Study protocol: understanding the pathophysiologic mechanisms underlying delirium in older people undergoing hip fracture surgery.

BACKGROUND: Postoperative delirium (POD) is a common complication of older people undergoing hip fracture surgery, which negatively affects clinical- and healthcare-related outcomes. Unfortunately, POD pathophysiology is still largely unknown, despite previous studies showing that neuroinflammation, neuroendocrine dysfunction, increased reactive oxidative stress (ROS), and endothelial dysfunctions may be involved. There is also evidence that many of the pathophysiological mechanisms which are involved in delirium are involved in sarcopenia too. This article describes the protocol of a pilot study to evaluate the feasibility of a larger one that will explore the pathophysiological mechanisms correlating POD with sarcopenia. We will analyse whether various biomarkers reflecting neuroinflammation, ROS, neuroendocrine disorders, and microvasculature lesions will be simultaneously expressed in in the blood, cerebrospinal fluid (CSF), and muscles of patients developing POD. METHODS: Two centres will be involved in this study, each recruiting a convenient sample of ten older patients with hip fracture. All of them will undergo a baseline Comprehensive Geriatric Assessment, which will be used to construct a Rockwood-based Frailty Index (FI). Blood samples will be collected for each patient on the day of surgery and 1 day before. Additionally, CSF and muscle fragments will be taken and given to a biologist for subsequent analyses. The presence of POD will be assessed in each patient every morning until hospital discharge using the 4AT. Delirium subtypes and severity will be assessed using the Delirium Motor Subtype Scale-4 and the Delirium-O-Meter, respectively. We will also evaluate the patient's functional status at discharge, using the Cumulated Ambulation Score. DISCUSSION: This study will be the first to correlate biomarkers of blood, CSF, and muscle in older patients with hip fracture.

Necdin enhances muscle reconstitution of dystrophic muscle by vessel-associated progenitors, by promoting cell survival and myogenic differentiation.

Improving stem cell therapy is a major goal for the treatment of muscle diseases, where physiological muscle regeneration is progressively exhausted. Vessel-associated stem cells, such as mesoangioblasts (MABs), appear to be the most promising cell type for the cell therapy for muscular dystrophies and have been shown to significantly contribute to restoration of muscle structure and function in different muscular dystrophy models. Here, we report that melanoma antigen-encoding gene (MAGE) protein necdin enhances muscle differentiation and regeneration by MABs. When necdin is constitutively overexpressed, it accelerates their differentiation and fusion in vitro and it increases their efficacy in reconstituting regenerating myofibres in the α-sarcoglycan dystrophic mouse. Moreover, necdin enhances survival when MABs are exposed to cytotoxic stimuli that mimic the inflammatory dystrophic environment. Taken together, these data demonstrate that overexpression of necdin may be a crucial tool to boost therapeutic applications of MABs in dystrophic muscle.

Duodenal expression of a putative stimulator of Fe transport and transferrin receptor in anemia and hemochromatosis.

BACKGROUND & AIMS: Stimulator of Fe Transport (SFT) and transferrin receptor (TfR) are proteins involved in iron transport. This study evaluated iron metabolism protein expression in duodenal biopsy specimens from controls and patients with abnormal iron metabolism. METHODS: Twelve controls, 8 patients with iron deficiency anemia, 7 with HFE-related hemochromatosis, and 6 with non-HFE-related iron overload were studied. Immunohistochemistry was performed on duodenal biopsy specimens with anti-TfR and anti-SFT antibodies which recognize a putative stimulator of Fe transport of ~80 kilodaltons. RESULTS: In controls, the putative stimulator of Fe transport was expressed in the middle and distal part of the villi in the subapical cytoplasmatic region. Its expression increased in anemics and, to a lesser degree, in HFE-related hemochromatotics, whereas it was reduced in patients with non-HFE-related iron overload. TfR expression showed a crypt-to-tip gradient in controls, but not in anemics, in whom it was uniformly overexpressed. TfR expression was intermediate in HFE-related hemochromatotics and similar to controls in non-HFE-related iron overload. CONCLUSIONS: Expression of the putative stimulator of Fe transport and TfR increases in iron deficiency. Increased expression of both proteins is present only in HFE-related hemochromatotics suggesting that other factors may be involved in determining non-HFE-related iron overload phenotype.

Iron overload and gene expression in HepG2 cells: analysis by differential display.

