation remain largely unknown. In the current study, we sequenced the exomes of 23 surgically resected pancreatic carcinomas with acinar differentiation. These analyses revealed a relatively large number of genetic alterations at both the individual base pair and chromosomal levels. There was an average of 119 somatic mutations/carcinoma. When three outliers were excluded, there was an average of 64 somatic mutations/tumour (range 12-189). The mean fractional allelic loss (FAL) was 0.27 (range 0-0.89) and heterogeneity at the chromosome level was confirmed in selected cases using fluorescence in situ hybridization (FISH). No gene was mutated in >30% of the cancers. Genes altered in other neoplasms of the pancreas were occasionally targeted in carcinomas with acinar differentiation; SMAD4 was mutated in six tumours (26%), TP53 in three (13%), GNAS in two (9%), RNF43 in one (4%) and MEN1 in one (4%). Somatic mutations were identified in genes in which constitutional alterations are associated with familial pancreatic ductal adenocarcinoma, such as ATM, BRCA2 and PALB2 (one tumour each), as well as in genes altered in extra-pancreatic neoplasms, such as JAK1 in four tumours (17%), BRAF in three (13%), RB1 in three (13%), APC in two (9%), PTEN in two (9%), ARID1A in two (9%), MLL3 in two (9%) and BAP1 in one (4%). Perhaps most importantly, we found that more than one-third of these carcinomas have potentially targetable genetic alterations, including mutations in BRCA2, PALB2, ATM, BAP1, BRAF and JAK1.

[Influence of Ca2+ on kinetic parameters of pancreatic acinar mitochondria in situ respiration].

The dependence of respiration rate of rat permeabilized acinar pancreacytes on oxidative substrates concentration was studied at various [Ca2+] - 10-8-10-6 M. Pancreacytes were permeabilized with 50 microg of digitonin per 1 million cells. Respiration rate was measured polarographically using the Clark electrode at oxidation of succinate or pyruvate either glutamate in the presence of malate. Parameters of Michaelis-Menten equation were calculated by the method of Cornish-Bowden or using Idi-Hofsti coordinates and parameters of Hill equation - using coordinates {v; v/[S]h}. In the studied range of [Ca2+] the kinetic dependence of respiration at pyruvate oxidation is described by the Michaelis-Menten equation, and at oxidation of succinate or glutamate - by Hill equation with h = 1.11-1.43 and 0.50-0.85, respectively. The apparent constant of respiration half-activation (K0.5) did not significantly change in the studied range of [Ca2+] while at 10-7 M Ca2+ it was 0.90 +/- 0.06 mM for succinate, 0.096 +/- 0.007 mM for pyruvate and 0.34 +/- 0.03 mM for glutamate. Maximum respiration rate Vax at pyruvate oxidation increased from 0.077 +/- 0.002 to 0.119 +/- 0.002 and 0.140 +/- 0.002 nmol O2/(s.million cells) due to the increase of [Ca2+] from 10-7 to 5x10-7 or 10-6 M, respectively. At oxidation of succinate or glutamate Ca2+ did not significantly affect Vmax Thus, the increase of [Ca2+] stimulates respiration of mitochondria in situ of acinar pancreacytes at oxidation of exogenous pyruvate (obviously due to pyruvate dehydrogenase activation), but not at succinate or glutamate oxidation.

Acinar cell cystadenoma of the pancreas: a benign neoplasm or non-neoplastic ballooning of acinar and ductal epithelium?

Acinar cell cystadenoma (ACA) of the pancreas was initially described as a non-neoplastic cyst of the pancreas and, at that time, referred to as "acinar cystic transformation." In subsequent studies, these lesions were given the designation of "-oma," despite the relative lack of evidence supporting a neoplastic process. To characterize these lesions further, we examined the clinical, pathologic, and immunohistochemical features of 8 ACAs. The majority of patients were female (7 of 8, 88%) and ranged in age from 18 to 57 years (mean, 43 y). Grossly, the cysts involved the head (n=5), body (n=1), or the entire pancreas (n=2). ACAs were either multilocular (n=4) or unilocular (n=4) and ranged in size from 1.8 to 15 cm (mean, 6.8 cm). Histologically, multilocular ACAs were lined by patches of acinar and ductal epithelium. Immunolabeling, including double-labeling for cytokeratin 19 and chymotrypsin, highlighted the patchy pattern of the ductal and acinar cells lining the cysts. In some areas, the cysts with patches of acinar and ductal differentiation formed larger locules with incomplete septa as they appeared to fuse with other cysts. In contrast, the unilocular cases were lined by 1 to 2 cell layers of acinar cells with little intervening ductal epithelium. Nuclear atypia, mitotic figures, necrosis, infiltrative growth, and associated invasive carcinoma were absent in all cases. In addition, we assessed the clonal versus polyclonal nature of ACAs, occurring in women, using X-chromosome inactivation analysis of the human androgen receptor (AR) gene. Five of 7 cases were informative and demonstrated a random X-chromosome inactivation pattern. Clinical follow-up information was available for all patients, and follow-up ranged from 10 months to 7.8 years (mean, 3.6 y), with no evidence of recurrence or malignant transformation. We hypothesize that early lesions are marked by acinar dilatation that expands into and incorporates smaller ductules and later larger ducts. As the cysts increase in size, they fuse forming larger cysts. Later lesions demonstrate a unilocular cyst lined by predominantly acinar epithelium with scattered ductal cells. The term cystadenoma, with its neoplastic connotation, does not seem to accurately reflect the histologic, immunohistochemical, or molecular features of these lesions. We suggest readopting the term "acinar cystic transformation" until the non-neoplastic versus neoplastic origin of these lesions can be resolved.

