Generation of antibodies to an extracellular region of the transporters Glut1/Glut4 by immunization with a designed antigen.

Monoclonal antibodies are one of the fastest growing class of drugs. Nevertheless, relatively few biologics target multispanning membrane proteins because of technical challenges. To target relatively small extracellular regions of multiple membrane-spanning proteins, synthetic peptides, which are composed of amino acids corresponding to an extracellular region of a membrane protein, are often utilized in antibody discovery. However, antibodies to these peptides often do not recognize parental membrane proteins. In this study, we designed fusion proteins in which an extracellular helix of the membrane protein glucose transporter 1 (Glut1) was grafted onto the scaffold protein Adhiron. In the initial design, the grafted fragment did not form a helical conformation. Molecular dynamics simulations of full-length Glut1 suggested the importance of intramolecular interactions formed by surrounding residues in the formation of the helical conformation. A fusion protein designed to maintain such intramolecular interactions did form the desired helical conformation in the grafted region. We then immunized an alpaca with the designed fusion protein and obtained VHH (variable region of heavy-chain antibodies) using the phage display method. The binding of these VHH antibodies to the recombinant Glut1 protein was evaluated by surface plasmon resonance, and their binding to Glut1 on the cell membrane was further validated by flow cytometry. Furthermore, we also succeeded in the generation of a VHH against another integral membrane protein, glucose transporter 4 (Glut4) with the same strategy. These illustrates that our combined biochemical and computational approach can be applied to designing other novel fusion proteins for generating site-specific antibodies.

TNF induces glycolytic shift in fibroblast like synoviocytes via GLUT1 and HIF1A.

TNF is a central cytokine in the pathogenesis of rheumatoid arthritis (RA). Elevated level of TNF causes local inflammation that affects immune cells and fibroblast-like synoviocytes (FLS). Nowadays, only 20-30% of patients experience remission after the standard of care therapy-antibodies against TNF. Interestingly, responders show reduced levels of GLUT1 and GAPDH, highlighting a potential link to cellular metabolism. The aim of the study was to investigate whether TNF directly affects the metabolic phenotype of FLS. Real-time respirometry displayed TNF-induced upregulation of glycolysis along with a modest increase of oxidative phosphorylation in FLS from healthy donors. In addition, TNF stimulation enhanced HIF1A and GLUT1 expression. The upregulation of HIF1A and GLUT1 reflects their enriched level in FLS from RA patients (RA-FLS). The inhibition of TAK1, HIF1a and hexokinase deciphered the importance of TNF/TAK1/HIF1A/glycolysis signaling axis. To prove that inhibition of glycolysis reduced the pathogenic phenotype, we showed that 2-deoxyglucose, a hexokinase inhibitor, partially decreased secretion of RA biomarkers. In summary, we identified a direct role of TNF on glycolytic reprogramming of FLS and confirmed the potency of immunometabolism for RA. Further studies are needed to evaluate the therapeutic impact especially regarding non-responder data.

Role of breast cancer-derived exosomes in metabolism of immune cells through PD1-GLUT1-HK2 metabolic axis.

Tumor cells modulate immune responses by secreting exosomes. Tumor exosomes can affect the metabolism of immune cells and increase immune inhibitory molecules such as programmed cell death protein 1 (PD-1). PD-1 inhibits the glycolysis pathway in immune cells. We investigated the role of tumor exosomes in how metabolic changes occur through the PD1-GLUT1-HK2 metabolic axisin peripheral blood mononuclear cells (PBMCs). The MDA-MB-231 cell line was cultured, serum samples from breast cancer patients were collected, and exosomes purified from serum samples and the MDA-MB-231 cell line. PBMCs were treated with purified exosomes for 72 h and, the expression of PD1-GLUT1-HK2 genes was measured by real-time PCR. Our study results showed relative expression of the HK2 gene in both groups treated with MDA-MB-231 cell line exosomes and serum exosomes of breast cancer patients was significantly increased compared to the control group (p < 0.0001). Also, the relative expression of the PD1 gene and GLUT1 gene showed a significant increase compared to the control group only in the group treated with MDA-MB-231 cell line exosomes (p < 0.0001). Therefore, Breast cancer exosomes increased the expression of key genes in the glycolysis pathway, increasing the glycolysis pathway in PBMCs. Increased expression of PD-1 could not prevent the expression of critical genes in the glycolysis pathway as in previous studies.