The aim of the present study was to evaluate the effect of iron overload on gene expression in HepG2 cells by differential display. Iron-treated cells showed a 50% decrease in apolipoprotein B100 (Apo B100) and a 2- and 3-fold increase in semaphorin cd100 and aldose reductase mRNA, respectively, with parallel variations in Apo B100 and aldose reductase proteins. These effects were time-dependent. Vitamin E prevented the increase in aldose reductase expression, but had no effect on Apo B100 and semaphorin cd100. Treatment with hydrogen peroxide and 4-hydroxy-2,3-nonenal increased only aldose reductase mRNA. These data suggest that iron can affect mRNA levels by lipid peroxidation-dependent and -independent pathways.

Absence of mutations in the highest mutability region of the p53 gene in tumour-derived CHEF18 Chinese hamster cells.

A series of independent tumour-derived Chinese hamster CHEF18 cell lines was analyzed for the presence of mutations in the cDNA region (exons 5-9) of the p53 gene, where the great majority of p53 mutations of human tumours accumulate. Since the gene is highly conserved among species, we used two primers designed on the basis of the human cDNA sequence to isolate the cDNA region from total RNA of Chinese hamster cells. The amplified fragments of 614 bp were digested with cleavase I endonuclease and fragment length polymorphism analysis showed that the restriction pattern of the p53 exons 5-9 region of tumour-derived cell lines was identical to that of diploid Chinese hamster CHL fibroblasts. Sequencing of the amplified fragments showed 100% homology between sequences, which demonstrated the absence of p53 mutations in the exons 5-9 cDNA region expected to have the highest mutability. Nevertheless, the antibody DO-7 recognized the presence of a stabilized p53 protein only in tumour-derived cell lines, which indicated that p53 expression correlated with transformed status.

Assignment of Chinese hamster p53 gene (TP53) to chromosome band 2p31, a region not involved in the karyotypic changes of a tumorigenic cell line.

The Chinese hamster tumor-derived cell line 835T2 exhibits specific karyotypic changes, including the loss and/or translocation of genetic material. To investigate whether the p53 tumor suppressor gene was involved in the exchanges, cDNA from primary Chinese hamster cells was isolated by using sense and antisense primers of the human p53 gene. The cDNA was sequenced, and the sequence was compared with the Syrian and human p53 cDNA reported sequences. The sequence homology was very elevated, demonstrating that the cloned fragment contained part of the Chinese hamster p53 gene. The corresponding genomic fragment was also cloned and used as a biotin-labeled probe for in situ hybridization on Chinese hamster chromosome spreads. Hybridization was visualized by avidin-FITC, and the assignment was done comparing the banding obtained with BamHI restriction enzyme and the location of the fluorescent signals pattern of the same metaphase. The signals revealed that the p53 gene (TP53) is localized on Chinese hamster chromosome band 2p31, which is not involved in the karyotypic changes specific to the 835T2 cell line.

Molecular characterization of a variant of proviral bovine leukaemia virus (BLV).

Southern-blot hybridization and partial sequencing of the pol and env genes were used to characterize BLV-integrated provirus of seropositive cattle from two dairy herds in northern Italy. Comparison of the data obtained with those of previously characterized BLV strains from other geographic areas (Australia, Belgium, Japan and USA) revealed the presence of a viral variant (BLV-12), which showed both conserved and unique features. Regarding the gp51 envelope glycoprotein, the BLV-12 variant showed: 1. A high extent of conservation, which included potential glycosylation sites and cysteine residues; 2. Three unique amino acid residues not present in any of the other BLV strains analysed; and 3. Some variability at the level of one (G) of the three (F, G and H) conformational epitopes, which is probably important in the process of infection. These results agree with the suggestion that the sequence variability of the gp51 glycoprotein preferentially involves structures whose change is thought to underlie the phenomenon of escape from immune surveillance.

Distinctive features of the alpha 1-domain alpha helix of HLA-C heavy chains free of beta 2-microglobulin.

