Inhibition of recall responses through complementary therapies targeting CD8+ T-cell- and alloantibody-dependent allocytotoxicity in sensitized transplant recipients.

Allospecific T memory cell responses in transplant recipients arise from environmental exposure to previous transplantation or cross-reactive heterologous immunity. Unfortunately, these memory responses pose a significant barrier to the survival of transplanted tissue. We have previously reported that concurrent inhibition of CD154 and LFA-1 suppresses primary CD8-dependent rejection responses that are not controlled by conventional immunosuppressive strategies. We hypothesized that CD154- and LFA-1-mediated inhibition, by targeting activation as well as effector functions, may also be efficacious for the control of alloreactive CD8+ T-cell responses in sensitized hosts. We found that treatment with anti-LFA-1 mAb alone enhanced transplant survival and reduced CD8-mediated cytotoxicity in sensitized CD4 KO recipients. However, treatment with anti-CD154 mAb alone did not have an effect. Notably, when both CD4- and CD8-dependent rejection pathways are operative (wild-type sensitized recipients), LFA-1 significantly inhibited CD8-mediated in vivo allocytotoxicity but did not correspond with enhanced hepatocyte survival. We hypothesized that this was due to alloantibody-mediated rejection. When anti-LFA-1 mAb treatment was combined with macrophage depletion, which we have previously reported impairs alloantibody-mediated parenchymal cell damage, in vivo cytotoxic effector function was significantly decreased and was accompanied by significant enhancement of hepatocyte survival in sensitized wild-type recipients. Therefore, LFA-1 is a potent therapeutic target for reduction of CD8-mediated cytotoxicity in sensitized transplant recipients and can be combined with other treatments that target non-CD8-mediated recall alloimmunity.

ATP-binding cassette transporter ABCB5 gene is expressed with variability in malignant melanoma.

BACKGROUND: Melanoma is a malignant neoplasm with high metastatic disease risk and elevated mortality. Incidence of melanoma varies according to geographic region and genetic BACKGROUND: Epidemiological studies indicate that acral melanoma (AM) is among the most common melanomas in the Mexican population. While extensive studies have identified genes associated with melanoma, little is known about the genes involved in the pathogenesis of AM. OBJECTIVE: To compare the gene expression patterns between primary melanoma and normal skin. METHODS: We used 10 samples of fresh acral melanomas and normal skin for the study of differential gene expression and 22 samples of melanoma for in situ hybridization. RESULTS: We first identified a gene that was present in a sample of AM and absent in normal skin. DNA sequencing of this differentially expressed gene revealed that it corresponded to ABCB5, a gene recently implicated in the regulation of progenitor cell fusion. Furthermore, we detected ABCB5 expression in other melanoma specimens by RT-PCR. We showed that nine out of ten melanomas were positive for ABCB5 while only one melanoma and normal skin samples were negative. All ABCB5 expressing melanomas had variable gene expression according to in situ hybridization studies, suggesting that the ABCB5 gene may be differentially regulated by individual melanomas. CONCLUSIONS: The ABCB5 gene may be related to the properties of chemoresistance and aggressiveness of melanoma. The high expression found in samples of acral melanoma may provide more insight on the pathogenesis of this common type of melanoma in the Mexican population, frequently associated with poor prognosis.

Conformational changes and cleavage of tau in Pick bodies parallel the early processing of tau found in Alzheimer pathology.

Neuronal protein inclusions are a common feature in Alzheimer disease (AD) and Pick disease. Even though the inclusions are morphologically different, flame-shape structure for AD vs. spherical structure for Pick disease, both have filaments mainly composed of tau protein. In AD, a well-defined pattern of conformational changes and truncation has been described. In this study, we used laser scanning confocal microscopy to characterize and compare the processing of tau protein during Pick disease with that found in AD. We found that tau protein of Pick disease preserves most of the relevant epitopes found in AD, the conformational foldings labelled by Alz-50 and Tau-66, the cleavage sites D(421) and E(391), as well as many phosphorylated sites, such as Ser(199/202), Thr(205) and Ser(396/404). We found a strong pattern of association between phosphorylation and cleavage at site D(421), as well as the phosphorylation and the conformational Alz-50 epitope. When we used late AD markers such as the conformational Tau-66 epitope and MN423 (cleavage at site E(391)) in Pick bodies (PBs), the overlap was significantly less. Furthermore, following morphological quantification, we found significantly higher numbers of phosphorylated tau in PBs. Overall, our findings suggest that phosphorylation is an early event, likely preceding the cleavage of tau at D(421). Despite this consistency with AD, we found a major distinction, namely that PBs lack beta-sheet conformation. We propose a scheme of early tau processing in these structures, similar to neurofibrillary tangles of AD.

Accumulation of C-terminally truncated tau protein associated with vulnerability of the perforant pathway in early stages of neurofibrillary pathology in Alzheimer's disease.

