Frequent PD-L1 expression in primary and metastatic penile squamous cell carcinoma: potential opportunities for immunotherapeutic approaches.

BACKGROUND: Despite aggressive multimodal therapy, locally advanced and/or metastatic penile squamous cell carcinoma (SqCC) is associated with significant morbidity and mortality, indicating a need for new therapeutic options. Given the emerging clinical utility of immunotherapeutics, we sought to assess the incidence and potential clinical significance of PD-L1 expression in penile SqCC. PATIENTS AND METHODS: Using an anti-PD-L1 primary antibody (clone 5H1), immunohistochemistry was carried out on whole tumor sections from 37 patients with penile SqCC treated at our institution between 2005 and 2013. PD-L1-positive tumors were defined as those with membranous staining in ≥5% of tumor cells. Association between PD-L1 expression and clinicopathologic parameters was examined using Fisher's exact test. Correlation between PD-L1 expression in primary tumors and matched metastases was assessed using the Spearman rank correlation coefficient (ρ). The difference in cancer-specific mortality between PD-L1-positive and -negative groups was examined using the log-rank test. RESULTS: Twenty-three (62.2%) of 37 primary tumors were positive for PD-L1 expression, and there was strong positive correlation of PD-L1 expression in primary and metastatic samples (ρ = 0.72; 0.032 < P < 0.036). Primary tumor PD-L1 expression was significantly associated with usual type histology (P = 0.040) and regional lymph node metastasis (P = 0.024), as well as decreased cancer-specific survival (P = 0.011). CONCLUSIONS: The majority of primary penile SqCC tumors express PD-L1, which is associated with high-risk clinicopathologic features and poor clinical outcome. These data provide a rational basis for further investigation of anti-PD-1 and anti-PD-L1 immunotherapeutics in patients with advanced penile SqCC.

Transcriptional regulation of the neuronal L-type calcium channel alpha 1D subunit gene.

1. The transcriptional regulation of the rat brain L-type calcium channel alpha 1D subunit (RB alpha 1D) gene was investigated using NG108-15 neuroblastoma-glioma cells. 2. Differentiation of NG108-15 cells in the presence of prostaglandin E1 or retinoic acid resulted in the appearance of mRNA encoding the RB alpha 1D subunit detected using Northern blot analysis. 3. A rat genomic DNA library was screened, and a 15.2-kb clone was isolated and partially sequenced which included part of the 5' upstream sequence through the initial part of intron 2 of the RB alpha 1D gene. 4. Deletion analysis, using a CAT reporter gene and transfected NG108-15 cells, revealed that the 1.2-kb 5'-upstream sequence from the RB alpha 1D gene contains cis-acting positive and negative regulatory elements. A deletion of the 3' end of exon 1 also suggested the presence of regulatory elements in the first exon. 5. DNase footprinting of exon 1 of the RB alpha 1D gene revealed two regions protected from digestion by specific protein binding, and the second region included an (ATG)7 trinucleotide repeat sequence. Electrophoretic mobility shift assays confirmed nuclear protein(s) binding to the (ATG)7 sequence. 6. The (ATG)7 sequence functions as a enhancer when linked to a thymidine kinase promoter and a CAT reporter gene. 7. These results provide the initial description of the transcriptional regulation of the RB alpha 1D gene and identify a novel enhancer that consists of an (ATG)7 trinucleotide repeat sequence.

Problems in research integrity arising from misconceptions about the ownership of research.