Pharmacological inhibition of GLUT1 as a new immunotherapeutic approach after myocardial infarction.

Myocardial infarction (MI) is one of the major contributors to cardiovascular morbidity and mortality. Excess inflammation significantly contributes to cardiac remodeling and heart failure after MI. Accumulating evidence has shown the central role of cellular metabolism in regulating the differentiation and function of cells. Metabolic rewiring is particularly relevant for proinflammatory responses induced by ischemia. Hypoxia reduces mitochondrial oxidative phosphorylation (OXPHOS) and induces increased reliance on glycolysis. Moreover, activation of a proinflammatory transcriptional program is associated with preferential glucose metabolism in leukocytes. An improved understanding of the mechanisms that regulate metabolic adaptations holds the potential to identify new metabolic targets and strategies to reduce ischemic cardiac damage, attenuate excess local inflammation and ultimately prevent the development of heart failure. Among possible drug targets, glucose transporter 1 (GLUT1) gained considerable interest considering its pivotal role in regulating glucose availability in activated leukocytes and the availability of small molecules that selectively inhibit it. Therefore, we summarize current evidence on the role of GLUT1 in leukocytes (focusing on macrophages and T cells) and non-leukocytes, including cardiomyocytes, endothelial cells and fibroblasts regarding ischemic heart disease. Beyond myocardial infarction, we can foresee the role of GLUT1 blockers as a possible pharmacological approach to limit pathogenic inflammation in other conditions driven by excess sterile inflammation.

Gout and pseudo-gout-related crystals promote GLUT1-mediated glycolysis that governs NLRP3 and interleukin-1ß activation on macrophages.

OBJECTIVE: Macrophage activation by monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals mediates an interleukin (IL)-1ß-dependent inflammation during gout and pseudo-gout flare, respectively. Since metabolic reprogramming of macrophages goes along with inflammatory responses dependently on stimuli and tissue environment, we aimed to decipher the role of glycolysis and oxidative phosphorylation in the IL-1ß-induced microcrystal response. METHODS: Briefly, an in vitro study (metabolomics and real-time extracellular flux analysis) on MSU and CPP crystal-stimulated macrophages was performed to demonstrate the metabolic phenotype of macrophages. Then, the role of aerobic glycolysis in IL-1ß production was evaluated, as well in vitro as in vivo using 18F-fluorodeoxyglucose positron emission tomography imaging and glucose uptake assay, and molecular approach of glucose transporter 1 (GLUT1) inhibition. RESULTS: We observed that MSU and CPP crystals led to a metabolic rewiring toward the aerobic glycolysis pathway explained by an increase in GLUT1 plasma membrane expression and glucose uptake on macrophages. Also, neutrophils isolated from human synovial fluid during gout flare expressed GLUT1 at their plasma membrane more frequently than neutrophils isolated from bloodstream. Both glucose deprivation and treatment with either 2-deoxyglucose or GLUT1 inhibitor suppressed crystal-induced NLRP3 activation and IL-1ß production, and microcrystal inflammation in vivo. CONCLUSION: In conclusion, we demonstrated that GLUT1-mediated glucose uptake is instrumental during the inflammatory IL-1ß response induced by MSU and CPP crystals. These findings open new therapeutic paths to modulate crystal-related inflammation.

Diagnostic Value of Glucose Transporter 1 (GLUT-1) Expression in Nested Variant of Urothelial Carcinoma.

