Helmet continuous positive airway pressure for patients' transport using a single oxygen cylinder: A bench study.

BACKGROUND: Continuous positive airway pressure (CPAP) is frequently used to treat patients with acute respiratory failure in out-of-hospital settings. Compared to a facemask, the helmet has many advantages for the patient but requires a minimum gas flow of 60 L/min to avoid CO2 rebreathing. The aim of the present bench study was to evaluate the performance of four Venturi devices, connected to a single oxygen cylinder, in delivering helmet-CPAP with clinically relevant gas flow, fraction of inspired oxygen (FiO2), and positive end-expiratory pressure (PEEP) values. METHODS: Three double-inlet Venturi systems (EasyVent, Ventuplus, Compact-HAR) were connected to full 5-L oxygen cylinders using a double flowmeter, and their oxygen requirements to reach different setups (flow 60-80 L/min; FiO2 0.4-0.5-0.6, PEEP 7.5-10-12.5 cmH2O) were tested. The fourth Venturi system (O2-MAX) was directly attached to the tank, and the flow and FiO2 delivered at preset FiO2 0.3 and 0.6 were recorded. The runtime of the cylinder was assessed. RESULTS: EasyVent, Ventuplus, and O2-MAX were able to deliver helmet-CPAP with clinically useful setups when connected to a single oxygen cylinder, while Compact-HAR did not. The runtime of the cylinders ranged between 28 and 60 minutes according to the preset flow and FiO2. The delivered gas flow decreased slowly and linearly with the drop in cylinder pressure until its exhaustion. CONCLUSIONS: Helmet-CPAP might be provided using portable Venturi systems connected to an oxygen cylinder, but not all of them are able to deliver it. The use of a double flowmeter allows delivery of both high flow and high FiO2 when double-inlet Venturi systems are used. Due to the flow drop observed during the cylinder consumption, a flow >60 L/min should be set when helmet-CPAP is started. Considering the flow drop phenomenon, the estimated duration of the tank runtime can be used with a margin of safety when planning patient transport.

Persistence of psychosine in brain lipid rafts is a limiting factor in the therapeutic recovery of a mouse model for Krabbe disease.

Sphingolipids are intrinsic components of membrane lipid rafts. The abnormal accumulation of these molecules may introduce architectural and functional changes in these domains, leading to cellular dysfunction. Galactosylsphingosine (psychosine) is a pathogenic lipid raft-associated molecule whose accumulation leads to brain deterioration and irreversible neurological handicap in the incurable leukodystrophy Krabbe disease (KD). The relevance of clearing excessive levels of pathogenic psychosine from lipid rafts in therapy for KD has not been investigated. The work presented here demonstrates that psychosine inhibits raft-mediated endocytosis in neural cells. In addition, although in vitro enzyme reconstitution is sufficient for the reversal of related endocytic defects in affected neural cells, traditional in vivo enzyme therapies in the mouse model of KD appear to be insufficient for complete removal of pathogenic levels of raft-associated psychosine. This work describes a mechanism that may contribute to limiting the in vivo efficacy of traditional therapies for KD.

Combined hematopoietic and lentiviral gene-transfer therapies in newborn Twitcher mice reveal contemporaneous neurodegeneration and demyelination in Krabbe disease.

This study characterized the therapeutic benefits of combining hematogenous cell replacement with lentiviral-mediated gene transfer of galactosylceramidase (GALC) in Twitcher mice, a bona fide model for Krabbe disease. Bone marrow cells and GALC-lentiviral vectors were administered intravenously without any preconditioning to newborn Twitcher pups before postnatal day 2. Treated Twitchers survived up to 4 months of age. GALC activity remained less than 5% of normal values in the nervous system for the first 2 months after treatment and reached approximately 30% in long-term-surviving mice. Long-term reconstitution of GALC activity in the nervous system was provided primarily by infiltrating macrophages and to a lesser extent by direct lentiviral transduction of neural cells. Treated Twitchers had significant preservation of myelin, with a G-ratio (ratio of the axon diameter to the diameter of the myelinated fiber) in sciatic nerve myelin of 0.75 +/- 0.08 compared with 0.85 +/- 0.10 in untreated mutants. Although treated mutants had improved locomotor activities during their long-term survival, they died with symptoms of progressive neurological degeneration, indistinguishable from those seen in untreated Twitchers. Examination of long-lived Twitchers showed that treated mutants were not protected from developing degeneration of axons throughout the neuroaxis. These results suggest that GALC deficiency not only affects myelinating glia but also leads to neuronal dysfunction. The contemporaneous neuropathology might help to explain the limited efficacy of current gene and cell therapies.