Many allegations of scientific misconduct result from activities that are perceived by the complainants as the "theft" of ideas, experimental results, or other intellectual property. The authors' thesis is that many of these allegations originate in misconceptions about the ownership of publicly supported scientific research. Some universities and medical schools may have their own codes for authorship, and journals and professional societies have codes or guidelines. In the NIH intramural programs, research data are considered to be the property of the institutes, not the individual researchers. In contrast, the training and experience of most scientists lead them to consider research data as being theirs. The paper discusses the origins of this attitude toward data and the ways that the structures of university laboratories and training programs lead to confusion and misunderstandings of researchers' "rights" to data. Also, emotional and personality factors often complicate these issues and lead to confrontations. Other misconceptions widely held among researchers: the false concepts of "my grant" and the "co-principal" investigator, ideas about who is and is not qualified to be an author, and ideas about sharing data. The authors emphasize the importance of scientifically literate legal advisers and the necessity for graduate students, postdoctoral fellows, and professors to understand their institutions' and grantors' guidelines and their obligations as scientists. At the heart of these obligations at all levels of research is honesty.

Effects of jet lag on factors related to sport performance.

Three studies were performed to evaluate the effects of jet lag on factors associated with sport performance. In Study 1, members of the USA Women's Soccer Team traveled to Taiwan; in Study 2, North American students and faculty traveled to Western Europe; and in Study 3, European students traveled to North America. After travel, there was disruption of mood state and a reduction in dynamic strength; peak 5-s power and 30-s work capacity were reduced for 2 days (5-s power: 9.8 vs. 9.0 vs. 9.0 W.kg-1; 30-s work capacity: 213 vs. 199 vs. 201 J.kg-1). In these studies, mood state, anaerobic power and capacity, and dynamic strength were affected by rapid transmeridianal travel, and even highly trained athletes suffered from jet lag. However, effects of travel on the variables tested were essentially eliminated after 3 or 4 days.

Distribution of phosphoprotein p19 in rat brain during ontogeny: stage-specific expression in neurons and glia.

p19 is an evolutionarily highly conserved 19-kDa cytosolic protein that undergoes hormonally regulated phosphorylation in a variety of mammalian cells. Its expression is abundant in brain and testis and is developmentally regulated. Here we have used immunocytochemistry to define the cell types expressing p19 in the rat CNS during pre- and postnatal development. p19-like immunoreactivity appears in young postmitotic neurons in the mantle zone of the neural tube on embryonic day 12-13. Subsequently, it is abundant in most, if not all, early immature forms of both neurons and glia and declines to undetectable levels in fully differentiated cells. In adult brain, strong p19-like immunoreactivity remains detectable in selective regions, primarily where production of glia and neurons is known to persist, such as the subventricular zone of olfactory bulb and lateral ventricle, and the dentate gyrus. The abundance of p19 mRNA, determined by Northern blot analysis of selected brain regions, parallels the distribution of p19 assessed by immunocytochemistry, suggesting that control of p19 expression is pretranslational. Together with previous findings on the transient expression of p19 during spermatogenesis, the present data suggest that expression of p19 occurs in a number of cell lineages in a differentiation stage-dependent manner. In brain, p19 represents a new marker that may prove valuable for defining immature cell populations.

Stage-specific expression of phosphoprotein p19 during spermatogenesis in the rat.

The expression of phosphoprotein p19, a 19-kDa cytosolic substrate for cyclic adenosine monophosphate (cAMP)-dependent protein kinase, occurs abundantly in brain and testis and is developmentally regulated. In the present study we have identified the cell types of adult rat testis that contain p19. Using cryostat sections, which were first incubated with rabbit anti-p19 for immunohistochemistry followed by counterstaining with periodic acid-Schiff (PAS)-hematoxylin to reveal nuclear morphology, we demonstrate that immunoreactive p19 is detectable only in germ cells and is restricted to a limited stage of spermatogenesis. Expression first appears after the differentiating gametes have entered the prophase of meiosis, is abundant in spermatocytes until meiosis is completed, and declines to undetectable levels in maturing spermatids. We have ruled out immunocross-reactivity with SCG10, a 22-kDa protein that is closely related in structure to p19, by demonstrating, using Northern blot analysis, that RNA transcripts encoding SCG10 are not detectable in adult rat testis, whereas p19 is abundantly expressed. The transient expression of p19 during spermatogenesis suggests that the protein plays a role during male gamete differentiation.

Characterization of opioid receptors in cultured neurons.

