Gene therapy for pituitary tumors

Pituitary tumors are the most common primary intracranial neoplasms. Although most pituitary tumors are considered typically benign, others can cause severe and progressive disease. The principal aims of pituitary tumor treatment are the elimination or reduction of the tumor mass, normalization of hormone secretion and preservation of remaining pituitary function. In spite of major advances in the therapy of pituitary tumors, for some of the most difficult tumors, current therapies that include medical, surgical and radiotherapeutic methods are often unsatisfactory and there is a need to develop new treatment strategies. Gene therapy, which uses nucleic acids as drugs, has emerged as an attractive therapeutic option for the treatment of pituitary tumors that do not respond to classical treatment strategies if the patients become intolerant to the therapy. The development of animal models for pituitary tumors and hormone hypersecretion has proven to be critical for the implementation of novel treatment strategies and gene therapy approaches. Preclinical trials using several gene therapy approaches for the treatment of anterior pituitary diseases have been successfully implemented. Several issues need to be addressed before clinical implementation becomes a reality, including the development of more effective and safer viral vectors, uncovering novel therapeutic targets and development of targeted expression of therapeutic transgenes. With the development of efficient gene delivery vectors allowing long-term transgene expression with minimal toxicity, gene therapy will become one of the most promising approaches for treating pituitary adenomas.     

Immunoblot analysis of Leishmania panamensis antigens in sera of patients with American cutaneous leishmaniasis.

Sera from 86 Colombian patients with parasitologically confirmed cutaneous leishmaniasis were studied by immunoblot analysis in order to identify a specific pattern for Leishmania infection. A soluble extract of Leishmania panamensis was used as the antigen. Sera from patients with Chagas' disease and sera from patients with no record of infection with trypanosomatids were also studied. The sera from patients with cutaneous leishmaniasis specifically recognized fractions of 120 kDa (76.7%), 123 and 129 kDa (69.7%), 138 kDa (61.6%), 141 kDa (53.4%), and 78 kDa (44.1%). No band common to all patients infected with Leishmania parasites was found at the time of diagnosis. Likewise, the pattern of immunoblot change after the patients were treated and apparently cured with meglumine antimoniate (Glucantime) was studied by evaluating sera pretreatment and 1 year posttreatment. Only minor changes in the color intensity at the same serum dilution between pre- and posttreatment sera were found, although the antibody titers by indirect immunofluorescence were negative for the posttreatment sample. This study shows that Western blot analysis is a more sensitive test for the detection of anti-Leishmania antibodies. However, the importance of whether the presence of antibodies correlates with the presence of Leishmania antigens could not be resolved by the data obtained from this study.     

Neuroendocrine carcinomas (carcinoid tumor) of the testis. A clinicopathologic and immunohistochemical study of ten cases

We studied 10 cases of primary pure testicular neuroendocrine carcinoma. Patients were between 16 and 48 years old and had testicular swelling with pain or a painless testicular mass and no history of neuroendocrine carcinoma or other malignant neoplasm. All underwent orchiectomy. The tumors were low (n = 9) and intermediate (n = 1) grades with a variegated histologic appearance characterized by a nesting pattern, cords of neoplastic cells with rosettes, or sheets of neoplastic cells. Mitotic activity was lacking in 9 cases. In 1 case, mitotic figures ranged from 7 to 8 per 10 high-power fields, and cellular atypia and comedo-like necrosis were present. Immunohistochemical studies using a keratin cocktail, chromogranin, synaptophysin, epidermal growth factor, p53, placental-like alkaline phosphatase, and CD117 (c-kit) were performed in all cases. Keratin, chromogranin, and synaptophysin were positive in all tumors. Clinical follow-up information was obtained for 6 patients (range, 12-60 months): 5 with low-grade tumors were alive 24 to 60 months after diagnosis; 1 with an intermediate-grade tumor died of tumor 12 months after initial diagnosis. The behavior of these tumors, while in the testicular region, correlates well with the histologic grade. We propose replacing the term testicular carcinoid with neuroendocrine carcinoma, which better reflects the nature of these neoplasms.     

NK-active cytokines IL-2, IL-12, and IL-15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells.

Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)-2, IL-12, and IL-15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus-infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL-2, IL-12, and IL-15 on PKCalpha and PKCepsilon--a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKCalpha and PKCepsilon activities; 2) whereas PKCepsilon activity is induced by cytokine stimulation, PKCalpha activity is inhibited; and 3) both the induction of PKCepsilon and the inhibition of PKCalpha functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine-induced NK cell stimulation.     

Four-color flow cytometric analysis of peripheral blood donor cell chimerism

Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.     

Heat shock proteins (HSPs) 27, 72 and 73 in normal and pre-ulcerative mucosa of the gastric pars oesophagea in swine

Heat shock proteins (HSPs), known to play a key role in cellular homeostasis, may also play a role in the defensive mechanisms of gastric mucosa. By means of appropriate immunohistochemical and immunobiochemical techniques, the expression of HSP27, HSP72 and HSP73 within the epithelium of normal and pre-ulcerative (hyperkeratinized) mucosa of the pars oesophagea of abattoir pigs was assessed. In normal mucosa, HSP72 and HSP73 expression was mainly limited to the basal epithelial cell layer, whereas HSP27 expression was consistently detected within the superficial epithelial cell layers. In hyperkeratinized mucosa, HSP72 and HSP73 immunoreactivity appeared to be more widespread, becoming very intense within epithelial cells affected by hydropic degeneration. Hyperkeratinized mucosa also showed HSP27 immunoreactivity, which was particularly intense in epithelial areas affected by hydropic degeneration. Western blot analysis confirmed HSP27, HSP72 and HSP73 expression in normal and in pre-ulcerative mucosa of the pars oesophagea. Semi-quantitative analysis showed that for all three HSPs the immunoreactivity was more intense in pre-ulcerative mucosa than in normal mucosa. The different expression patterns observed may have functional significance; further studies are needed, however, to define the role of HSPs in swine oesophagogastric lesions, the aetiology and pathogenesis of which are largely unknown.     

Nkx2.5‐negative myocardium of the posterior heart field and its correlation with podoplanin expression in cells from the developing cardiac pacemaking and conduction system

Recent advances in the study of cardiac development have shown the relevance of addition of myocardium to the primary myocardial heart tube. In wild‐type mouse embryos (E9.5–15.5), we have studied the myocardium at the venous pole of the heart using immunohistochemistry and 3D reconstructions of expression patterns of MLC‐2a, Nkx2.5, and podoplanin, a novel coelomic and myocardial marker. Podoplanin‐positive coelomic epithelium was continuous with adjacent podoplanin‐ and MLC‐2a‐positive myocardium that formed a conspicuous band along the left cardinal vein extending through the base of the atrial septum to the posterior myocardium of the atrioventricular canal, the atrioventricular nodal region, and the His‐Purkinje system. Later on, podoplanin expression was also found in the myocardium surrounding the pulmonary vein. On the right side, podoplanin‐positive cells were seen along the right cardinal vein, which during development persisted in the sinoatrial node and part of the venous valves. In the MLC‐2a‐ and podoplanin‐positive myocardium, Nkx2.5 expression was absent in the sinoatrial node and the wall of the cardinal veins. There was a mosaic positivity in the wall of the common pulmonary vein and the atrioventricular conduction system as opposed to the overall Nkx2.5 expression seen in the chamber myocardium. We conclude that we have found podoplanin as a marker that links a novel Nkx2.5‐negative sinus venosus myocardial area, which we refer to as the posterior heart field, with the cardiac conduction system. Anat Rec, 290:115–122, 2007. © 2006 Wiley‐Liss, Inc.     

The pulmonary biology of isoprostanes

Isoprostanes were first recognized as convenient markers of oxidative stress, but their powerful effects on a variety of cell functions are now also being increasingly appreciated. This is particularly true of the lung, which is comprised of a wide variety of different cell types (smooth muscle, innervation, epithelium, lymphatics, etc.), all of which have been shown to respond to exogenously applied isoprostanes. In this review, we summarize these biological responses in the lung, and also consider the roles that isoprostanes might play in a range of pulmonary clinical disorders.