An unusual parotid gland in the tent-building bat,Uroderma bilobatum: Possible correlation of interspecific ultrastructural differences with differences in salivary pH and buffering capacity

The tent-building bat, Uroderma bilobatum, is a small, frugivorous phyllostomid bat with a broad neotropical distribution. Generally found in humid forest, this bat lives in small groups that create daytime “roosts” from large leaves of a variety of tropical plants. Fruit eating engenders a variety of ecological and physiological challenges for bats, some of which could require adaptive features in their salivary glands. The parotid salivary glands of Uroderma bilobatum were prepared for transmission electron microscopy by using methods that have become standard for field work. The parotid gland is extremely unusual in structure. Although the secretory endpieces still produce serous granules with a complex substructure, they are modified into quasi striated ducts. Their basal folds, which are extensive, occasionally harbor some vertically oriented mitochondria, imparting a resemblance to striated ducts. Other evidence for the endpiece origin of these parenchymal components is a well-developed system of intercellular canaliculi, structures that never occur in bona fide striated ducts. The long but sparse intercalated ducts consist of two types of cells, each of which elaborates a modest number of secretory granules of differing substructure. Striated ducts are of conventional morphology, except that a few dark cells shaped like wine glasses are present in their walls. The striated duct cells produce no secretory granules, but their apical cytoplasm may contain some small, empty vesicles. Capillaries lie in longitudinal grooves in the base of the duct cells, an arrangement that might enhance electrolyte exchange. Excretory ducts consist of simple cuboidal epithelium composed of cytologically unspecialized cells that sometimes includes a dark cell. It was concluded that salivary glands could have a major role in adapting species to acquire nutrients from marginal sources, such as tropical fruits, which have a low protein and sodium content. The unusual parotid acinar cells in Uroderma bilobatum are discussed in the context of salivary pH and buffering capacity. Comparisons are made with four other bat species, including an insectivorous species with a salivary pH > 8.0 and a very high buffering capacity, an intermediate species, and a fruit bat with acidic-stimulated saliva and very low buffering capability. Such interspecific comparisons provide a foundation for hypothesizing that ultrastructural features of the acinar cell basolateral membranes and intercellular canaliculi correlate with differences involving Na⁺/H⁺ exchangers and release of HCO₃⁻ and, thus, are associated with the species differences that are important to diet and nutrient acquisition. Anat. Rec. 252:290–300, 1998. © 1998 Wiley-Liss, Inc.     

Patterns of reactivity with the monoclonal antibodies HMFG1 and HMFG2 in normal endometrium, endometrial hyperplasia and adenocarcinoma

Using an indirect immunoperoxidase technique the expression of the epitopes in human milk fat globule (HMFG) membranes detected by the monoclonal antibodies HMFG1 and HMFG2 was studied in the normal endometrium and in cases of cystic glandular hyperplasia, glandular hyperplasia with architectural atypia (complex hyperplasia), glandular hyperplasia with cytological atypia (atypical hyperplasia) and invasive adenocarcinoma. Luminal reactivity with HMFG1 was seen in cases of normal endometrium, cystic glandular hyperplasia and glandular hyperplasia with architectural atypia. In contrast most cases of glandular hyperplasia with cytological atypia and invasive adenocarcinoma also showed areas of cytoplasmic reactivity. Reactivity with HMFG2 was scanty.     

Relative paucity of gross genetic alterations in myoepitheliomas and myoepithelial carcinomas of salivary glands