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.

Regulation of dopamine receptors in the MtT/W15 transplantable pituitary tumor by estrogen.

We investigated the effects of estrogens on the regulation of dopamine receptors in the MtT/W15 transplantable rat pituitary tumor. Diethylstilbestrol (DES) and 17beta-estradiol treatment in female rats significantly decreased the number of dopamine binding sites (B max) from 85 +/- 3.9 fmol/mg protein in untreated rats to 9.2 +/- 1.2 and 8.2 +/- 2.8 fmol/mg protein in DES and 17beta-estradiol-treated rats, respectively, while the binding affinities (Kd) did not change significantly. Testosterone treatment did not change the B max, while ovariectomy resulted in a significant increase in the B max (146.3 +/- 6.7 fmol/mg protein). The effects of DES on the B max were reversible, since removal of the DES for one week before sacrificing the animals led to a marked increase in the B max (54.9 +/- 3.1 fmol/mg protein). Pituitaries from normal female rats treated with DES for 6 and 9 weeks had a significant decrease in the B max. These results show that the number of dopamine binding sites in the membranes of MtT/W15 tumors is decreased by estrogen treatment and that this effect is reversible after removal of the estrogenic stimulus.

Anti-complement immunofluorescence establishes nuclear localization of human cytomegalovirus matrix protein.

A monospecific, polyclonal antiserum to the 69-kDa matrix protein of human cytomegalovirus (HCMV) was prepared in a guinea pig and used to determine the intracellular distribution of this viral antigen. The resulting antiserum was specific for infected cells as tested by immunofluorescence, and specific for the HCMV matrix protein as determined by "nitrocellulose immunoassay" of electrophoretically separated, infected-cell proteins. Antibodies were reacted with fixed, infected human fibroblasts, and visualized by the anti-complement immunofluorescence procedure to avoid complications arising from the strong IgG Fc binding activity of the infected-cell-specific cytoplasmic inclusion. Results establish that the matrix protein is located in the nucleus, and indicate that it is concentrated in the nucleoplasm rather than within the intranuclear inclusions.

A monoclonal antibody equivalent to anti-rat neural antigen-1 as a marker for Schwann cells.

The monoclonal antibody 217c, raised by Peng et al. [(1982) Science, Wash. 215, 1102-1104] in mice against the rat glioma cell line C6, can be used as a marker for normal Schwann cells. In mixed cultures of Schwann cells and fibroblasts from neonatal rat sciatic nerve, this monoclonal antibody, detected by indirect immunofluorescence, bound to the surface of cells with the same elongated morphology as those that express a previously described surface antigen, rat neural antigen-1 (Ran-1), defined by polyclonal mouse antisera. In these experiments Ran-1 and the antigenic determinant recognized by monoclonal 217c were both found on normal rat Schwann cells and on the rat glial tumor cell lines C6, 33B and 21A and the pheochromocytoma PC12. Neither anti-Ran-1 nor the monoclonal antibody bound to neurons, fibroblasts or glial cells in newborn rat cerebellum cultures, the rat muscle cell line L6, the transformed rat fibroblast cell line Rat 1, the rat brain tumor cell line B28 or the mouse Schwannoma cell line TR6B. Thus the monoclonal 217c behaved as if it were detecting Ran-1 by binding to normal rat Schwann cells and to those tumor cells that have this antigen. Our data show that this monoclonal antibody is a reliable and convenient marker for rat Schwann cells in culture.

A subset of Schwann cells in peripheral nerves contain a 50-kDa protein antigenically related to astrocyte intermediate filaments.