Subcellular distribution of melatonin receptors in human parotid glands.

The hormone melatonin influences oral health through a variety of actions, such as anti-inflammatory, anti-oxidant, immunomodulatory and antitumour. Many of these melatonin functions are mediated by a family of membrane receptors expressed in the oral epithelium and salivary glands. Using immunoblotting and immunohistochemistry, recent studies have shown that the melatonin membrane receptors, MT1 and MT2, are present in rat and human salivary glands. To date, no investigation has dealt with the ultrastructural distribution of the melatonin receptors. This was the aim of the present study, using the immunogold method applied to the human parotid gland. Reactivity to MT1 and, with less intensity, to MT2 appeared in the secretory granules of acinar cells and in the cytoplasmic vesicles of both acinar and ductal cells. Plasma membranes were also stained, albeit slightly. The peculiar intracytoplasmic distribution of these receptors may indicate that there is an uptake/transport system for melatonin from the circulation into the saliva.

Stacked endoplasmic reticulum sheets are connected by helicoidal membrane motifs.

The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used improved staining and automated ultrathin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell.

A novel explant outgrowth culture model for mouse pancreatic acinar cells with long-term maintenance of secretory phenotype.

The development of in vitro models able to support the long-term viability and function of acinar cells is critical for exploring pancreatic pathophysiology. Despite considerable efforts, no long-term culture models for non-transformed pancreatic acini exist. Our aim was to develop and validate culture conditions for this purpose. An explant outgrowth culture design was established in which mouse pancreatic explants were cultured at the gas-liquid interphase. An enriched culture medium, pH 7.8, was employed to promote the selective outgrowth of acinar cells and to support their differentiated phenotype. After 7 days, the outgrown primary acinar cells were subcultured and maintained up to an additional 7 days as secondary monolayers on tissue culture plastic. Measurements of basal and caerulein-induced amylase secretion, phase-contrast microscopy and immunohistochemical analyses were used to characterize the cultures. Explants retained their pancreatic cytoarchitecture for 2 days in vitro. A triphasic dose response to caerulein was detected in 7-day primary cultures. The maximal rate of secretion was 1.2-fold versus basal (p=0.009) and 1.7-fold versus 1 pM caerulein (p=0.014). In secondary cultures the response was biphasic with maximal rates of secretion being 1.9-fold in 3- to 4-day cultures at 0.01 nM (p=0.049) and 2-fold in 6- to 7-day cultures at 0.1 nM (p=0.003). The present culture model provides a means to obtain functionally competent normal mouse acinar cells for long-term in vitro experimentation.

Postnatal expression of sialin in the mouse submandibular gland.

OBJECTIVE: Sialin has been identified as a sialic acid and aspartate/glutamate transporter. Both cytoplasmic localization and the plasma membrane labelling pattern suggested that sialin may possess multiple transport functions in different cell types. In mouse embryos, sialin expression was primarily detected in the central nervous system. However, sialin shows widespread and high-level expression in adult tissues. Despite its ubiquitous expression and important functions, the postnatal expression profile and subcellular localization of sialin in the salivary gland remains elusive. The aim of the present study was to investigate the expression and subcellular distribution of sialin during postnatal development in the mouse submandibular gland (SMG). DESIGN: Six SMGs from both female and male C57BL/6 mice were collected at P10, P30 and P90, and the material from each littermate of either sex was pooled to extract total RNA and tissue protein. The remaining tissues were immediately fixed in 10% neutral buffered formalin for histological analysis. The mRNA and protein expression levels of sialin were examined by quantitative real-time RT-PCR and Western blot analysis. The subcellular distribution of sialin was analysed by immunohistochemistry and immunofluorescence. RESULTS: The postnatal expression level of sialin in the mouse SMG was comparable with that in brain at each time point tested. The temporal expression of sialin in the SMG gradually increased during postnatal maturation. Immunohistochemical and immunofluorescence analysis demonstrated that sialin was predominantly expressed on the basal cytoplasmic membrane of acini and ducts, as well as in some myoepithelial cells in the SMG. CONCLUSIONS: The high-level expression and subcellular distribution pattern of sialin in the SMG suggest that sialin may play an important role in the transport and secretion of saliva.