Neurofibrillary pathology is a characteristic hallmark of Alzheimer's disease that is closely correlated with cognitive decline. We have analysed the density and distribution of neurofibrillary tangles (NFTs) that are immunoreactive with the monoclonal antibody (mAb) 423 in a prospectively analysed population of Alzheimer's disease (AD) cases and age-matched controls. NFTs were examined in allocortical and isocortical areas and correlated with Braak pathological stage and clinical severity of dementia. The mAb 423 was used as it recognises a C-terminally truncated tau fragment that is a major constituent of NFTs. Our results show that extracellular NFTs and, to a lesser extent, intracellular NFTs, correlated significantly with both Braak stages and the clinical index of severity. Furthermore, a differential distribution of the two types of tangles indicates that layer II of the entorhinal cortex and the transentorhinal area are particularly vulnerable to neurofibrillary degeneration. These areas serve as a point of connection between isocortex and hippocampus. Our findings, therefore, suggest that the perforant pathway may be substantially affected by the accumulation of truncated tau protein in AD and that this represents a neuropathological predictor for the clinical severity of dementia. When neurofibrillary pathology was examined by combined labelling with mAbs 423 and Alz-50 and the dye thiazin red, we were able to demonstrate various stages of tau aggregation. The different stages may represent a sequence of conformational changes that tau proteins undergo during tangle formation in the allocortex during the early development of dementia in AD.

Tau-66: evidence for a novel tau conformation in Alzheimer's disease.

We have characterized a novel monoclonal antibody, Tau-66, raised against recombinant human tau. Immunohistochemistry using Tau-66 reveals a somatic-neuronal stain in the superior temporal gyrus (STG) that is more intense in Alzheimer's disease (AD) brain than in normal brain. In hippocampus, Tau-66 yields a pattern similar to STG, except that neurofibrillary lesions are preferentially stained if present. In mild AD cases, Tau-66 stains plaques lacking obvious dystrophic neurites (termed herein 'diffuse reticulated plaques') in STG and the hippocampus. Enzyme-linked immunosorbent assay (ELISA) analysis reveals that Tau-66 is specific for tau, as there is no cross-reactivity with MAP2, tubulin, Abeta(1-40), or Abeta(1-42), although Tau-66 fails to react with tau or any other polypeptide on western blots. The epitope of Tau-66, as assessed by ELISA testing of tau deletion mutants, appears discontinuous, requiring residues 155-244 and 305-314. Tau-66 reactivity exhibits buffer and temperature sensitivity in an ELISA format and is readily abolished by SDS treatment. Taken together these lines of evidence indicate that the Tau-66 epitope is conformation-dependent, perhaps involving a close interaction of the proline-rich and the third microtubule-binding regions. This is the first indication that tau can undergo this novel folding event and that this conformation of tau is involved in AD pathology.

Alternative splicing regulates the nuclear or cytoplasmic localization of dystrophin Dp71.

The subcellular distribution of Dp71 isoforms alternatively spliced for exon 71 and/or 78 was examined. The cDNA sequence of each variant was fused to the C-terminus of the green fluorescent protein and the constructs were transfected transiently in the cell lines HeLa, C2C12 and N1E-115. The subcellular distribution of the fused proteins was determined by confocal microscope analysis. The Dp71 isoform lacking the amino acids encoded by exons 71 and 78 was found exclusively in the cytoplasm whereas the variants containing the amino acids encoded by exon 71 and/or exon 78 show a predominant nuclear localization. The nuclear localization of Dp71 provides a new clue towards the establishment of its cellular function.

The extent of neurofibrillary pathology in perforant pathway neurons is the key determinant of dementia in the very old.

Neurofibrillary pathology as found in Alzheimer's disease (AD) is also found in the normal elderly, suggesting that these changes may be part of the aging process. In this study, we assessed the densities and distribution of structures recognized by the monoclonal antibody (mAb) to phosphorylated tau (AT8) in the hippocampal formation and medial temporal isocortex of 19 centenarians. Of these, 4 cases were demented and 15 non-demented. AT8 immunoreactivity correlated with the global deterioration scale (GDS). The density of both intraneuronal neurofibrillary tangles (I-NFTs) and neuritic clusters (NCs) significantly correlated with the GDS in the layer II of the entorhinal cortex (r = 0.66, P = 0.005 and r= 0.611, P = 0.01, respectively). Density of I-NFTs in the subiculum (r = 0.491; P = 0.034) also correlated significantly. No other area was found to be statistically significant. Importantly, no correlation was found when demented and non-demented centenarian cases were analyzed separately, suggesting that the difference marks a fundamental shift between AD and non-demented individuals. This assertion is supported by the significantly higher densities of I-NFTs and NCs in the transentorhinal (P = 0.043 and P = 0.011, respectively) and layer II of the entorhinal cortex (P = 0.02 and P = 0.007, respectively), and I-NFTs in the subiculum (P < 0.001) and CAI (P = 0.011) in the demented group when compared with the non-demented cases. Granular diffuse deposits, an early stage parameter of the neurofibrillary pathology involving accumulation of non-fibrillar abnormally phosphorylated tau protein did not correlate with the GDS or between the two groups studied. This study, combining morphometric and confocal analyses, not only provides further evidence that, in the brains of patients with AD, the perforant pathway is highly sensitive to tau pathology but also that involvement is distinct from the changes of normal aging, even of the oldest old.