OBJECTIVE: Nested variant is bland-looking but aggressive subtype of urothelial carcinoma (UC). Cases having significant muscle invasion do not cause problems but small and superficial biopsies may be challenging due to morphological similarities between nested variant urothelial carcinoma and benign urothelial lesions. MATERIAL AND METHOD: We studied Glucose transporter 1 (GLUT-1), which is an integral membrane protein providing glucose pass through plasma membrane down its concentration gradient, to see if it is useful for the differential diagnosis. Twenty five cases of nested variant urothelial carcinoma and a control group consisting of 12 cases of cystitis glandularis, cystitis cystica and 4 cases of inverted papilloma were stained with GLUT-1 immunohistochemically. Membranous staining was scored on a scale of 0 to +3. RESULTS: Eleven of 25 nested variant UC cases showed a score of 2 and 14 of them showed a score of 3 on immunostaining with GLUT-1. Two cases showed a score of 1 and 10 cases did not show any staining in the control group. CONCLUSION: Our results showed that GLUT-1 may be a helpful marker when morphological separation cannot be made between nested variant UC and benign urothelial leisons. We also think that anti-GLUT-1 antibody treatment may be an option in the targeted treatment of nested variant.

Polymorphism rs1385129 Within Glut1 Gene SLC2A1 Is Linked to Poor CD4+ T Cell Recovery in Antiretroviral-Treated HIV+ Individuals.

Untreated HIV infection is associated with progressive CD4+ T cell depletion, which is generally recovered with combination antiretroviral therapy (cART). However, a significant proportion of cART-treated individuals have poor CD4+ T cell reconstitution. We investigated associations between HIV disease progression and CD4+ T cell glucose transporter-1 (Glut1) expression. We also investigated the association between these variables and specific single nucleotide polymorphisms (SNPs) within the Glut1 regulatory gene AKT (rs1130214, rs2494732, rs1130233, and rs3730358) and in the Glut1-expressing gene SLC2A1 (rs1385129 and rs841853) and antisense RNA 1 region SLC2A1-AS1 (rs710218). High CD4+Glut1+ T cell percentage is associated with rapid CD4+ T cell decline in HIV-positive treatment-naïve individuals and poor T cell recovery in HIV-positive individuals on cART. Evidence suggests that poor CD4+ T cell recovery in treated HIV-positive individuals is linked to the homozygous genotype (GG) associated with SLC2A1 SNP rs1385129 when compared to those with a recessive allele (GA/AA) (odds ratio = 4.67; P = 0.04). Furthermore, poor response to therapy is less likely among Australian participants when compared against American participants (odds ratio: 0.12; P = 0.01) despite there being no difference in prevalence of a specific genotype for any of the SNPs analyzed between nationalities. Finally, CD4+Glut1+ T cell percentage is elevated among those with a homozygous dominant genotype for SNPs rs1385129 (GG) and rs710218 (AA) when compared to those with a recessive allele (GA/AA and AT/TT respectively) (P < 0.04). The heterozygous genotype associated with AKT SNP 1130214 (GT) had a higher CD4+Glut1+ T cell percentage when compared to the dominant homozygous genotype (GG) (P = 0.0068). The frequency of circulating CD4+Glut1+ T cells and the rs1385129 SLC2A1 SNP may predict the rate of HIV disease progression and CD4+ T cell recovery in untreated and treated infection, respectively.

Metabolically active CD4+ T cells expressing Glut1 and OX40 preferentially harbor HIV during in vitro infection.

High glucose transporter 1 (Glut1) surface expression is associated with increased glycolytic activity in activated CD4+ T cells. Phosphatidylinositide 3-kinases (PI3K) activation measured by p-Akt and OX40 is elevated in CD4+Glut1+ T cells from HIV+ subjects. TCR engagement of CD4+Glut1+ T cells from HIV+ subjects demonstrates hyperresponsive PI3K-mammalian target of rapamycin signaling. High basal Glut1 and OX40 on CD4+ T cells from combination antiretroviral therapy (cART)-treated HIV+ patients represent a sufficiently metabolically active state permissive for HIV infection in vitro without external stimuli. The majority of CD4+OX40+ T cells express Glut1, thus OX40 rather than Glut1 itself may facilitate HIV infection. Furthermore, infection of CD4+ T cells is limited by p110γ PI3K inhibition. Modulating glucose metabolism may limit cellular activation and prevent residual HIV replication in 'virologically suppressed' cART-treated HIV+ persons.