Only a few monoclonal antibodies are available with a restricted specificity to HLA-C products. In the present report, we demonstrate that antibody L31, previously shown to react with beta 2m-less (free) class I MHC heavy chains, binds to an epitope (residues 66-68 of the alpha 1 domain alpha helix) present on all the HLA-C alleles corresponding to the accepted (CW1 through CW8) serologic specificities, and on a few HLA-B heavy chains sharing with HLA-C an aromatic residue at position 67. Extensive IEF blot testing of HLA homozygous, EBV-transformed B-lymphoid cells indicates that HLA-C molecules are present at significantly lower levels than HLA-B polypeptides not only at cell surface, as previously demonstrated, but also in total cellular extracts. Testing of metabolically labeled HLA-CW1, -CW5, and -CW6 transfectants and HLA homozygous lymphoid cells, particularly HLA-CW1-expressing cells, demonstrates that the L31 epitope is present on a subpopulation of naturally occurring HLA-C molecules distinct from that identified by antibody W6/32 to beta 2m-associated heavy chains. Pulse-chase experiments demonstrate that this epitope is transiently made available to antibody binding at early biosynthetic stages, but becomes hidden upon assembly with beta 2m. Thus, free HLA-C and other Y/F67+ heavy chains are characterized by distinctive antibody binding features in a region (residues 66-68) included in a previously identified HLA-C restricted motif, which has been suggested to be the primary cause of distinctive features of the antigen-binding groove, low affinity for endogenous peptide antigens and beta 2m, and preferential uptake of exogenous peptides, possibly of viral origin. We also show that HLA-CW1 heavy chains, both free and beta 2m associated, acquire sialilation. Free HLA-CW1 heavy chains are expressed at the cell surface even when unsialilated, albeit at low levels.

Expression of distinct conformations of free HLA-Cw4 heavy chains in transfected neuroblastoma cells.

Epitope mapping of HLA-Cw4 indicates that the two monoclonal antibodies (mAbs) L31 and M38, specific for beta 2-microglobulin (beta2m)-free HLA-C heavy chains, react preferentially with the KYK motif, located in the binding groove (alpha1 domain). Transfection of HLA-Cw4 cDNA into a neuroblastoma cell line, which normally expresses negligible HLA class I, resulted in the constitutive surface expression of molecules displaying different reactivities with the two mAbs. This cellular system was used to determine whether L31 and M38 recognize distinct conformations of beta2m-free HLA-C proteins, and to investigate their mechanism of expression. Interferon-gamma greatly enhanced the expression of L31-reactive free chains, while abolishing that of M38-reactive molecules. The cytokine-induced expression of L31-reactive molecules was inhibited by anti-sense oligonucleotides specific for beta2m mRNA, while constitutive expression of L31-reactive molecules was only partially affected. Exogenous beta2m resulted in a reduction of constitutive L31 reactivity, and in a concomitant increase of M38 reactivity. These results indicate that: 1) at the cell surface, L31 and M38 react with two distinct conformations of HLA-Cw4 beta2m-free heavy chains, of which the L31-reactive conformation is the least folded; 2) the expression of both conformers can be modulated by endogenous or exogenous beta2m; and (3) L31-reactive molecules exposed at the cell surface are likely to derive from the dissociation of empty HLA-Cw4/beta2m complexes.

Expression of beta 2m-free HLA class I heavy chains in neuroblastoma cell lines.

Flow cytometry with the specific monoclonal antibody (MoAb) L31 was used to analyse the expression of HLA class I heavy chains not bound with beta 2-microglobulin (beta 2m) by neuroblastoma (NB) cell lines IMR-32 and LA-N-1. The cells, which express barely detectable amounts of beta 2m-free (L31-positive molecules) and beta 2m-complexed HLA class I antigens (W6.32- and BBM.1-reactive molecules), expressed MHC class I molecules not bound to light chains upon differentiation with either retinoic acid or serum starvation. The expression was not accompanied by an increase of surface heterodimers. Conversely, recombinant interferon-gamma (rIFN-gamma) treatment led IMR-32 and LA-N-1 cells to almost exclusively express beta 2m-complexes HLA class I heavy chains. Surface beta 2m-free MHC class I molecules displayed a molecular mass of approximately 45 kDa and did not bind exogenously added beta 2m. No changes in the synthesis of either HLA class I and beta 2m mRNAs or of L31 proteins were observed in differentiated NB cells, thus suggesting that the surface exposure of unusual HLA class I antigens is regulated post-translationally. These findings indicate that, in addition to activated lymphocytes, the surface expression of beta 2m-free class I heavy chains is a feature of other cell types, such as NB cells.

Use of polymerase chain reaction to diagnose bovine leukemia virus infection in calves at birth.

