Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease.

There is growing evidence that oxidative stress and mitochondrial respiratory failure with attendant decrease in energy output are implicated in nigral neuronal death in Parkinson disease (PD). It is not known, however, which cellular elements (neurons or glial cells) are major targets of oxygen-mediated damage. 4-Hydroxy-2-nonenal (HNE) was shown earlier to react with proteins to form stable adducts that can be used as markers of oxidative stress-induced cellular damage. We report here results of immunochemical studies using polyclonal antibodies directed against HNE-protein conjugates to label the site of oxidative damage in control subjects (ages 18-99 years) and seven patients that died of PD (ages 57-78 years). All the nigral melanized neurons in one of the midbrain sections were counted and classified into three groups according to the intensity of immunostaining for HNE-modified proteins--i.e., no staining, weak staining, and intensely positive staining. On average, 58% of nigral neurons were positively stained for HNE-modified proteins in PD; in contrast only 9% of nigral neurons were positive in the control subjects; the difference was statistically significant (Mann-Whitney U test; P < 0.01). In contrast to the substantia nigra, the oculomotor neurons in the same midbrain sections showed no or only weak staining for HNE-modified proteins in both PD and control subjects; young control subjects did not show any immunostaining; however, aged control subjects showed weak staining in the oculomotor nucleus, suggesting age-related accumulation of HNE-modified proteins in the neuron. Our results indicate the presence of oxidative stress within nigral neurons in PD, and this oxidative stress may contribute to nigral cell death.

Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.

High levels of the p53 protein are immunohistochemically detectable in a majority of human nonmelanoma skin cancers and UVB-induced murine skin tumors. These increased protein levels are often associated with mutations in the conserved domains of the p53 gene. To investigate the timing of the p53 alterations in the process of UVB carcinogenesis, we used a well defined murine model (SKH:HR1 hairless mice) in which the time that tumors appear is predictable from the UVB exposures. The mice were subjected to a series of daily UVB exposures, either for 17 days or for 30 days, which would cause skin tumors to appear around 80 or 30 weeks, respectively. In the epidermis of these mice, we detected clusters of cells showing a strong immunostaining of the p53 protein, as measured with the CM-5 polyclonal antiserum. This cannot be explained by transient accumulation of the normal p53 protein as a physiological response to UVB-induced DNA damage. In single exposure experiments the observed transient CM-5 immunoreactivity lasted for only 3 days and was not clustered, whereas these clusters were still detectable as long as 56 days after 17 days of UVB exposure. In addition, approximately 70% of these patches reacted with the mutant-specific monoclonal antibody PAb240, whereas transiently induced p53-positive cells did not. In line with indicative human data, these experimental results in the hairless mouse model unambiguously demonstrate that constitutive p53 alterations are causally related to chronic UVB exposure and that they are a very early event in the induction of skin cancer by UVB radiation.

Mutation detection by highly sensitive methods indicates that p53 gene mutations in breast cancer can have important prognostic value.

Human cancer cells with a mutated p53 tumor-suppressor gene have a selective growth advantage and may exhibit resistance to ionizing radiation and certain chemotherapeutic agents. To examine the prognostic value of mutations in the p53 gene, a cohort of 90 Midwestern Caucasian breast cancer patients were analyzed with methodology that detects virtually 100% of all mutations. The presence of a p53 gene mutation was by far the single most predictive indicator for recurrence and death (relative risks of 4.7 and 23.2, respectively). Direct detection of p53 mutations had substantially greater prognostic value than immunohistochemical detection of p53 overexpression. Analysis of p53 gene mutations may permit identification of a subset of breast cancer patients who, despite lack of conventional indicators of poor prognosis, are at high risk of early recurrence and death.

The generation, migration, and differentiation of olfactory neurons in the adult primate brain

In adult rodents, neural progenitor cells in the subependymal (SZ) zone of the lateral cerebral ventricle generate neuroblasts that migrate in chains via the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into interneurons. However, the existence of this neurogenic migratory system in other mammals has remained unknown. Here, we report the presence of a homologue of the rodent SZ/RMS in the adult macaque monkey, a nonhuman Old World primate with a relatively smaller OB. Our results—obtained by using combined immunohistochemical detection of a marker for DNA replication (5-bromodeoxyuridine) and several cell type-specific markers—indicate that dividing cells in the adult monkey SZ generate neuroblasts that undergo restricted chain migration over an extended distance of more than 2 cm to the OB and differentiate into granule interneurons. These findings in a nonhuman primate extend and support the use of the SZ/RMS as a model system for studying neural regenerative mechanisms in the human brain.

In vivo detection and imaging of phosphatidylserine expression during programmed cell death

One of the earliest events in programmed cell death is the externalization of phosphatidylserine, a membrane phospholipid normally restricted to the inner leaflet of the lipid bilayer. Annexin V, an endogenous human protein with a high affinity for membrane bound phosphatidylserine, can be used in vitro to detect apoptosis before other well described morphologic or nuclear changes associated with programmed cell death. We tested the ability of exogenously administered radiolabeled annexin V to concentrate at sites of apoptotic cell death in vivo. After derivatization with hydrazinonicotinamide, annexin V was radiolabeled with technetium 99m. In vivo localization of technetium 99m hydrazinonicotinamide-annexin V was tested in three models: fuminant hepatic apoptosis induced by anti-Fas antibody injection in BALB/c mice; acute rejection in ACI rats with transplanted heterotopic PVG cardiac allografts; and cyclophosphamide treatment of transplanted 38C13 murine B cell lymphomas. External radionuclide imaging showed a two- to sixfold increase in the uptake of radiolabeled annexin V at sites of apoptosis in all three models. Immunohistochemical staining of cardiac allografts for exogenously administered annexin V revealed intense staining of numerous myocytes at the periphery of mononuclear infiltrates of which only a few demonstrated positive apoptotic nuclei by the terminal deoxynucleotidyltransferase-mediated UTP end labeling method. These results suggest that radiolabeled annexin V can be used in vivo as a noninvasive means to detect and serially image tissues and organs undergoing programmed cell death.

Detection of JC virus DNA sequence and expression of the viral oncoprotein, tumor antigen, in brain of immunocompetent patient with oligoastrocytoma.

We describe molecular and clinical findings in an immunocompetent patient with an oligoastrocytoma and the concomitant presence of the human papovavirus, JC virus (JCV), which is the etiologic agent of the subacute, debilitating demyelinating disease, progressive multifocal leukoencephalopathy. Histologic review revealed a glial neoplasm consisting primarily of a moderately cellular oligodendroglioma with distinct areas of a fibrillary astrocytoma. Immunohistochemical analysis revealed nuclear staining of tumor cells with antibodies against the viral oncoprotein [tumor antigen (T antigen)], the proliferation marker (Ki67), and the cellular proliferation regulator (p53). Using primers specific to the JCV control region, PCR yielded amplified DNA that was identical to the control region of the Mad-4 strain of the virus. PCR analysis demonstrated the presence of the genome for the viral oncoprotein, T antigen, and results from primer extension studies revealed synthesis of the viral early RNA for T antigen in the tumor tissues. The presence of viral T antigen in the tumor tissue was further demonstrated by immunoblot assay. To our knowledge, this is the first report of the presence of JCV DNA, RNA, and T antigen in tissue in which viral T antigen is localized to tumor cell nuclei and suggests the possible association of JCV with some glial neoplasms.