Effect of cysteine-rich whey protein (immunocal®) supplementation in combination with resistance training on muscle strength and lean body mass in non-frail elderly subjects: a randomized, double-blind controlled study.

OBJECTIVES: The purpose of the present study was to examine the effect of a cysteine-rich whey protein (Immunocal®) supplementation in combination with resistance training on muscle strength and lean body mass (LBM) in elderly individuals. We hypothesized that the cysteine-rich whey protein (Immunocal®) group would experience a greater increase in muscle strength and lean body mass versus the control group (casein). DESIGN: Randomized double-blind controlled intervention study. SETTING: Institut de Recherches Cliniques de Montréal in Montreal, Canada. PARTICIPANTS: Ninety-nine non-frail elderly subjects were recruited. INTERVENTION: Participants were randomly assigned into two groups. The experimental group received a cysteine-rich whey protein isolate (Immunocal®) (20 g/day) and the control group received casein (20 g/day) during a 135-day period. In addition, both groups performed the same resistance training program (3 times per week). MEASUREMENTS: Body composition (DXA) and muscle strength (leg press) were measured. RESULTS: Of the 99 recruited participants, 84 completed the 135-day study period. Of these, 67 subjects (33 in the casein group and 34 in the Immunocal® group) complied and used at least 80 % of the study product and completed at least 80 % of their training sessions. Results in this selected group show an increase in all three muscle strength variables (absolute, normalized by BW and by LBM) by 31.0 %, 30.9 % and 30.0 %, respectively in the casein group as well as 39.3 %, 39.9 % and 43.3 %, respectively in the Immunocal® group after the intervention (p < 0.05). The increases in muscle strength favored Immunocal® versus casein by approximately 10 % when expressed in kg per kg BW and in kg per kg LBM (p < 0.05). No significant changes were found between pre-and-post intervention in both groups for total LBM. CONCLUSIONS: Our findings showed increases in muscle strength in both groups after resistance training, however, significant additional increases were observed in muscle strength with the addition of a cysteine-rich whey protein (Immunocal®) versus casein.

[Hemophagocytic syndrome and toxoplasmic primo-infection]. / Syndrome d'activation macrophagique et primo-infection toxoplasmique.

Hemophagocytic syndrome (HPS) is a clinical entity that combines the clinical, biological and histological symptoms. The physiopathological mechanism involves the interaction between T lymphocytes/NK cells and macrophages, at the origin of an uncontrolled activation of the macrophages. The consequence is a hemophagocytosis extending to numerous organs, preferentially bone marrow. Clinical symptoms include cytopenia, fever unresponsive to antibiotics and multiple organ dysfunctions. Infections, lymphoproliferative disorders, cancers, systemic diseases are the most prevalent triggers or etiologies of HPS. Because of its high risk of mortality, HPS constitutes a diagnostic and therapeutic urgency. The search for an aetiology, in particular by serological testing, is essential because it conditions the treatment and thus the evolution of the disease. We report here the case of a 12 years-old boy presenting a HPS secondary to a toxoplasmic primo-infection. The objective of this work is to present the step of the biological diagnosis of HPS. Moreover, this observation allows the study of a very rare clinical presentation of toxoplasmic primo-infection, in an immunocompetant patient.

Intracellular rate-limiting steps of gene transfer using glycosylated polylysines in cystic fibrosis airway epithelial cells.

To identify the intracellular barriers to efficient gene transfer, we studied the intracellular trafficking of biotinylated plasmid DNA complexed with either fluorescein-conjugated lactosylated or mannosylated polylysine by confocal microscopy. Both are known to be taken up by cystic fibrosis airway epithelial cells (SigmaCFTE29o- cells), but their gene transfer efficiencies differ markedly: lactosylated polylysine is the most efficient glycosylated polylysine while mannosylated polylysine is quite inefficient for gene transfer. Mannosylated complexes appeared to stay longer in endosomes labeled by anti-transferrin receptor antibody than lactosylated complexes (from 30 min to 3 h and from 10 min to 30 min, respectively). At 24 h, higher percentages of mannosylated than lactosylated complexes were localized inside lysosomes labeled by anti-LAMP-1 antibody (41.8 +/- 6.6% versus 19.8 +/- 5.2%, respectively, P < 0.05). Between 30 min and 8 h, complexes reached the nuclei labeled by anti-lamin A/C antibody and no difference was observed between the nuclear amounts of mannosylated and lactosylated complexes. However, as analyzed by a nuclease S1 transcription assay, initiation of transcription was prevented when plasmid DNA was complexed to mannosylated polylysine. Our results indicate that the major limiting steps for mannosylated versus lactosylated polylysine transfer of plasmid DNA are delayed exit from endosomes, high accumulation in lysosomes and limited transcription of the complexed plasmid DNA.