The diagnosis of salivary gland myoepithelioma, an entity with heterogeneous cytomorphology and inconsistent immunophenotype, rests on conventional histology. However, the clinical course cannot be predicted reliably from cytomorphological and immunophenotypic analysis. The present study determined the immunophenotype of a representative series of 12 myoepitheliomas and 21 malignant myoepitheliomas. Among the seven markers tested, antibodies against cytokeratins 5/6, S-100 protein, and vimentin produced the most consistent reactivity profile. Comparative genomic hybridization (CGH) profiles of 12 myoepitheliomas showed chromosomal losses in three of 12 cases. In myoepithelial carcinomas, however, ten of 19 tissues investigated by CGH lacked detectable cytogenetic aberrations. In five cases, aberrations involved chromosome 8, in line with observations in salivary gland carcinomas of other differentiation. One case that was represented in three separately localized manifestations of the disease proved informative as to the relevance of gross aberration for tumour development, as these tumours differed in their CGH profiles. Staining for cytokeratins 5/6 is a useful addition to the established immunohistological marker panel in the work-up of myoepitheliomas, because of its reliable expression in most cases and because it may underline the epithelial nature of the lesion. CGH proved to be of limited value as a diagnostic adjunct; the presence of numerous gross cytogenetic aberrations should raise the suspicion of malignancy. The low frequency of aberrations detectable by CGH in overtly malignant myoepithelial neoplasms suggests that gross cytogenetic alterations were acquired in the course of tumour progression and points to the relevance of genetic changes not resolved by CGH.     

Electron microscopic immunogold localization of salivary mucin MUC5B in human buccal and palatal glands

In recent years, minor salivary glands, due to their involvement in the health and homeostasis of the oral cavity, have been the focus of several research investigations. Despite the fact that a considerable amount of data has been collected, many aspects of their functional features, including the secretory components they produce, remain to be ascertained. In this study we have analyzed the ultrastructural distribution of the MUC5B mucin in human palatal and buccal glands by means of post-embedding immunoelectron microscopy.Thin sections of normal human buccal and palatal glands obtained at surgery, were treated with polyclonal antibodies to human salivary MUC5B. Intense MUC5B reactivity was observed in the secretory granules of mucous cells of all glands examined. The present results provide new data regarding the secretory pattern of MUC5B in human buccal and palatal glands, indicating their significant contribution to the maintenance of the mucous biofilm that protects buccal and palatal mucosal areas.     

Phenotypes in canalicular adenoma of human minor salivary glands reflect the interplay of altered secretory product, absent neuro-effector relationships and the diversity of the microenvironment

AIMS: Uncertainty about the factors influencing phenotypes in salivary canalicular adenoma prompted the present investigation. METHODS AND RESULTS: Specimens of canalicular adenoma from 15 patients were examined with the use of histology, histochemistry for protein, mucosubstances and pigments, nerve staining and immunocytochemistry for cytoskeleton components. The tumours consisted largely of simple cells lining tubules that were occasionally cystic or branching and budding, and were set in loose, vascular and often haemorrhagic stroma. Other phenotypes recognized were mucous cells, apocrine-like cells, pigmented cells, microliths and stromal macrophages, detected in 26.6%, 20%, 33.3%, 20% and 53. 3% of the patients, respectively. Simple cells showed moderate levels of -SH groups and strong immunoreactivity for 'simple' epithelial phenotype cytokeratin. The simple cells lining cystic tubules showed additional immunoreactivity for 'stratified' epithelial phenotype cytokeratin, possibly an adaptation to mechanical pressure. Lumina showed variable levels of neutral and carboxylated glycoproteins, and chondroitin sulphate. Stroma showed high levels of chondroitin sulphate and hyaluronic acid. Mucous cells showed high levels of -SS- groups and nonsulphated glycoproteins. Apocrine-like cells contained lipofuscin. Pigmented cells contained haemosiderin, possibly a consequence of localized iron overload. Microliths contained mucosubstances. Macrophages often contained lipofuscin. No nerves were found in relation to the tumours. CONCLUSIONS: The results suggest that, contrary to popular belief, phenotypes in canalicular adenoma do not reflect histogenetic concepts but rather may derive from the interplay between an altered secretory product, consisting of glycosaminoglycan and an immature form of glycoprotein, the lack of neuro-effector relationships and the different microenvironments throughout the tumour.     

Cytokeratin expression in normal salivary glands and in cystadenolymphomas demonstrated by monoclonal antibodies against selective cytokeratin polypeptides

Summary The distribution of selective cytokeratin polypeptides, vimentin, and glial fibrillary protein (GFP) in 5 human cystadenolymphomas of the parotid gland was compared with normal human parotid (n=5) and submandibular (n=4) glands using a panel of monoclonal antibodies against diverse and selective cytokeratin polypeptides, vimentin and glial fibrillary protein (GFP). A biotin-streptavidin method was used on cryostat sections. The immunocytochemical finding of identical cytokeratin polypeptides Nos. 7, 8, 18 and 19 and basal cells selectively labeled by the monoclonal antibody KS 8.58, in both the epithelial part of the cystadenolymphomas and in the duct epithelium of the parotid gland, confirms the hypothesis that the epithelial compartment of cystadenolymphomas is derived from the duct system. The triple expression of cytokeratin, vimentin and GFP in myoepithelial cells of the parotid gland is discussed.     