Phenotype and function of dendritic cells and T-lymphocyte polarization in the human colonic mucosa and adenocarcinoma.

AIM: To evaluate the status of activation of the intestinal dendritic cells (DCs) and T lymphocytes (T cells) from surgical specimens of human colon and adenocarcinoma, and the potential effect of administration of interleukin 2 (IL-2). METHODS: Patients undergoing colectomy for cancer were randomized to receive subcutaneous IL-2 (12million UI/day) (treated group; n=10) for 3days before operation or no treatment (control group, n=10). DCs and T cells were isolated and purified from the lamina propria (LP) of segments of normal colon and adenocarcinoma of both groups. Cell phenotype was determined by expression of membrane receptors. Interaction between DC and T cells was assesses by a mixed leukocyte reaction using naïve T cells co-cultured with DCs. CD4+ T-cell polarization was studied by intracellular staining with monoclonal antibodies for interleukin-4 and interferon-gamma. RESULTS: CD4+ T cells were significantly less in tumour than in LP (p<0.05) in both treated and control groups. IL-2 did not modify the number of any of the T-cell subsets analysed. In contrast, T cells isolated from LP and neoplasm of treated patients produced more interferon-gamma and less interleukin-4 (p<0.05 vs. controls). IL-2 administration significantly increased (p<0.05) the number of mature, myeloid and plasmocytoid DCs compared to controls. Allogeneic naïve T cells were polarized toward a Th1 type of response which appeared to be mediated by IL-2 activated DCs. CONCLUSIONS: systemic IL-2 treatment may have immunomodulatory properties on intestinal DC maturation and drive a Th1 mediated anti-neoplastic response.

Design and optimization of lentiviral vectors for transfer of GALC expression in Twitcher brain.

BACKGROUND: Demyelination in globoid cell leukodystrophy (GLD) is due to a deficiency of galactocerebrosidase (GALC) activity. Up to now, in vivo brain viral gene transfer of GALC showed modest impact on disease development in Twitcher mice, an animal model for GLD. Lentiviral vectors, which are highly efficient to transfer the expression of therapeutic genes in neurons and glial cells, have not been evaluated for direct cerebral therapy in GLD mice. METHODS: Lentiviral vectors containing the untagged cDNA or the hemagglutinin (HA)-tagged cDNA for the full-length mouse GALC sequence were generated and validated in vitro. In vivo therapeutic efficacy of these vectors was evaluated by histology, biochemistry and electrophysiology after transduction of ependymal or subependymal layers in young Twitcher pups. RESULTS: Both GALC lentiviral vectors transduced neurons, oligodendrocytes and astrocytes with efficiencies above 75% and conferred high levels of enzyme activity. GALC accumulated in lysosomes of transduced cells and was also secreted to the extracellular medium. Conditioned GALC medium was able to correct the enzyme deficiency when added to non-transduced Twitcher glial cultures. Mice that received intraventricular injections of GALC vector showed accumulation of GALC in ependymal cells but no diffusion of the enzyme from the ependymal ventricular tree into the cerebral parenchyma. Significant expression of GALC-HA was detected in neuroglioblasts when GALC-HA lentiviral vectors were injected in the subventricular zone of Twitcher mice. Life span and motor conduction in both groups of treated Twitcher mice were not significantly ameliorated. CONCLUSIONS: Lentiviral vectors showed to be efficient for reconstitution of the GALC expression in Twitcher neural cells. GALC was able to accumulate in lysosomes as well as to enter the secretory pathway of lysosomal enzymes, two fundamental aspects for gene therapy of lysosomal storage diseases. Our in vivo results, while showing the capacity of lentiviral vectors to transfer expression of therapeutic GALC in the Twitcher brain, did not limit progression of disease in Twitchers and highlight the need to evaluate other routes of administration.

Myelin deterioration in Twitcher mice: motor evoked potentials and magnetic resonance imaging as in vivo monitoring tools.