Targeting of cell receptors and gene transfer efficiency: a balancing act.

Vectors conjugated with ligands recognized by cell surface receptors are of interest for cystic fibrosis gene therapy since these vectors would allow cell-specific targeting. However, an efficient and specific uptake may be abrogated by a subsequent intracellular trafficking leading to an inefficient gene transfer. This has been shown for polylysine substituted with mannose residues. While mannose-specific membrane lectins are predominantly expressed at the surface of airway cells and mannosylated complexes are the most efficiently incorporated glycosylated complexes in these cells, mannosylated complexes lead to a low gene transfer efficiency because of an inefficient exit from endosomal compartments, a high accumulation in lysosomes and an inefficient nuclear import. In contrast, the entry of low amounts of lactosylated complexes is balanced by more efficient intracellular trafficking, leading to an efficient gene transfer. This emphasizes that for a successful gene transfer, it is necessary to find the balance between efficient and specific uptake, and intracellular trafficking that overcomes the various cellular barriers and enables the plasmid to reach the nucleus.

Hsp104 interacts with Hsp90 cochaperones in respiring yeast.

The highly abundant molecular chaperone Hsp90 functions with assistance from auxiliary factors, collectively referred to as Hsp90 cochaperones, and the Hsp70 system. Hsp104, a molecular chaperone required for stress tolerance and for maintenance of [psi(+)] prions in the budding yeast Saccharomyces cerevisiae, appears to collaborate only with the Hsp70 system. We now report that several cochaperones previously thought to be dedicated to Hsp90 are shared with Hsp104. We show that the Hsp90 cochaperones Sti1, Cpr7, and Cns1, which utilize tetratricopeptide repeat (TPR) domains to interact with a common surface on Hsp90, form complexes with Hsp104 in vivo and that Sti1 and Cpr7 interact with Hsp104 directly in vitro. The interaction is Hsp90 independent, as further emphasized by the fact that two distinct TPR domains of Sti1 are required for binding Hsp90 and Hsp104. In a striking parallel to the sequence requirements of Hsp90 for binding TPR proteins, binding of Sti1 to Hsp104 requires a related acidic sequence at the C-terminal tail of Hsp104. While Hsp90 efficiently sequesters the cochaperones during fermentative growth, respiratory conditions induce the interaction of a fraction of Hsp90 cochaperones with Hsp104. This suggests that cochaperone sharing may favor adaptation to altered metabolic conditions.

A human immunodeficiency virus Env inducible transcription system to examine consequences of gp120 expression.

According to several studies, the HIV-1 envelope gp120 protein and the co-receptor CXCR4 play an essential role in HIV-1 induced cell toxicity. Characterisation of the CD4-independent m7NDK isolate provided the opportunity of studying the effects of direct interactions between m7NDK gp120 and CXCR4. Therefore, an inducible expression system was designed enabling synthesis of HIV-1 Env proteins upon doxycycline induction. Analysis of the expression of the env gene of the m7NDK HIV-1 isolate revealed, unexpectedly, that even long-term expression of m7NDK gp120 did not result in cytotoxycity in CXCR4-positive or -negative cell lines. This is the first report of a CD4-independent HIV-1-protein inducible expression regulated through the Tet-On system and by an alternative splicing. Env inducible expression cell lines could constitute a useful cellular tool to undertake analysis of HIV Env protein expression.

Growth inhibition and growth stimulation by estradiol of estrogen receptor transfected human breast epithelial cell lines involve different pathways.