Increased expression of cyclooxygenase-2 in human salivary gland tumors

We examined the immunohistochemical localization of cyclooxygenase (COX)-2 in human salivary gland tumors. Thirty salivary gland adenomas (SGA), 40 salivary gland carcinomas (SGC) and 15 normal salivary glands (NSG) were studied. NSG showed restricted COX-2 staining only in the epithelial cells of salivary ducts. In contrast, COX-2 protein was detected in 27 cases of SGA (90%), except for three myoepitheliomas, and in all cases of SGC (100%) at various intensities and in various fashions. Thirteen SGA (43%) and 36 SGC (90%) cases showed strong COX-2 staining predominantly in tumor cells containing ductal components, as did serous and mucous acinic components of acinic cell carcinomas, mucoepidermoid carcinomas and mucinous carcinomas. These findings may suggest that COX-2 in salivary gland tumors is expressed in tumor cells derived from pluripotential ductal epithelium that can histologically develop into either serous or mucinous acinar cells.     

Deregulated expression of p16INK4a and p53 pathway members in benign and malignant myoepithelial tumours of the salivary glands

Aims: Myoepithelial salivary gland tumours are uncommon and follow an unpredictable biological course. The aim was to examine their molecular background to acquire a better understanding of their clinical behaviour. Methods and results: Expression of protein (E2F1, p16^INK4a, p53, cyclin D1, Ki67 and Polycomb group proteins BMI‐1, MEL‐18 and EZH2) was investigated in 49 benign and 30 primary malignant myoepithelial tumours and five histologically benign recurrences by immunohistochemistry and the findings correlated with histopathological characteristics. Benign tumours showed a higher percentage of cells with expression of p16^INK4a pathway members [p16^INK4a and E2F1 (both P < 0.001), and cyclin D1, P = 0.002] compared with normal salivary gland. Furthermore, malignant tumours expressed p53 (P = 0.003) and EZH2 (P = 0.09) in a higher percentage. Recurrences displayed more p53 + tumour cells (P = 0.02) than benign primaries. Amongst the benign tumours, the clear cell type had the highest proliferation fraction (P = 0.05) and a higher percentage of EZH2 was detected in the plasmacytoid cell type (P = 0.002). Conclusions: This study is the first to demonstrate that deregulation of the p16^INK4a senescence pathway is involved in the development of myoepithelial tumours. We propose that additional inactivation of p53 in malignant primaries and benign recurrences contributes to myoepithelial neoplastic transformation and aggressive tumour growth.     

Localization of phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K) α, β, γ in the three major salivary glands in situ of mice and their response to β‐adrenoceptor stimulation

Phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K), which is composed of three isozymes (α, β and γ), catalyzes the production of phosphatidylinositol bisphosphate (PIP2). This phospholipid functions in membrane trafficking, as an anchor for actin cytoskeletons and as a regulator of intramembranous channels/transporters. It is also a precursor of such second messengers as diacylglycerol, inositol triphosphate and phosphatidylinositol (3,4,5)‐triphosphate. In the present study, the expression and localization of endogenous PIP5Ks were examined in the three major salivary glands of young adult mice in situ. In western blotting of normal control glands, immunoreactive bands for individual PIP5Ks were detectable, with the highest density in the parotid gland and the weakest density in the submandibular gland. In immuno‐light microscopy under non‐stimulated condition, weak immunoreactivity for PIP5Kα was confined to the apical plasmalemma in parotid, but not sublingual or submandibular, acinar cells. Immunoreactivity for PIP5Kβ was weak to moderate and confined to ductal cells but not acinar cells, whereas that for PIP5Kγ was selectively and intensely detected in myoepithelial cells but not acinar cells, and it was weak in ductal cells in the three glands. In western blot of the parotid gland stimulated by isoproterenol, a β‐adrenoceptor agonist, no changes were seen in the intensity of immunoreactive bands for any of the PIP5Ks. In contrast, in immuno‐light microscopy, the apical immunoreactivity for PIP5Kα in parotid acinar cells was transiently and distinctly increased after the stimulation. The increased immunoreactivity was ultrastructurally localized on most apical microvilli and along contiguous plasma membrane, where membranous invaginations of various shapes and small vesicles were frequently found. It was thus suggested that PIP5Kα is involved in post‐exocytotic membrane dynamics via microvillous membranes. The present finding further suggests that each of the three isoforms of PIP5K functions through its product PIP2 discretely in different cells of the glands to regulate saliva secretion.     