Antisera raised to the astrocyte intermediate filament structural protein stained elements in the peripheral nerves of several species. These elements were not associated with myelinated nerve fibers, were more common in splenic and vagus nerves than in the sciatic nerve, and persisted after nerve transection. In teased nerve preparations antigen-positive cells appeared to be the Schwann cells that surround small diameter, unmyelinated axons. Absorption of the antiserum with purified rat spinal cord 50-kDa protein or with bovine splenic nerve cytoskeletal extract blocked the reaction with CNS astrocyte processes or with PNS nerve fibers. Immunoblots of cytoskeletal preparations of bovine splenic nerve or rat sciatic nerve showed that the antigen from peripheral nerves comigrated at 50 kDa with antigen from bovine or rat spinal cord or cultured rat astrocytes. The CNS and PNS 50-kDa proteins from bovine tissues were subjected to limited digestion with Staphylococcus aureus protease V8. After separation on SDS-gels, antigenic peptides were detected by immunoblotting. The pattern of antigenic peptides for the CNS and PNS proteins were identical. We conclude that Schwann cells associated with nonmyelinated axons contain a cytoskeletal protein that is the same size and has the same peptide map as the major structural protein of astrocyte intermediate filaments.

Schwann cells cultured from adult rats contain a cytoskeletal protein related to astrocyte filaments.

Cells that bound antibody to the astrocyte intermediate filament protein were cultured from adult rat sciatic nerve. The antigen was intracellular, finely filamentous, and formed perinuclear caps in response to colchicine, all properties of intermediate filaments. Cytoskeletal proteins of these cultures were separated by SDS-gel electrophoresis, transferred to nitrocellulose paper, and shown to bind the glial-specific antiserum to a protein of 50,000 daltons. All the cells that bound this serum had a Schwann cell surface antigen, Ran-1, whereas fibroblasts from the nerve had Thy-1 surface antigen and did not contain the astrocyte filament antigen. These results prove that some Schwann cells from adult nerve, in contrast to fibroblasts or immature Schwann cells, have an intermediate filament protein that shares antigenic determinants with, or may be identical to, the astrocyte filament protein.

Identification of a calcium-regulated insulinoma cell phosphoprotein as an islet cell keratin.

This report describes the cytoskeleton nature of a 60,000-mol-wt protein, P60, previously shown to undergo Ca2+ influx-induced phosphorylation concomitant with insulin release in hamster insulinoma cells. Four lines of evidence suggest that P60 is an intermediate filament protein of the keratin class. (a) As previously described (Schubart, U.K., 1982, J. Biol. Chem. 257:12231-12238), Triton X-100-insoluble cytoskeletons are enriched for P60; (b) these cytoskeletons contain 7-11-nm filaments as determined by negative staining; (c) immunoblot analysis revealed that all proteins detected in the insulinoma cell cytoskeletons are recognized by a monoclonal antibody that interacts with a common determinant in all intermediate filament proteins; and (d) P60 was shown, by its identical migration on two-dimensional electrophoresis and by its immunologic relatedness, to be analogous to a known keratin present in HeLa cells. An antibody specific for P60, as judged by immunoblotting, was developed in a rabbit. In indirect immunofluorescence studies on insulinoma cells, this anti-P60 antibody produced a filamentous staining pattern. The antibody also permitted the identification of P60 in normal pancreatic islets as determined both by immunoblotting of hamster islet proteins resolved by two-dimensional electrophoresis and by indirect immunofluorescence microscopy on cryostat sections of hamster pancreas. In addition, the antibody recognized an antigen in the epithelial layer of pancreatic exocrine ducts, as determined by indirect immunofluorescence. The data have implications for the embryonic origin of pancreatic islets. Together with the phosphorylation data, these findings suggest that this islet cell cytokeratin may be involved in the regulation of insulin release.

Monoclonal antibodies binding to nuclear structures and axons in adult avian dorsal root ganglia.

Four monoclonal antibodies raised against embryonic chick dorsal root ganglia (5) recognize epitopes in neuronal and supporting cell nuclei, and in axons and contiguous cytoplasmic elements in some neurons of the adult chicken. Binding, analyzed at the ultrastructural level, is to reaction sites on the nuclear matrix, nucleolar complex, nuclear bodies, and filamentous elements in axons and some perikaryal regions.

The long term culture of bulk-isolated bovine oligodendroglia from adult brain.