Epidermal growth factor (EGF) and estradiol (E2) are important mitogens in breast epithelial cells, and expression of epidermal growth factor receptor (EGFR) and estrogen receptor (ER) is often inversely correlated in human breast cancer cells. Stable transfection of ER-negative cells with ER cDNA is not sufficient to restore E2-mediated growth stimulation, on the contrary, E2 often inhibits growth of ER-transfected cell lines. In this study we used the ER-transfected human breast epithelial cell lines HMT-3522F9, growth inhibited by E2 in the presence of EGF, and HMT-3522F9/S3B, growth stimulated by E2 in the absence of EGF. In S3B cells, no active MAP kinase could be detected in response to E2, suggesting that signalling through the MAP kinase is not the major pathway in the E2-mediated growth stimulation. Interestingly, a decreased level of active MAP kinase was observed in HMT-3522F9 cells in response to E2, indicating that in these cells cross-talk between the ER and the MAP kinase signalling pathway could be due to the E2-mediated growth inhibition. Moreover, we found that EGF-induced signalling also could be reduced by E2 in S3B cells, suggesting a general mechanism of action by E2 in cells concomitantly expressing ER and EGFR.

Proliferation and remodeling of the peritubular microcirculation after nephron reduction: association with the progression of renal lesions.

Little is known about the serial changes that might occur in renal capillaries after reduction of renal mass. In the current study, our aim was to document potential alterations in the morphology and proliferation of the renal cortical peritubular microcirculation at specific time points (7 and 60 days) after experimental 75% surgical nephron reduction using two strains of mice that we here demonstrate react differently to the same initial insult: one strain (C57BL6xDBA2/F1 mice) undergoes compensatory growth alone, whereas the other (FVB/N mice) additionally develops severe tubulo-interstitial lesions. Our data demonstrate that significant remodeling and proliferation occur in renal cortical peritubular capillaries after experimental nephron reduction, as assessed by microangiography using infusion of fluorescein isothiocyanate-labeled dextran, expression of the endothelial markers CD34 and Tie-2, and co-expression of CD34 and proliferating cell nuclear antigen, a surrogate marker of cell proliferation. This was accompanied by an increase of renal vascular endothelial growth factor protein levels and a change in distribution of this protein within the kidney itself. Moreover, most of these responses were accentuated in FVB/N mice in the presence of progressive renal disease and positively correlated with tubular epithelial cell proliferation. Hence, we have made three significant novel observations that illuminate the complex pathophysiology of chronic kidney damage after nephron reduction: 1) cortical peritubular capillaries grow by proliferation and remodeling, 2) vascular endothelial growth factor expression is altered, and 3) the development of tubulo-interstitial disease is genetically determined.

Identification of two estrogen regulated genes associated with growth regulation of human breast cancer.

We have identified two estrogen regulated gene products in the E(2) growth inhibited human breast cancer xenograft, T61; one showing 100% homology to the human BAC clone RP11-112E16, the other 100% homology to the human CPR3/DNJ3 gene. Verification by Northern blot analyses showed an up-regulation of the BAC clone RP11-112E16 and the CPR3/DNJ3 mRNAs upon E(2) treatment. Treatment of T61 tumors with tamoxifen, leading to static tumor growth, also increased the expression of the BAC clone RP11-112E16 and the CPR3/DNJ3 mRNAs. A similar association between growth inhibition and BAC clone RP11-112E16 and CPR3/DNJ3 mRNA induction was observed in MCF-7 cells treated with ICI 182.780. In MCF-7 cells, treatment with E(2) resulted in growth stimulation concomitant with a decrease in the BAC clone RP11-112E16 and CPR3/DNJ3 mRNA expression. Treatment with a combination of E(2) and ICI 182.780 abolished the anti-estrogen induced increase in BAC clone RP11-112E16 and CPR3/DNJ3 mRNA expression, indicating that regulation of the gene products is mediated through the ER. The association between growth inhibition and BAC clone RP11-112E16 or CPR3/DNJ3 mRNA expression was supported by high expression of both gene products in brain tissue. Further investigations are ongoing to clarify the biological function of these two gene products.

Hepatic expression of SV40 small-T antigen blocks the in vivo CD95-mediated apoptosis.

We have previously demonstrated that CD95-mediated apoptosis of hepatocytes is blocked in a murine model of hepatocarcinogenesis due to the expression of SV40 early sequences encoding the large-T and small-t antigens. In this study, we set out to pinpoint the sequences involved in this apoptosis-resistant phenotype, and tested several mutants of the SV40 early region for their ability to confer protection against CD95-induced apoptosis in transgenic mice. We show that resistance to apoptosis is independent of the transforming character of the mutants and demonstrate that the expression of the small-t antigen alone in transgenic mice is sufficient to confer this resistance. Our data also reveal an increased level of activated Akt kinase in these transgenic mice, and this could account for this hitherto unknown function of the SV40 small-t antigen.