Vascular endothelial growth factor is constitutively expressed in normal human salivary glands and is secreted in the saliva of healthy individuals

The expression of vascular endothelial growth factor (VEGF), a specific mitogen for endothelial cells, was examined in salivary glands and in normal saliva. In normal salivary glands, VEGF mRNA and protein were strongly present in acinar cells, whereas little or no VEGF was found in ductal cells. In chronically inflamed glands, VEGF protein was in addition present in ductal elements and in infiltrating mononuclear cells. No difference of VEGF expression was observed between benign and malignant salivary gland tumours. By ELISA, whole saliva of 24 healthy individuals contained up to 2·5 ng/ml (mean 1·4 ng/ml; SD 0·77 ng/ml) of VEGF, confirming the constitutive secretion of this cytokine by human salivary glands. Western blot analysis of normal saliva under non-reducing conditions detected anti-VEGF reactive protein moieties of ≈46 kD, corresponding to VEGF secreted by cells in tissue culture. Additional anti-VEGF reactive proteins of ≈60 and 90 kD were detected in the saliva of some individuals. The presence of considerable quantities of VEGF in normal human saliva suggests an important role for this cytokine in the maintenance of the homeostasis of mucous membranes, with rapid induction of neoangiogenesis by salivary VEGF helping to accelerate wound healing within the oral cavity. Moreover, salivary VEGF may permeabilize intraglandular capillaries and thus participate in the regulation of saliva production itself. Copyright © 1998 John Wiley & Sons, Ltd.     

MUC1 and MUC2 expression in salivary gland tumors and in non-neoplastic salivary gland tissue

The expression of MUC1 and MUC2 was studied in salivary gland tumors and non-neoplastic salivary gland tissue. Formalin-fixed paraffin-embedded specimens from 101 patients (21 pleomorphic adenomas (PA), 22 Warthin's tumors (WT), 26 adenoid cystic carcinomas (ACC), 13 acinic cell adenocarcinomas (ACA), 9 mucoepidermoid carcinomas (MC), and 10 specimens of non-neoplastic parotid and submandibular gland tissue) were immunostained. All salivary gland tumors expressed MUC1. A strong immunoreactivity was noted in WT and MC, a moderate in ACC and ACA, and a weak in PA. Strong expression of MUC2 was noted in all WT, moderate expression in MC, and weak expression in PA and ACA. All cases of ACC except for two were negative for MUC2. In general, MUC1 expression was stronger than that of MUC2. Non-neoplastic salivary gland tissue revealed a moderate MUC1 and MUC2 expression in excretory ducts and a strong expression in striated ducts. The apical plasma membrane of some serous acini expressed MUC1. Mucous acini were negative for both antigens. No change in immunoreactivity was noted in cases of chronic sclerosing sialadenitis. In conclusion, the different expression pattern of MUC1 and MUC2 in salivary gland neoplasia may be of additional value for the classification of salivary gland tumors.     