We have used magnetic resonance imaging (MRI) and motor evoked potentials (MEPs) for monitoring disease progression within the CNS of the Twitcher mouse, the murine model for globoid cell leukodystrophy (GLD). GLD is a lysosomal storage disorder, resulting from galactocerebrosidase deficiency, causing central and peripheral myelin impairment, leading to death, usually during early infancy. Neuroradiological, electrophysiological, and pathological parameters of myelin maturation were evaluated in Twitcher mice between postnatal days 20 and 45. Healthy controls showed a gradual-appearance MRI T2-weighted hypointensity of the corpus callosum (CC) starting at about P30 and ending at about P37, whereas MRI of age-matched Twitcher mice showed a complete loss of the CC-related MRI signal. MEPs allowed the functional assessment of myelin maturation within corticospinal motor pathways and showed a progressive deterioration of MEPs in Twitcher mice with increased central conduction time (CCT; 5.12 +/- 0.49 msec at P27 to 6.45 +/- 1.96 msec at P32), whereas physiological CCT shortening was found in healthy controls (3.01 +/- 0.81 msec at P27 to 2.5 +/- 0.27 msec at P32). These findings were not paralleled by traditional histological stainings. Optical observation of Bielchowsky and Luxol fast blue-PAS stainings showed mild axonal/myelin deterioration of the Twitcher brain within this time frame. Our results demonstrate that serial MRI and MEP readings are sensitive evaluation tools for in vivo monitoring of dysmyelination in Twitcher mice and underscore their potential use for longitudinal evaluation of the therapeutic impact of gene and cell therapies on these animals.

The favorable prognostic significance of surgery-induced hyperprolactinemia in node-positive breast cancer patients: ten-year disease-free survival results.

It has been shown that each manipulation of the mammary region, including breast surgery, may stimulate prolactin secretion. However, it has also been observed that in more than 50% of breast cancer patients surgical removal of the tumor is not followed by enhanced prolactin secretion. This might be indicative of an altered psychoneuroendocrine control of the mammary gland, which could lead to the onset of more biologically aggressive breast cancer. In fact, surgery-induced hyperprolactinemia has been proven to be associated with a better prognosis in terms of survival in node-negative breast cancer patients. The present study was performed to investigate the impact of postoperative hyperprolactinemia on the disease-free survival (DFS) of breast cancer patients with axillary node involvement. The study included 100 consecutive node-positive breast cancer patients who were followed for at least 10 years. Surgery-induced hyperprolactinemia occurred in 45/100 (45%) patients without any significant correlation with the main prognostic variables including number of involved nodes and ER status. The two groups of patients received the same adjuvant therapies. After a median follow-up of 151 months, the recurrence rate in patients with surgery-induced hyperprolactinemia was significantly lower than in patients with no postoperative hyperprolactinemia (23/45 vs 43/55, p<0.01). Moreover, DFS was significantly longer in hyperprolactinemic patients than in patients who had no enhanced secretion of prolactin postoperatively. In agreement with the results described previously in node-negative breast cancer, our study demonstrates the favorable prognostic significance of surgery-induced hyperprolactinemia in terms of DFS duration also in breast cancer patients with axillary node involvement, independent of the other well-known prognostic variables, thereby confirming that the psychoneuroendocrine status of cancer patients may influence the prognosis of their disease.

Analysis of galactocerebrosidase activity in the mouse brain by a new histological staining method.

Gene therapy of galactocerebrosidase (GALC) deficient mice (Twitcher mutants) requires a fast and sensitive assay to detect transduced cells in vitro and in vivo. We have developed a new rapid histochemical method that specifically detects GALC activity in situ in neural cells using 5-Br-3Cl-beta-galactopiranoside (X-Gal) in the presence of taurodeoxycholic and oleic acids to enhance suspension of the substrate at low pH. Using this method, we observed robust X-Gal staining in diverse neuronal populations and interfascicular oligodendrocytes in sections from normal mouse brain. In contrast, sections of Twitcher brain did not show a specific staining pattern in neurons or glial cells. The availability of this new sensitive and rapid in situ detection assay is fundamental for the follow-up of Twitcher mice under gene or cellular therapies to correct central GALC deficiency.

Caveolinopathies: mutations in caveolin-3 cause four distinct autosomal dominant muscle diseases.

The caveolin-3 protein is expressed exclusively in muscle cells. Caveolin-3 expression is sufficient to form caveolae-sarcolemmal invaginations that are 50 to 100 nm in diameter. Monomers of caveolin-3 oligomerize to form high molecular mass scaffolding on the cytoplasmic surface of the sarcolemmal membrane. A mutation in one caveolin-3 allele produces an aberrant protein product capable of sequestering the normal caveolin-3 protein in the Golgi apparatus of skeletal muscle cells. Improper caveolin-3 oligomerization and membrane localization result in skeletal muscle T-tubule system derangement, sarcolemmal membrane alterations, and large subsarcolemmal vesicle formation. To date, there have been eight autosomal dominant caveolin-3 mutations identified in the human population. Caveolin-3 mutations can result in four distinct, sometimes overlapping, muscle disease phenotypes: limb girdle muscular dystrophy, rippling muscle disease, distal myopathy, and hyperCKemia. Thus, the caveolin-3 mutant genotype-to-phenotype relation represents a clear example of how genetic background can influence phenotypic outcome. This review examines in detail the reported cases of patients with caveolin-3 mutations and their corresponding muscle disease phenotypes.