Regression of primary hepatocarcinoma in cancer-prone transgenic mice by local interferon-gamma delivery is associated with macrophages recruitment and nitric oxide production.

The clinical potential of tumor therapies must be evaluated using animal models closely resembling human cancers. We investigated the impact of locally delivered interferon-gamma (IFN-gamma) on primary hepatocarcinoma spontaneously developed by T-SV40 transgenic mice. A single intratumor injection of adenovirus IFN-gamma was sufficient enough to induce in vivo production of biologically active IFN-gamma, as assessed by STAT1 activation. IFN-gamma secretion led to the regression of primary tumor, principally by apoptosis of tumor hepatocytes. The lack of T-cells infiltrates in the liver upon treatment excluded a role of a specific immune response. In contrast, indirect pathways may include tumoricidal function of macrophages. Indeed, they were massively recruited in the entire liver under IFN-gamma treatment; transmigration through hepatic blood vessels could be observed and co-localization with damaged hepatocytes was obvious. This correlated with nonparenchymal liver cell iNOS expression and high level of NO in hepatic extracts. Moreover, in vitro experiments showed that NO releasing agents induced cell death of freshly isolated tumor hepatocytes, suggesting that NO could be one of the major effector molecules. Altogether, these observations defined an important role of IFN-gamma in controlling tumor development in a model of primary hepatocarcinoma.

Determination of essential amino acids involved in the CD4-independent tropism of the X4 human immunodeficiency virus type 1 m7NDK isolate: role of potential N glycosylations in the C2 and V3 regions of gp120.

Seven mutations in the C2, V3, and C3 regions of gp120 are implicated in the tropism of the first CD4-independent human immunodeficiency virus type 1 isolate, m7NDK. Site-directed mutagenesis revealed that three amino acids are essential to maintain this tropism, one in the C2 region and two in the V3 loop. Two mutations implied N glycosylation modifications.

An in vitro model of human breast carcinogenesis: epigenetic aspects.

A review is given of 12 years of research on a human breast epithelial cell line, HMT-3522, which has undergone malignant transformation in vitro without being exposed to known carcinogenic agents. Epigenetic aspects of the malignant transformation have been considered and the results have been viewed in a clinical context. It has been concluded that the history and characteristics of the cell line resembles the comedocarcinoma of the human breast. It is hypothesized that progression from benign lesion to comedo in situ carcinoma and invasive carcinoma occurs if low levels of epidermal growth factor are prevailing in the microenvironment. Our data also suggest that breast cancer developed under high epidermal growth factor receptor activity is estrogen receptor negative, while suppression of epidermal growth factor receptor activity promotes estrogen responsive breast cancer.

Engraftment of autologous retrovirally transduced hepatocytes after intraportal transplantation into nonhuman primates: implication for ex vivo gene therapy.

The main impediment to effective ex vivo liver gene therapy of metabolic diseases is the lack of experimental work on large animals to resolve such important issues as effective gene delivery, cell-processing techniques, and the development of appropriate vectors. We have used a nonhuman primate, as a preclinical model, to analyze the limiting steps of this approach using recombinant retroviruses. Seven monkeys (Macaca fascicularis) underwent the complete protocol: their left liver lobe was resected, a catheter was placed in the inferior mesenteric vein and connected to an infusion chamber, and the hepatocytes were isolated, cultured, and transduced with a retroviral vector containing the beta-galactosidase gene. The hepatocytes were harvested and returned to the host via the infusion chamber. Biopsies were taken 4-40 days later. No animal was killed in the course of the experiments. They all tolerated the procedure well. We have developed and defined conditions that permit the proliferation and transduction of up to 90% of the plated hepatocytes. A significant proportion of genetically modified cells, representing up to 3% of the liver mass, were safely delivered to the liver via the chamber. Polymerase chain reaction analysis detected integrated viral DNA sequences and quantitative analysis of the in situ beta-Gal-expressing hepatocytes indicated that a significant amount of transduced hepatocytes, up to 2%, had become integrated into the liver and were functional. These results represent substantial advances in the development of the ex vivo approach and suggest that this approach is of clinical relevance for liver-directed gene therapy.