Immunohistochemical and histochemical characterization of intraosseous arteriovenous malformations of the jaws: analysis of 16 cases with emphasis on GLUT-1 immunophenotype.

OBJECTIVES: Intraosseous vascular lesions of the craniofacial region are rare and may cause diagnostic and therapeutic challenges. The purpose of this study was to characterize 16 cases of intraosseous arteriovenous malformations (AVMs) affecting the jaws. STUDY DESIGN: Immunohistochemical evaluation was performed using antibodies against α-smooth muscle actin (α-SMA), desmin, CD31, D2-40, and glucose transporter 1 (GLUT-1). Staining with elastic Verhoeff-Van Gieson and Masson trichrome histochemical stains was also performed. RESULTS: No gender predilection (female:male ratio = 1:1) was observed, with patients' mean age being 50.8 years (SD of ±13.9; range 28-71 years). Predilection for the mandible was observed (12 of 16 [75%]). Immunohistochemically, diffuse endothelial CD31 staining was noted, and α-SMA strongly highlighted smooth muscle cells and pericytes. Desmin-positive vessels were identified in 9 of 16 AVMs (56.3%). D2-40 was uniformly negative in all specimens. AVMs were negative for GLUT-1 (11 of 14 [78.6%]) except for 2 cases (2 of 14 [14.3%]) exhibiting focal limited cytoplasmic GLUT-1 immunoreactivity. One case was equivocal for GLUT-1. Masson trichrome highlighted smooth muscle cells, and elastic fibers were identified in thick-walled arteries. CONCLUSIONS: AVMs of the jaws generally lack expression of GLUT-1, similar to soft tissue vascular malformations. Clinicoradiographic features of intraosseous AVMs in the present study were consistent with the findings of previous studies, although mean age was higher.

Antibody to human α-fetoprotein inhibits cell growth of human hepatocellular carcinoma cells by resuscitating the PTEN molecule: in vitro experiments.

It has been proposed that α-fetoprotein (AFP) is a new member of the intracellular signaling molecule family of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway via interaction with the phosphatase and tensin homolog (PTEN). In this study, the effects of anti-human AFP antibody on the functions of PTEN were examined using an AFP-producing human hepatoma cell line. The antibody caused significant inhibition of cell growth, compared to a normal IgG control, with the accumulation of intracellular immune complexes followed by significant reduction of cytosolic functional AFP. Decrease in the amount of AKT phosphorylated on serine (S) 473 indicated that PI3K/AKT signaling was suppressed in the cells. S380-phosphorylated PTEN increased markedly by the second day after antibody treatment, with slight but significant increase in the PTEN protein level. Since phosphorylation at S380 is critical for PTEN stability, the increase in S380-phosphorylated PTEN indicated maintenance of the number of PTEN molecules and the related potential to control PI3K/AKT signaling. p53 protein (P53) significantly, but slightly increased during antibody treatment, because PTEN expression increased the stability and function of P53 via both molecular interactions. P53 phosphorylated at S20 or at S392 dramatically increased, suggesting an increase in the stability, accumulation and activation of P53. Glucose transporter 1 (GLUT1) increased immediately after antibody treatment, pointing to a deficiency of glucose in the cells. Immunofluorescence cytology revealed that antibody-treatment re-distributed GLUT1 molecules throughout the cytoplasm with a reduction of their patchy localization on the cell surface. This suggested that translocation of GLUT1 depends on the PI3K/AKT pathway, in particular on PTEN expression. Antibody therapy targeted at AFP-producing tumor cells showed an inhibitory effect on the PI3K/AKT pathway via the liberation, restoration and functional stabilization of PTEN. PTEN simultaneously induced both P53 activation and intracellular translocation of GLUT1, since these are closely associated with PTEN.