A specific polymerase chain reaction (PCR) assay was devised, allowing detection of 1 bovine leukemia virus (BLV)-infected cell in 10(4) bovine lymphocytes. The efficacy of field application of the developed method was verified by evaluating the rate of viral transmission to calves from infected cows, whether they have persistent lymphocytosis. With this objective, 43 calves were simultaneously tested at birth and at 6 months of age for viral antibodies in serum and for proviral DNA in lymphocytes. At birth, 36 calves were BLV-negative and 3 were BLV-positive by results of serologic and DNA-based assays. Conversely, results for 4 calves had lack of correlation between the diagnostic methods. In particular, 2 calves were DNA-positive and antibody-negative for BLV and 2 other calves had the opposite test results. At 6 months of age, when the immunologic pattern more closely reflects the status of calves' immune response, independent of maternal antibodies, all calves DNA-negative for BLV at birth (n = 38), were consistently PCR- and antibody-negative for BLV. On the contrary, the cattle DNA-positive for BLV at birth (n = 5), whether seropositive or not, were PCR- and antibody-positive for BLV, at the time of the second screening. Thus, these results indicate reliability of the PCR to diagnose perinatal BLV infection. Furthermore, the observation that all calves found to be infected at birth were born to BLV-positive cows with persistent lymphocytosis, indicates that the persistent lymphocytosis status of the cow may represent a factor associated with BLV infection in utero.

Molecular organization and chromosomal location of human GC-rich heterochromatic blocks.

From the sequencing of three genomic DNA fragments and PCR amplification products from total human DNA, we have derived the sequence of a 545-bp Sau3A fragment (68% GC), representative of a family of human DNA repeats. Since previous studies suggested its linkage with unrelated Sau3A repeats of 68 bp (54% GC) (beta-satellite sequences), this feature was further investigated by in situ hybridization experiments and by Southern blot analysis of a panel of DNAs from human-Chinese hamster somatic cell hybrids. Both DNA repeats are preferentially localized on the heterochromatic regions of acrocentric chromosomes, on the pericentromeric heterochromatin of chromosome 1, 3 and 9, and on the proximal euchromatic region of the chromosome Y q arm. On chromosome 9, both repeats are part of a 2.7-kb higher-order repeat unit. These results and the Southern blot analysis on partial digests of total DNA, suggest that the linkage between the two repetitive DNA sequences is a constant feature throughout the genome. Furthermore, Southern blot analysis of HpaII-digested and MspI-digested DNA from different human tissues and tumor cell lines indicates that the investigated heterochromatic blocks appear to be subjected to changes in their methylation pattern.

Detection of bovine leukaemia virus (BLV) infection by DNA probe technology.

Classical serological methods and Southern blot hybridization for the diagnosis of bovine leukaemia virus (BLV) infection have been compared during the first nine months of life of offspring from BLV serum-negative and serum-positive dams belonging to a Friesian dairy herd in Italy. At birth, 9/13 calves analysed showed serum positivity for anti-gp60 BLV antibodies by agar immunodiffusion and/or by ELISA. However, only two calves were positive for BLV integrated proviruses in their lymphocyte DNA. At six months of age, anti-gp60 BLV antibodies and proviral DNA positivities were simultaneously shown only by the two cattle identified as DNA-positive at birth. This pattern remained constant up to nine months of age. Furthermore, analysis of the molecular characteristics of BLV integrated proviruses, carried out by using, as probes, the almost complete proviral genome (Belgian isolate) or a subclone of the env gene radioactively labelled or chemically modified, revealed that the calves under study were infected by a different isolate (Japanese isolate) and that, in one of the cattle, the majority of integrated proviruses was characterized by deletions probably located in the 5' half of the proviral genome.

Replication pattern of human repeated DNA sequences.

Either aphidicolin- or thymidine-synchronized human HL-60 cells were used to study the replication pattern of a family of human repetitive DNA sequences, the Eco RI 340 bp family (alpha RI-DNA), and of the ladders of fragments generated in total human DNA after digestion with XbaI and HaeIII (alpha satellite sequences). DNAs replicated in early, middle-early, middle-late and late S periods were labelled with BUdR or with [3H]thymidine. The efficiency of the cell synchronization procedure was confirmed by the transition from a high-GC to a high-AT average base composition of the DNA synthesized going from early to late S periods. By hybridizing EcoRI 340 bp repetitive fragments to BUdR-DNAs it was found that this family of sequences is replicated throughout the entire S period. Comparing fluorograph densitometric scans of [3H]DNAs to the scans of ethidium bromide patterns of total HL-60 DNA digested with XbaI and HaeIII, it was observed that DNA synthesized in different S periods is characterized by approximately the same ladder of fragments, while the intensity of each band may vary through the S phase; in particular, the XbaI 2.4 kb fragment becomes undetectable in late S.