Gene therapy of cystic fibrosis: the glycofection approach.

Many cells express surface membrane lectins that selectively bind and carry glycoconjugates into intracellular endosomes; in addition, various intracellular membrane and soluble lectins act as shuttles between different compartments. On this basis, we developed glycosylated polycations, now called glycofectins (glycosylated polylysine and polyethyleneimine). Recently, we set up a simple way to transform oligosaccharides into glycosynthons suitable to substitute proteins or polymers. Glycofectins bind plasmid DNA leading to compact glycoplexes. Glycoplexes prepared with glycofectins were found to be much more active than naked plasmid to transfer genes to various types of cells including human airway epithelial and serous cells. The gene transfer efficiency was found to depend on the nature of the sugars borne by glycofectins. It appeared that the sugar-dependent efficiency was not only related to the uptake but also to the intracellular traffic of glycoplexes.

CXCR4 is down-regulated in cells infected with the CD4-independent X4 human immunodeficiency virus type 1 isolate m7NDK.

Macrophages and T cells infected in vitro with CD4-dependent human immunodeficiency virus type 1 (HIV-1) isolates have reduced levels of CD4 protein, a phenomenon involved in retroviral interference. We have previously characterized the first CD4-independent HIV-1 X4 isolate m7NDK, which directly interacts with CXCR4 through its mutated gp120. We thus investigate CXCR4 expression in cells infected with this m7NDK CXCR4-dependent HIV-1 mutant. We present evidence of the down-regulation of CXCR4 membrane expression in CD4-positive or -negative cells chronically infected with the HIV-1 m7NDK, a phenomenon which is not observed in the CD4-dependent HIV-1 NDK parental strain. This down-regulation of CXCR4 was demonstrated by fluorescence-activated cell sorter analysis and was confirmed by the absence of CXCR4 functionality in m7NDK-infected cells, independently of the presence of CD4 protein. Furthermore, a drastic reduction of the intracellular level of CXCR4 protein was also observed. Reduced levels of CXCR4 mRNA transcripts were found in m7NDK-infected HeLa and CEM cells, reduced levels that could not be attributed to a reduced stability of CXCR4 mRNA. Down-regulation of CXCR4 on m7NDK-infected cells may thus be explained by transcriptional regulation.

Histidylated polylysine as a synthetic vector for gene transfer into immortalized cystic fibrosis airway surface and airway gland serous cells.

BACKGROUND: We recently designed a cationic polymer called histidylated polylysine made of polylysine partially substituted with histidyl residues which become protonated at slightly acidic pH. This polymer is thought to induce the leakage of acidic vesicles containing plasmid/histidylated polylysine complexes. METHODS: and results Here, we have analyzed the ability of histidylated polylysine to transfer reporter or CFTR genes into immortalized cystic fibrosis airway surface epithelial cells (sigmaCFTE29o- cells) and airway gland serous cells (CF-KM4 cells) which are both important targets for cystic fibrosis gene therapy. The luciferase reporter gene expression measured after gene transfer with histidylated polylysine into both cell lines was quite high and similar to that obtained with commercially available vectors. In addition, the level of expression was not dependent on the presence of a membrane disrupting agent such as chloroquine. Histidylated complexes were present in slightly acidic non-lysosomal cellular compartments as shown by a cytological approach using biotinylated plasmid, lysosome-specific antibodies and confocal microscopy. Histidylated complexes appeared to be of small size when prepared at low ionic strength and formed aggregates upon increasing the ionic strength. However, aggregate formation was prevented by the addition of 10% fetal bovine serum. Gene transfer efficiency varied with the size of the complexes and decreased when small particles were used. CONCLUSIONS: These results suggest that histidylated polylysine may be an efficient non-viral vector for gene transfer into cystic fibrosis airway surface epithelial cells and airway gland serous cells.

The consequence of p53 overexpression for liver tumor development and the response of transformed murine hepatocytes to genotoxic agents.