Secretory defects induced by immunosuppressive agents on human pancreatic beta-cells.

Despite the considerable interest for islet and pancreas transplantation, remarkably little is known about the direct effects of immunosuppressive drugs on human beta-cell function. We measured different insulin secretory parameters and insulin gene expression of human islets cultured for 5 days in the presence of mycophenolate mofetil (MMF), cyclosporin A (CsA), tacrolimus (FK506) or a mixture of 3 cytokines. Basal insulin release after exposure to cytokines and FK506 was significantly higher than in control islets. Responsiveness to an acute glucose stimulus did not differ significantly between control and treated islets. However, absolute incremental insulin responses (delta-AUCs) of islets exposed to cytokines or FK506 were significantly higher compared to islets exposed to CsA or MMF, mainly because of the higher basal release. Indeed, maximal over basal release (stimulation index, SI) tended to be lower in islets exposed to FK506 than in control islets. Insulin gene expression was significantly reduced only in islets exposed to CsA. FK506 was, among those tested, the immunosuppressive drug that most profoundly altered the normal insulin secretory pattern of human beta-cells, whereas CsA was the only inhibiting insulin gene expression. Although the abnormalities induced by the immunosoppressive drugs utilized in this study were modest, these in vitro data are consistent with the reported in vivo diabetogenicity of CsA and FK506 and point to MMF as the ideal immunosuppressive agent from a pancreatic beta-cell point of view.

[Case report: rheumatoid arthritis and large granular lymphocytes syndrome]. / Artrite reumatoide e leucemia a "large granular lymphocytes": descrizione di un caso clinico.

Felty's syndrome (FS) is a rare complication (less than 1%) of rheumatoid arthritis (RA), with the clinical feature of splenomegaly and neutropenia. Approximately 10-40% of FS patients have an expansion of peripheral blood large granular lymphocytes (LGL). This cell population mainly consists of two subsets: cytotoxic T cells (CD8+, CD57+) and natural killer cells (CD3-,CD8-,CD56+). It has been hypothesised that LGL expansion could be responsible for neutropenia by suppressing neutrophil precursors in the bone marrow, but various mechanisms have been proposed to explain this association. We report a case of a 60-year-old woman with rheumatoid factor positive RA who developed LGL expansion responsible for splenomegaly, but without neutropenia. In conclusion, LGL expansion is an uncommon complication of RA and may be responsible for both FS and clinical pictures resembling FS.

Caveolae and caveolin-3 in muscular dystrophy.

Caveolae are vesicular invaginations of the plasma membrane, and function as 'message centers' for regulating signal transduction events. Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolar membrane domains in skeletal muscle and in the heart. Several mutations within the coding sequence of the human caveolin-3 gene (located at 3p25) have been identified. Mutations that lead to a loss of approximately 95% of caveolin-3 protein expression are responsible for a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans. By contrast, upregulation of the caveolin-3 protein is associated with Duchenne muscular dystrophy (DMD). Thus, tight regulation of caveolin-3 appears essential for maintaining normal muscle health and homeostasis.

Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism.

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether caveolin-1 plays any role in regulating cell cycle progression. Here, we directly demonstrate that caveolin-1 expression arrests cells in the G(0)/G(1) phase of the cell cycle. We show that serum starvation induces up-regulation of endogenous caveolin-1 and arrests cells in the G(0)/G(1) phase of the cell cycle. Moreover, targeted down-regulation of caveolin-1 induces cells to exit the G(0)/G(1) phase. Next, we constructed a green fluorescent protein-tagged caveolin-1 (Cav-1-GFP) to examine the effect of caveolin-1 expression on cell cycle regulation. We directly demonstrate that recombinant expression of Cav-1-GFP induces arrest in the G(0)/G(1) phase of the cell cycle. To examine whether caveolin-1 expression is important for modulating cell cycle progression in vivo, we expressed wild-type caveolin-1 as a transgene in mice. Analysis of primary cultures of mouse embryonic fibroblasts from caveolin-1 transgenic mice reveals that caveolin-1 induces 1) cells to exit the S phase of the cell cycle with a concomitant increase in the G(0)/G(1) population, 2) a reduction in cellular proliferation, and 3) a reduction in the DNA replication rate. Finally, we demonstrate that caveolin-1-mediated cell cycle arrest occurs through a p53/p21-dependent pathway. Taken together, our results provide the first evidence that caveolin-1 expression plays a critical role in the modulation of cell cycle progression in vivo.