Use of Anti-Noxa Antibody for Differential Diagnosis between Epithelioid Mesothelioma and Reactive Mesothelial Hyperplasia.

OBJECTIVES: The histological differential diagnosis between epithelioid mesothelioma (EM) and reactive mesothelial hyperplasia (RMH) is not always straightforward. The aim of the present study was to search for new immunohistochemical markers to distinguish EM from RMH. METHODS: We evaluated and compared the expression of apoptosis-related genes in EM and RMH by real-time RT-PCR array analysis followed by clustering of significant gene expression. Immunohistochemical staining and statistical analysis of Noxa expression in 81 cases of EM and 55 cases of RMH were performed and compared with the utility of other previously reported antibodies such as Desmin, EMA, GLUT-1, IMP-3 and CD146. RESULTS: Noxa mRNA expression levels were found to be increased in EM when compared to RMH by RT-PCR array analysis. In the immunohistochemical analysis, Noxa showed sensitivity of 69.0%, specificity of 93.6% and positive predictive value of 93.0% as a positive marker of EM in distinguishing it from RMH, and these values were almost similar to IMP-3. CONCLUSION: Noxa is a marker with relatively high specificity, and can be used to distinguish EM from RMH. It would be a valuable addition to the current antibody panel used for the differential diagnosis of EM and RMH.

Metabolic mysteries of the inflammatory response: T cell polarization and plasticity.

While simultaneously maintaining homeostasis and reducing further harm to the host, the immune system is equipped to eliminate both tumors and pathogenic microorganisms. Bifurcated into cell-mediated and humoral immunity, the adaptive immune system requires a series of complex and coordinated signals to drive the proliferation and differentiation of appropriate subsets. These include signals that modulate cellular metabolism. When first published in the 1920s, "the Warburg effect" was used to describe a phenomenon in which most cancer cells relied on aerobic glycolysis to meet their biosynthetic demands. Despite the early observations of Warburg and his colleagues, targeting cancer cell metabolism for therapeutic purposes still remains theoretical. Notably, many T cells exhibit the same Warburg metabolism as cancer cells and the therapeutic benefit of targeting their metabolic pathways has since been reexamined. Emerging evidence suggests that specific metabolic alterations associated with T cells may be ancillary to their subset differentiation and influential in their inflammatory response. Thus, T cell lymphocyte activation leads to skewing in metabolic plasticity, and issue that will be the subject of this review.

Foxp3-mediated inhibition of Akt inhibits Glut1 (glucose transporter 1) expression in human T regulatory cells.

CD4(+)CD25(+)Foxp3(+) Tregs have a diminished capacity to activate the PI3K/Akt pathway. Although blunted Akt activity is necessary to maintain Treg function, the consequences of this altered signaling are unclear. Glut1 is a cell-surface receptor responsible for facilitating glucose transport across plasma membranes, whose expression is tightly coupled to costimulatory signals and Akt phosphorylation. Freshly isolated human Tregs were unable to up-regulate Glut1 in response to TCR and costimulatory signals compared with Tconv. Consequently, the ability of Tregs to use glucose was also reduced. Introduction of Foxp3 into Tconv inhibited Akt activation and Glut1 expression, indicating that Foxp3 can regulate Glut1. Finally, pharmacologic activation of Akt in Tregs can induce Glut1, overcoming the effects of Foxp3. Together, these results illustrate the molecular basis behind differential glucose metabolism in Tregs.

Metabolic requirements for neutrophil extracellular traps formation.