To analyse the effect of p53 on liver tumor development, we generated transgenic mice overexpressing wild-type p53 in the liver and crossed them with transgenic mice in which the expression of the SV40 large T antigen (TAg) induces hepatic tumors. Remarkably, whereas preneoplastic TAg liver exhibited anisocaryosis and anisocytosis, TAg/p53 liver never presented any dysplastic cells. Moreover, whereas expression of p53 did not affect hepatic development, its constitutive expression in tumorigenic livers resulted in a significantly enhanced apoptosis once nodules had appeared. In contrast, p53 overexpression did not modify the elevated proliferation of TAg-transformed hepatocytes and had no effect on hepatocarcinoma progression. In vitro analysis of primary hepatocytes exposed to various genotoxic agents showed that p53 failed to sensitize normal or TAg-transformed hepatocytes to apoptosis, except when high doses of doxorubicin, UV-B and UV-C radiation were used. Our results confirmed that the hepatocyte cell type is very resistant to genotoxic agents and showed that constitutive expression of p53 failed to improve their responsiveness. In addition, our results showed that suppression of dysplastic cells, probably by restoring normal cytokinesis and karyokinesis, and enhancement of apoptosis by means of p53 overexpression were insufficient to counteract or delay the TAg-induced liver tumoral progression. Oncogene (2000) 19, 3498 - 3507

Targeted expression of a dominant-negative EGF-R in the kidney reduces tubulo-interstitial lesions after renal injury.

The role of EGF in the evolution of renal lesions after injury is still controversial. To determine whether the EGF expression is beneficial or detrimental, we generated transgenic mice expressing a COOH-terminal-truncated EGF-R under the control of the kidney-specific type 1 gamma-glutamyl transpeptidase promoter. As expected, the transgene was expressed exclusively at the basolateral membrane of proximal tubular cells. Under basal conditions, transgenic mice showed normal renal morphology and function. Infusion of EGF to transgenic animals revealed that the mutant receptor behaved in a dominant-negative manner and prevented EGF-signaled EGF-R autophosphorylation. We next evaluated the impact of transgene expression on the development of renal lesions in two models of renal injury. After 75% reduction of renal mass, tubular dilations were less severe in transgenic mice than in wild-type animals. After prolonged renal ischemia, tubular atrophy and interstitial fibrosis were reduced in transgenic mice as compared with wild-type mice. The beneficial effect of the transgene included a reduction of tubular cell proliferation, interstitial collagen accumulation, and mononuclear cell infiltration. In conclusion, functional inactivation of the EGF-R in renal proximal tubular cells reduced tubulo-interstitial lesions after renal injury. These data suggest that blocking the EGF pathway may be a therapeutic strategy to reduce the progression of chronic renal failure.

Intracellular pH regulation in a nonmalignant and a derived malignant human breast cell line.

Tumor cells in vivo often exist in an ischemic microenvironment that would compromise the growth of normal cells. To minimize intracellular acidification under these conditions, these cells are thought to upregulate H(+) transport mechanisms and/or slow the rate at which metabolic processes generate intracellular protons. Proton extrusion has been compared under identical conditions in two closely related human breast cell lines: nonmalignant but immortalized HMT-3522/S1 and malignant HMT-3522/T4-2 cells derived from them. Only the latter were capable of tumor formation in host animals or long-term growth in a low-pH medium designed to mimic conditions in many solid tumors. However, detailed study of the dynamics of proton extrusion in the two cell lines revealed no significant differences. Thus, even though the ability to upregulate proton extrusion in a low pH environment (pH(e)) may be important for cell survival in a tumor, this ability is not acquired along with the capacity to form solid tumors and is not unique to the transformed cell. This conclusion was based on fluorescence measurements of intracellular pH (pH(i)) on cells that were plated on extracellular matrix, allowing them to remain adherent to proteins to which they had become attached 24 to 48 h earlier. Proton translocation under conditions of low pH(e) was observed by monitoring pH(i) after exposing cells to an acute acidification of the surrounding medium. Proton translocation at normal pH(e) was measured by monitoring the recovery after introduction of an intracellular proton load by treatment with ammonium chloride. Even in the presence of inhibitors of the three major mechanisms of proton translocation (sodium-proton antiport, bicarbonate transport, and proton-lactate symport) together with acidification of their medium, cells showed only about 0.4 units of reduction in pH(i). This was attributed to a slowing of metabolic proton generation because the inhibitors were shown to be effective when the same cells were given an intracellular acidification.