Palmitoylation of caveolin-1 at a single site (Cys-156) controls its coupling to the c-Src tyrosine kinase: targeting of dually acylated molecules (GPI-linked, transmembrane, or cytoplasmic) to caveolae effectively uncouples c-Src and caveolin-1 (TYR-14).

Caveolin-1 was initially identified as a phosphoprotein in Rous sarcoma virus-transformed cells. Previous studies have shown that caveolin-1 is phosphorylated on tyrosine 14 by c-Src and that lipid modification of c-Src is required for this phosphorylation event to occur in vivo. Phosphocaveolin-1 (Tyr(P)-14) localizes within caveolae near focal adhesions and, through its interaction with Grb7, augments anchorage-independent growth and epidermal growth factor-stimulated cell migration. However, the cellular factors that govern the coupling of caveolin-1 to the c-Src tyrosine kinase remain largely unknown. Here, we show that palmitoylation of caveolin-1 at a single site (Cys-156) is required for coupling caveolin-1 to the c-Src tyrosine kinase. Furthermore, upon evaluating a battery of nonreceptor and receptor tyrosine kinases, we demonstrate that the tyrosine phosphorylation of caveolin-1 by c-Src is a highly selective event. We show that Src-induced tyrosine phosphorylation of caveolin-1 can be inhibited or uncoupled by targeting dually acylated proteins (namely carcinoembryonic antigen (CEA), CD36, and the NH(2)-terminal domain of Galpha(i1)) to the exoplasmic, transmembrane, and cytoplasmic regions of the caveolae membrane, respectively. Conversely, when these proteins are not properly targeted or lipid-modified, the ability of c-Src to phosphorylate caveolin-1 remains unaffected. In addition, when purified caveolae preparations are preincubated with a myristoylated peptide derived from the extreme N terminus of c-Src, the tyrosine phosphorylation of caveolin-1 is abrogated; the same peptide lacking myristoylation has no inhibitory activity. However, an analogous myristoylated peptide derived from c-Yes also has no inhibitory activity. Thus, the inhibitory effects of the myristoylated c-Src peptide are both myristoylation-dependent and sequence-specific. Finally, we investigated whether phosphocaveolin-1 (Tyr(P)-14) interacts with the Src homology 2 and/or phosphotyrosine binding domains of Grb7, the only characterized downstream mediator of its function. Taken together, our data identify a series of novel lipid-lipid-based interactions as important regulatory factors for coupling caveolin-1 to the c-Src tyrosine kinase in vivo.

Elevated expression of caveolin-1 in adenocarcinoma of the colon.

Caveolins 1, 2, and 3 are the principal proteins of caveolae, the vesicular invaginations of the plasma membrane. Several reports have suggested that caveolin-1 may have a role in cellular transformation and tumorigenesis. We studied the expression of caveolin-1 and caveolin-2 in normal epithelium, adenoma, and adenocarcinoma of the colon and their possible role in tumorigenesis. Formalin-fixed, paraffin-embedded sections of 41 cases of adenocarcinoma and 13 cases of adenoma of the colon were stained immunohistochemically with anti-caveolin-1 and anti-caveolin-2 antibodies. The expression of caveolin-1 was elevated in the overwhelming majority of the adenocarcinomas, while most normal colonic epithelium and adenomas showed little or no staining. There was significant statistical correlation of the expression of caveolin-1 with adenocarcinoma but not with tumor stage. Expression of caveolin-2 was undetectable in all of the normal colonic glands, adenomas, and carcinomas. We discuss the possible clinical implications of our findings within the context of caveolins and signal transduction.

IFN-alpha and IL-10 induce the differentiation of human type 1 T regulatory cells.