As part of the innate immune response, neutrophils are at the forefront of defence against infection, resolution of inflammation and wound healing. They are the most abundant leucocytes in the peripheral blood, have a short lifespan and an estimated turnover of 10(10) to 10(11) cells per day. Neutrophils efficiently clear microbial infections by phagocytosis and by oxygen-dependent and oxygen-independent mechanisms. In 2004, a new neutrophil anti-microbial mechanism was described, the release of neutrophil extracellular traps (NETs) composed of DNA, histones and anti-microbial peptides. Several microorganisms, bacterial products, as well as pharmacological stimuli such as PMA, were shown to induce NETs. Neutrophils contain relatively few mitochondria, and derive most of their energy from glycolysis. In this scenario we aimed to analyse some of the metabolic requirements for NET formation. Here it is shown that NETs formation is strictly dependent on glucose and to a lesser extent on glutamine, that Glut-1, glucose uptake, and glycolysis rate increase upon PMA stimulation, and that NET formation is inhibited by the glycolysis inhibitor, 2-deoxy-glucose, and to a lesser extent by the ATP synthase inhibitor oligomycin. Moreover, when neutrophils were exposed to PMA in glucose-free medium for 3 hr, they lost their characteristic polymorphic nuclei but did not release NETs. However, if glucose (but not pyruvate) was added at this time, NET release took place within minutes, suggesting that NET formation could be metabolically divided into two phases; the first, independent from exogenous glucose (chromatin decondensation) and, the second (NET release), strictly dependent on exogenous glucose and glycolysis.

How useful is GLUT-1 in differentiating mesothelial hyperplasia and fibrosing pleuritis from epithelioid and sarcomatoid mesotheliomas? An international collaborative study.

OBJECTIVE: Mesothelial hyperplasia (MH) and fibrosing pleuritis (FP) can be difficult to distinguish from epithelioid (MM-E) and sarcomatoid (MM-S) malignant pleural mesotheliomas. GLUT-1 has shown variable results regarding its sensitivity and specificity when used to evaluate mesothelial proliferations. We evaluated the utility of GLUT-1 immunostaining in differentiating MH and FP from MM-E and MM-S. MATERIALS AND METHODS: In this retrospective study, diagnostically well-characterized cases (MH=31, FP=29, MM-E=41, MM-S=29) were collected and manually stained for GLUT-1. All slides were visually scored by 2 pathologists; using the following system: 0%, 1+ 1-25%, 2+ 26-50% and 3+ >51% cells staining. RESULTS: All benign cases (n=60) were negative for GLUT-1 while 45 of 78 (58%) MM [21 of 41 (50%) MM-E, 21 of 29 (72%) MM-S and 3 of 3 biphasic mesothelioma (100%)] had 1+ to 3+ staining. Of the MM-E, 10 had 1+, and 11 had 2+ staining; of the MM-S 3 had 1+, 15 had 2+ and 3 had 3+ staining. Both sarcomatoid and epithelioid components of the 3 biphasic mesotheliomas revealed 1+ staining. All 5 desmoplastic MM were negative. CONCLUSIONS: Positive staining with GLUT-1 is helpful since it is present in half of MM-E and three-quarter of MM-S. Although all reactive mesothelial lesions were negative, the absence of immunoreactivity does not exclude the diagnosis of MM. As with all IHC stains used for diagnostic purposes, GLUT-1 has to be a part of a panel, and the results interpreted in the context of clinical, radiological and histological findings.

Molecular histopathology using gold nanorods and optical coherence tomography.