Oligodendroglia isolated from adult bovine brain by the method of Farooq et al. could be plated on polylysine-coated plastic dishes with an efficiency of 55-80%, and maintained in culture for as long as 4 months. The addition of cytosine arabinoside to the nutrient medium resulted in cultures that were approximately 90% oligodendroglia and 10% large fibroblasts. From 50 g of white matter 100-160 X 10(6) oligodendroglia, containing approximately 6-10 mg protein, could be obtained in culture. These small round cells started to send out processes at 5 days in vitro and by 2 weeks they formed an extensive network of processes. By immunofluorescence, all cells of this morphology were positive for galactocerebroside (GC) and myelin basic protein (MBP), and negative for glial filament protein and fibronectin. Most of the large flat cells were positive for fibronectin and negative for GC, MBP and glial filament protein. As the cultures aged the oligodendroglia tended to clump and blebs formed on the surface of both perikarya and processes. By 4 months they showed evidence of degeneration and detached from the substrate. Electron microscope examination showed that the cells had the appearance typical of oligodendroglia in situ. The somata were round to elliptical, with eccentrically placed nuclei, and were larger than freshly isolated cells. They grew directly on the substrate or on the surface of the fibroblasts. In older cultures the cells formed tight nests. The somata were enveloped by sheets of oligodendrocyte cytoplasm, sometimes having a myelin-like appearance. Gap junctions and small desmosomes were seen between oligodendroglial processes and between oligodendroglia and fibroblasts. The cytoplasm was characterized by a prominent Golgi apparatus, many mitochondria and lysosomes, scattered rough endoplasmic reticulum, free ribosomes, frequent centrioles and an abundance of microtubules. In cells from older cultures large vacuoles were common, and rarely they had multilamellar walls with alternating major and minor dense lines resembling myelin.

Development of Thy-1 antigen on cerebellar neurons in culture.

The Thy-1.1 cell surface antigen was demonstrated by indirect immunofluorescence on two types of neurons in dissociated cell cultures of developing rat cerebellum. In cultures from postnatal rats, small cells predominated. They bound tetanus toxin, a neuronal marker, and did not have the capacity to take up gamma-aminobutyric acid (GABA) as measured by autoradiography. From these properties, we conclude that they are granule cells. These neurons began to express the surface antigen Thy-1.1 by 2 weeks in culture, and by 4 weeks, the antigen was detected on about 40% of all of the surviving neurons. The second type of neuron, which may be a heterogeneous mixture of cerebellar neurons, was numerous only in cultures prepared from embryonic rats. They were larger than granule cells and expressed Thy-1.1 antigen after a few days in culture. Such cells bound tetanus toxin and were negative for the glial markers galactocerebroside and glial fibrillary acidic protein. Thy-1-positive cells and cells with high GABA uptake were most frequent in embryonic day 19 cultures, where 40 to 50% of all of the neurons were positive for either property. They survived about 1 week in culture. The size and frequency of Thy-1 cells and the frequency of similar cells with high GABA uptake suggest that the embryonic rat cultures included many Purkinje cells, which express Thy-1.

Ultrastructure and immunocytochemistry of rat Schwann cells and fibroblasts in vitro.

The ultrastructure of dissociated rat sciatic nerve Schwann cells and fibroblasts has been examined and correlated at the morphologic and immunocytochemical levels. In agreement with previous studies, the two cell types were readily distinguishable morphologically on the basis of cell shape and size but no unique structural feature could be found. Schwann cells in this system essentially lacked basal laminae but did show some membrane specializations reminiscent of the in vivo situation. Fibroblasts were exceedingly rich in actin webs beneath the plasmalemma. Immunocytochemically, only Schwann cells stained with anti-Ran-1 antisera and fibroblasts only with anti-Thy-1. Both staining patterns were exclusively associated with the surface membrane. The findings provide a basis for future studies with this system to which neuronal elements may be added in an attempt to initiate myelinative events.