Activation of pro-MMP-2 mediated by MT1-MMP in human salivary gland carcinomas: possible regulation of pro-MMP-2 activation by TIMP-2

Matrix metalloproteinases (MMPs), a family of extracellular matrix-degrading enzymes, are considered to play important roles in cancer invasion and metastasis. The present study examined the production levels of eight different MMPs (MMP-1, 2, 3, 7, 8, 9 and 13, and MT1-MMP) and two tissue inhibitors of metalloproteinases (TIMP-1 and 2) in homogenates of human salivary gland carcinomas [mucoepidermoid carcinomas (MECs), adenoid cystic carcinomas (ACCs), and adenocarcinomas (ADEs)] and non-neoplastic control salivary glands using sandwich enzyme immunoassay systems. The levels of MMP-1, MMP-2, MMP-13, MT1-MMP, and TIMP-1 were significantly higher in the carcinoma samples than in the controls (p < 0.05). Gelatin zymography demonstrated that the activation ratio of the MMP-2 zymogen (pro-MMP-2) was significantly higher in the carcinomas than in the controls (p < 0.05). In addition, the activation ratio in MECs was significantly higher than that in ACCs or ADEs (p < 0.01) and also correlated with histological grade and lymph node metastasis in MECs (p < 0.05), whereas the ratio showed no such correlation in ACCs or ADEs. Although the production levels of pro-MMP-2 and MT1-MMP were similar among these carcinoma groups, TIMP-2 levels were significantly higher in ACCs and ADEs than in MECs (p < 0.01). In carcinoma samples, the pro-MMP-2 activation ratio correlated directly with the MT1-MMP/TIMP-2 ratio (r = 0.736, n = 23; p < 0.01). Immunohistochemistry and in situ zymography demonstrated localization of MMP-2, MT1-MMP, and TIMP-2 to carcinoma cells, but only in MECs did carcinoma cell nests exhibit gelatinolytic activity, which was inhibited by 1,10-phenanthroline. These results suggest that enhanced activation of pro-MMP-2 mediated by MT1-MMP is implicated in the invasion and metastasis of MECs and that TIMP-2 may regulate pro-MMP-2 activation in salivary gland carcinomas.     

Controlling complement resistance in cancer by using human monoclonal antibodies that neutralize complement-regulatory proteins CD55 and CD59

Expression of the complement-regulatory proteins (CRP) CD46, CD55 and CD59 represents a strategy used by tumor cells to evade complement-dependent cell cytoxicity stimulated by monoclonal antibodies. We have isolated two single-chain variable fragments (scFv) to CD55 and CD59 from a human phage-display library and from these scFv we have produced two miniantibodies (MB), MB-55 (against CD55) and MB-59 (against CD59), containing the human hinge-CH2-CH3 domains of IgG1. The specificity of the two MB for the corresponding CRP was assessed by ELISA using purified CD46, CD55 and CD59. MB-55 and MB-59 neutralized the inhibitory activity of CD55 and CD59, respectively, restoring the complement-mediated lysis of sheep and guinea pig erythrocytes that was otherwise inhibited by the two CRP. FACS analysis revealed binding of MB-55 and MB-59 to the lymphoma cell line Karpas 422. The two MB induced a two-fold increase in the complement-dependent killing of these cells stimulated by Rituximab, a chimeric anti-CD20 monoclonal antibody. Transfection of HEK293T cells with vectors encoding MB-55 or MB-59 markedly reduced the expression of CD55 and CD59. We conclude that the human antibodies MB-55 and MB-59 may represent a therapeutic tool to increase the complement-dependent killing activity of Rituximab in the treatment of non-Hodgkin's lymphoma.     

Changes in systemic type 1 and type 2 immunity in normal pregnancy and pre-eclampsia may be mediated by natural killer cells

A bias of T cell immunity towards type 2 (Th2) is thought to be critical for normal pregnancy. Pathological pregnancies, such as pre-eclampsia, are characterised by cell-mediated (Th1) immune dominance. The Th1/Th2 paradigm, however, is too simplistic. Normal pregnancy is associated with a systemic inflammatory response which increases throughout gestation. This inflammatory response is exaggerated in pre-eclampsia, a syndrome of the third trimester. T helper (Th) cells are considered the primary mediators of these altered immune responses, and other T cells, i.e. T cytotoxic (Tc) cells, and lymphocytes of the innate immune system, i.e. natural killer (NK) and NKT cells, have been largely disregarded. In this study, we have used novel pan type 1 (IL-18 receptor) and pan type 2 (ST2L) lymphocyte function markers in four-colour flow cytometry to broadly characterise peripheral blood lymphocyte populations from non-pregnant, normal pregnant and pre-eclamptic women. There were no changes in the Th1/Th2 or Tc1/Tc2 cell ratios between the three groups; however, the NK1/NK2 and NKT1/NKT2 cell ratios were significantly decreased in normal pregnancy compared with non-pregnant (p <0.001 and p <0.01, respectively) and pre-eclamptic women (p <0.05). These results confirm that immunoregulation occurs in pregnancy, but suggest a dominant role of the innate rather than the adaptive immune system.     