PURPOSE: To examine the novel application of a commercially available optical coherence tomography (OCT) system toward molecular histopathology using gold nanorod (GNR) linked antibodies as a functionalized contrast agent to evaluate ocular surface squamous neoplasia (OSSN). METHODS: GNRs were synthesized and covalently attached to anti-glucose transporter-1 (GLUT-1) antibodies via carbodiimide chemistry. Three specimens from each of three distinct categories of human conjunctival tissue were selected for analysis, including conjunctiva without epithelial atypia (controls); conjunctival intraepithelial neoplasia, carcinoma in situ (CIS); and conjunctival squamous cell carcinoma (SCC). Tissue sections were incubated initially with GNR tagged anti-GLUT-1 antibodies and then with a fluorescent-tagged secondary antibody. Immunofluorescence and OCT imaging of the tissue was performed and the results were correlated to the light microscopic findings on traditional hemotoxyin and eosin stained sections. RESULTS: No binding of the functionalized GNRs was observed within the epithelium of three normal conjunctiva controls. While immunofluorescence disclosed variable binding of the functionalized GNRs to atypical epithelial cells in all six cases of OSSN, the enhancement of the OCT signal in three cases of CIS was insufficient to distinguish these specimens from normal controls. In two of three cases of SCC, binding of functionalized GNRs was sufficient to produce an increased scattering effect on OCT in areas correlating to atypical epithelial cells which stained intensely on immunofluorescence imaging. Binding of functionalized GNRs was sufficient to produce an increased scattering effect on OCT in areas correlating to regions of erythrocytes and hemorrhage which stained intensely on immunofluorescence imaging within all nine tested samples. CONCLUSIONS: We have demonstrated the use of OCT for molecular histopathology using functionalized gold nanorods in the setting of OSSN. Our results suggest a threshold concentration of functionalized GNRs within tissue is required to achieve a detectable enhancement in scattering of the OCT signal.

Comparison of conventional and novel PET tracers for imaging mesothelioma in nude mice with subcutaneous and intrapleural xenografts.

INTRODUCTION: Malignant mesothelioma is a highly aggressive tumor originating in the pleura, peritoneum and pericardium, and the prognosis of patients undergoing current treatment remains poor. To develop new therapies, it is important to have a noninvasive imaging system for evaluating the efficacy of such prospective treatments. We have established clinically relevant mouse models and evaluated conventional and novel positron emission tomography (PET) tracers. METHODS: Epithelioid and sarcomatoid mesothelioma cells were inoculated subcutaneously and intrapleurally into nude mice. Biodistribution and PET imaging studies were conducted by injecting [(18)F]fluoro-2-deoxy-D-glucose (FDG), 3'-[(18)F]fluoro-3'-doxythymidine (FLT) or 4'-methyl-[(11)C]thiothymidine (S-dThd) into the mouse models. In vitro cellular uptake of [(14)C]FDG and [(3)H]FLT and thymidine kinase 1 (TK(1)) activity in both cell lines were measured. Expression of glucose transporter 1 (GLUT-1) and Ki-67 in xenografted tumors was evaluated by immunohistochemical staining. RESULTS: In epithelioid mesothelioma models, biodistribution experiments showed that tumor uptake of [(11)C]S-dThd was significantly higher than that of [(18)F]FDG. On the other hand, in sarcomatoid models, [(18)F]FDG showed significantly higher accumulation than the other two tracers. These differential uptakes of the three tracers were confirmed by PET imaging. The cellular uptake of [(14)C]FDG and [(3)H]FLT and TK(1) activity in sarcomatoid cells were higher than those of epithelioid cells. GLUT-1 protein was strongly expressed in sarcomatoid but not in epithelioid tumor. We observed a high percentage of Ki-67-positive cells in both epithelioid and sarcomatoid tumors. CONCLUSIONS: We established nude mouse models of epithelioid and sarcomatoid subtypes of mesothelioma. PET tracers applicable for the evaluation of epithelioid and sarcomatoid mesothelioma would vary: [(18)F]FLT and [(11)C]S-dThd seemed suitable for the epithelioid subtype and [(18)F]FDG seemed suitable for the sarcomatoid subtype in our mouse models. Our results indicated that cellular uptake and TK(1) activity in vitro are not always consistent with tracer uptake of [(18)F]FLT and [(11)C]S-dThd in vivo. These mouse models and PET imaging might be useful tools for evaluating new and effective treatments in mesothelioma.

Abortive or minimal-growth hemangiomas: Immunohistochemical evidence that they represent true infantile hemangiomas.