CD4(+) T regulatory type 1 (Tr1) cells suppress Ag-specific immune responses in vitro and in vivo. Although IL-10 is critical for the differentiation of Tr1 cells, the effects of other cytokines on differentiation of naive T cells into Tr1 cells have not been investigated. Here we demonstrate that endogenous or exogenous IL-10 in combination with IFN-alpha, but not TGF-beta, induces naive CD4(+) T cells derived from cord blood to differentiate into Tr1 cells: IL-10(+)IFN-gamma(+)IL-2(-/low)IL-4(-). Naive CD4(+) T cells derived from peripheral blood require both exogenous IL-10 and IFN-alpha for Tr1 cell differentiation. The proliferative responses of the Tr1-containing lymphocyte populations, following activation with anti-CD3 and anti-CD28 mAbs, were reduced. Similarly, cultures containing Tr1 cells displayed reduced responses to alloantigens via a mechanism that was partially mediated by IL-10 and TGF-beta. More importantly, Tr1-containing populations strongly suppressed responses of naive T cells to alloantigens. Collectively, these results show that IFN-alpha strongly enhances IL-10-induced differentiation of functional Tr1 cells, which represents a first major step in establishing specific culture conditions to generate T regulatory cells for biological and biochemical analysis, and for cellular therapy to induce peripheral tolerance in humans.

Influence of caveolin-1 on cellular cholesterol efflux mediated by high-density lipoproteins.

Caveolin-1 is a principal structural component of caveolae membranes. These membrane microdomains participate in the regulation of signaling, transcytosis, and cholesterol homeostasis at the plasma membrane. In the present study, we determined the effect of caveolin-1 expression on cellular cholesterol efflux mediated by high-density lipoprotein (HDL). We evaluated this effect in parental NIH/3T3 cells as well as in two transformed NIH/3T3 cell lines in which caveolin-1 protein levels are dramatically downregulated. Compared with parental NIH/3T3 cells, these two transformed cell lines effluxed cholesterol more rapidly to HDL. In addition, NIH/3T3 cells harboring caveolin-1 antisense also effluxed cholesterol more rapidly to HDL. However, this effect was not due to changes in total cellular cholesterol content. We further showed that chronic HDL exposure reduced caveolin-1 protein expression in NIH/3T3 cells. HDL exposure also inhibited caveolin-1 promoter activity, suggesting a direct negative effect of HDL on caveolin-1 gene transcription. Moreover, we showed that HDL-induced downregulation of caveolin-1 prevents the uptake of oxidized low-density lipoprotein in human endothelial cells. These data suggest a novel proatherogenic role for caveolin-1, i.e., regarding the uptake and/or transcytosis of modified lipoproteins.

Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and t-tubule abnormalities.

Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cells. Recently, we identified a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans that is due to mutations within the coding sequence of the human caveolin-3 gene (3p25). These LGMD-1C mutations lead to an approximately 95% reduction in caveolin-3 protein expression, i.e. a caveolin-3 deficiency. Here, we created a caveolin-3 null (CAV3 -/-) mouse model, using standard homologous recombination techniques, to mimic a caveolin-3 deficiency. We show that these mice lack caveolin-3 protein expression and sarcolemmal caveolae membranes. In addition, analysis of skeletal muscle tissue from these caveolin-3 null mice reveals: (i) mild myopathic changes; (ii) an exclusion of the dystrophin-glycoprotein complex from lipid raft domains; and (iii) abnormalities in the organization of the T-tubule system, with dilated and longitudinally oriented T-tubules. These results have clear mechanistic implications for understanding the pathogenesis of LGMD-1C at a molecular level.

Caveolin-1 expression sensitizes fibroblastic and epithelial cells to apoptotic stimulation.

The potential role of caveolin-1 in apoptosis remains controversial. Here, we investigate whether caveolin-1 expression is proapoptotic or antiapoptotic using a well-defined antisense approach. We show that NIH/3T3 cells harboring antisense caveolin-1 are resistant to staurosporine-induced apoptosis, as assessed using cell morphology, DNA content, caspase 3 activation, and focal adhesion kinase cleavage. Importantly, sensitivity to apoptosis is recovered when caveolin-1 levels are restored. Conversely, recombinant stable expression of caveolin-1 in T24 bladder carcinoma cells sensitizes these cells to caspase 3 activation. Consistent with the observations using NIH/3T3 cells, downregulation of caveolin-1 in T24 cells substantially diminishes caspase 3-like activity. Loss of sensitivity to apoptotic stimulation is recovered by inhibition of the phosphatidylinositol 3-kinase pathway using LY-294002, suggesting a possible mechanism for the sensitizing effect of caveolin-1. Thus our results suggest that caveolin-1 may act as a coupling or sensitizing factor in signaling apoptotic cell death in both fibroblastic (NIH/3T3) and epithelial (T24) cells.