A broad range of mutations in HIV-1 neutralizing human monoclonal antibodies specific for V2, V3, and the CD4 binding site

The HIV vaccine-induced neutralizing antibodies (Abs) display low rates of mutation in their variable regions. To determine the range of neutralization mediated by similar human monoclonal Abs (mAbs) but derived from unselected chronically HIV-1 infected subjects, we tested a panel of 66 mAbs specific to V3, CD4 binding site (CD4bs) and V2 regions. The mAbs were tested against 41 pseudoviruses, including 15 tier 1 and 26 tier 2, 3 viruses, showing that the neutralization potency and breadth of anti-V3 mAbs were significantly higher than those of the anti-CD4bs and anti-V2 mAbs, and only anti-V3 mAbs were able to neutralize some tier 2, 3 viruses. The percentage of mutations in the variable regions of the heavy (VH) and light (VL) chains varied broadly in a range from 2% to 18% and correlated moderately with the neutralization breadth of tier 2, 3 viruses. There was no correlation with neutralization of tier 1 viruses as some mAbs with low and high percentages of mutations neutralized the same number of viruses. The electrostatic interactions between anti-V3 mAbs and the charged V3 region may contribute to their neutralization because the isoelectric points of the VH CDR3 of 48 anti-V3 mAbs were inversely correlated with the neutralization breadth of tier 2, 3 viruses. The results demonstrate that infection-induced antibodies to CD4bs, V3 and V2 regions can mediate cross-clade neutralization despite low levels of mutations which can be achieved by HIV-1 vaccine-induced antibodies.     

Immunohistochemical expression pattern of RIP5, FGFR1, FGFR2 and HIP2 in the normal human kidney development

AimPresent study analyses the co-localisation of RIP5 with FGFR1, FGFR2 and HIP2 in the developing kidney, as RIP5 is a major determinant of urinary tract development, downstream of FGF-signaling.MethodsParaffin embedded human kidney tissues of 16 conceptuses between the 6th-22th developmental week were analysed using double-immunofluorescence method with RIP5/FGFR1/FGFR2 and HIP2 markers. Quantification of positive cells were performed using Kruskal–Wallis test.ResultsIn the 6th week of kidney development RIP5 (89.6%) and HIP2 (39.6%) are strongly expressed in the metanephric mesenchyme. FGFR1 shows moderate/strong expression in the developing nephrons (87.3%) and collecting ducts (70.5%) (p < 0.05). RIP5/FGFR1 co-localized at the marginal zone and the ureteric bud with predominant FGFR1 expression. FGFR2 (26.1%) shows similar expression pattern as FGFR1 (70.5%) in the same kidney structures. RIP5/FGFR2 co-localized at the marginal zone and the collecting ducts (predominant expression of FGFR2). HIP2 is strongly expressed in collecting ducts (96.7%), and co-localized with RIP5. In 10th week, RIP5 expression decrease (74.2%), while the pattern of expression of RIP5 and FGFR1 in collecting ducts (33.4% and 91.9%) and developing nephrons (21.9% and 32.4%) (p < 0.05) is similar to that in the 6th developmental week. Ureter is moderately expressing RIP5 while FGFR1 is strongly expressed in the ureteric wall. FGFR2 is strongly expressed in the collecting ducts (84.3%) and ureter. HIP2 have 81.1% positive cells in the collecting duct. RIP5/FGFR1 co-localize in collecting ducts and Henley’s loop.ConclusionsThe expression pattern of RIP5, FGFR1, FGFR2 and HIP2 in the human kidney development might indicate their important roles in metanephric development and ureteric muscle layer differentiation through FGF signaling pathways.