BACKGROUND: Infantile hemangiomas have a characteristic natural history of rapid proliferation in the first weeks of life followed by spontaneous involution. At birth, they may be present as a precursor lesion. Sometimes one may see precursor lesions that never undergo a growth phase or that undergo minimal growth. It is unclear the exact nature of these precursor-like lesions. OBJECTIVE: We sought to describe the morphology and histopathology of these precursor-like lesions. METHODS: We describe 4 patients with macules resembling precursor lesions of hemangiomas that did not show proliferation phase or minimal growth. The histopathologic and immunohistochemical study with glucose transporter-1 was performed in all of these cases. RESULTS: The skin biopsy specimen showed superficial ectatic vessels that reacted with anti-glucose transporter-1 antibodies. All skin biopsy specimens exhibited capillary lobules in papillary dermis and, in two of them, in the reticular dermis and subcutis. LIMITATIONS: This text is limited by the number of cases reported. CONCLUSIONS: Precursor lesions of hemangioma that do not show proliferation phase or minimal growth represent, in the view of glucose transporter-1 immunoreactivity, true hemangiomas of infancy with an aborted or arrested growth cycle.

Glut-1 antibodies induce growth arrest and apoptosis in human cancer cell lines.

Glucose transporters (Gluts) facilitate glucose uptake and tumors frequently over express the Gluts, especially Glut-1. Breast cancer and lung cancer (NSCLC) cell lines were incubated with anti-Glut-1 antibodies alone or with cisplatin, paclitaxel or gefitinib. Inhibition of proliferation and apoptosis was assessed. Antibodies to Glut-1 inhibited proliferation by 50% and 75% in the tested NSCLC and breast cancer cell lines, respectively. They also potentiate the anti-proliferative effects of cisplatin, paclitaxel and gefitinib. Our results indicate that anti-Glut-1 antibodies inhibit proliferation and induce apoptosis in the evaluated cell lines and provide preliminary evidence of their anti-tumor activity.

GLUT-1-independent infection of the glioblastoma/astroglioma U87 cells by the human T cell leukemia virus type 1.

The human glucose transporter protein 1 (GLUT-1) functions as a receptor for human T cell leukemia virus (HTLV). GLUT-1 is a twelve-transmembrane cell surface receptor with six extracellular (ECL) and seven intracellular domains. To analyze HTLV-1 cytotropism, we utilized polyclonal antibodies to a synthetic peptide corresponding to the large extracellular domain of GLUT-1. The antibodies caused significant blocking of envelope (Env)-mediated fusion and pseudotyped virus infection of HeLa cells but had no significant effect on infection of U87 cells. This differential effect correlated with the detection of high-level surface expression of GLUT-1 on HeLa cells and very weak staining of U87 cells. To investigate this in terms of viral cytotropism, we cloned GLUT-1 cDNA from U87 cells and isolated two different versions of cDNA clones: the wild-type sequence (encoding 492 residues) and a mutant cDNA with a 5-base pair deletion (GLUT-1Delta5) between nucleotides 1329 and 1333. The deletion, also detected in genomic DNA, resulted in a frame-shift and premature termination producing a truncated protein of 463 residues. Transfection of the wild-type GLUT-1 but not GLUT-1Delta5 cDNA into CHO cells resulted in efficient surface expression of the human GLUT-1. Co-expression of GLUT-1 with GLUT-1Delta5 produces a trans-inhibition by GLUT-1Delta5 of GLUT-1-mediated HTLV-1 envelope (Env)-mediated fusion. Co-immunoprecipitation experiments demonstrated physical interaction of the wild-type and mutant proteins. Northern blot and RT-PCR analyses demonstrated lower GLUT-1 RNA expression in U87 cells. We propose two mechanisms to account for the impaired cell surface expression of GLUT-1 on U87 cells: low GLUT-1 RNA expression and the formation of GLUT-1/GLUT-1Delta5 heterodimers that are retained intracellularly. Significant RNAi-mediated reduction of endogenous GLUT-1 expression impaired HTLV-1 Env-mediated fusion with HeLa cells but not with U87 cells. We propose a GLUT-1-independent mechanism of HTLV-1 infection of U87 cells. The results may have important implications for HTLV-1 neurotropism and pathogenesis.