HIV-1 protease mutation 82M contributes to phenotypic resistance to protease inhibitors in subtype G.

OBJECTIVES: The purpose of this study was the qualitative and quantitative assessment of the in vitro effect of HIV-1 protease (PR) mutation 82M on replication capacity and susceptibility to the eight clinically available PR inhibitors (PIs).¦METHODS: The 82M substitution was introduced by site-directed mutagenesis in wild-type subtype B and G strains, as well as reverted back to wild-type in a therapy-failing strain. The recombinant viruses were evaluated for their replication capacity and susceptibility to PIs.¦RESULTS: The single 82M mutation within a wild-type subtype B or G background did not result in drug resistance. However, the in vitro effect of single PR mutations on PI susceptibility is not always distinguishable from wild-type virus, and particular background mutations and polymorphisms are required to detect significant differences in the drug susceptibility profile. Consequently, reverting the 82M mutation back to wild-type (82I) in a subtype G isolate from a patient that failed therapy with multiple other PR mutations did result in significant increases in susceptibility towards indinavir and lopinavir and minor increases in susceptibility towards amprenavir and atazanavir. The presence of the 82M mutation also slightly decreased viral replication, whether it was in the genetic background of subtype B or subtype G.¦CONCLUSIONS: Our results suggest that 82M has an impact on PI susceptibility and that this effect is not due to a compensatory effect on the replication capacity. Because 82M is not observed as a polymorphism in any subtype, these observations support the inclusion of 82M in drug resistance interpretation systems and PI mutation lists.

A novel and divergent role of granzyme a and B in resistance to helminth infection.

Granzyme (gzm) A and B, proteases of NK cells and T killer cells, mediate cell death, but also cleave extracellular matrices, inactivate intracellular pathogens, and induce cytokines. Moreover, macrophages, Th2 cells, regulatory T cells, mast cells, and B cells can express gzms. We recently reported gzm induction in human filarial infection. In this study, we show that in rodent filarial infection with Litomosoides sigmodontis, worm loads were significantly reduced in gzmA×B and gzmB knockout mice during the whole course of infection, but enhanced only early in gzmA knockout compared with wild-type mice. GzmA/B deficiency was associated with a defense-promoting Th2 cytokine and Ab shift, enhanced early inflammatory gene expression, and a trend of reduced alternatively activated macrophage induction, whereas gzmA deficiency was linked with reduced inflammation and a trend toward increased alternatively activated macrophages. This suggests a novel and divergent role for gzms in helminth infection, with gzmA contributing to resistance and gzmB promoting susceptibility.

On metapopulation resistance to drift and extinction.

The spatial configuration of metapopulations (numbers, sizes, and localization of patches) affects their ability to resist demographic extinction and genetic drift, but sometimes with opposite effects. Small and isolated patches, for instance, contribute marginally to demography but may play a large role in genetics by maintaining a sizeable amount of genetic variance among demes. In source-sink systems, similarly, connectivity may be beneficial in terms of effective size, but detrimental in terms of survival, by lowering the reproductive value of source populations. How to reconcile these opposite effects? Here we propose an analytical framework that integrates fixation time (ability to resist genetic drift) and extinction time (ability to resist demographic extinction) into a single index of resistance, measuring the ability of a metapopulation to maintain its demo-genetic integrity. We then illustrate with numerical examples how conflicting demands may be resolved.

A permeable cuticle in Arabidopsis leads to a strong resistance to Botrytis cinerea.

The plant cuticle composed of cutin, a lipid-derived polyester, and cuticular waxes covers the aerial portions of plants and constitutes a hydrophobic extracellular matrix layer that protects plants against environmental stresses. The botrytis-resistant 1 (bre1) mutant of Arabidopsis reveals that a permeable cuticle does not facilitate the entry of fungal pathogens in general, but surprisingly causes an arrest of invasion by Botrytis. BRE1 was identified to be long-chain acyl-CoA synthetase2 (LACS2) that has previously been shown to be involved in cuticle development and was here found to be essential for cutin biosynthesis. bre1/lacs2 has a five-fold reduction in dicarboxylic acids, the typical monomers of Arabidopsis cutin. Comparison of bre1/lacs2 with the mutants lacerata and hothead revealed that an increased permeability of the cuticle facilitates perception of putative elicitors in potato dextrose broth, leading to the presence of antifungal compound(s) at the surface of Arabidopsis plants that confer resistance to Botrytis and Sclerotinia. Arabidopsis plants with a permeable cuticle have thus an altered perception of their environment and change their physiology accordingly.

Résistances thérapeutiques dans les mouvements anormaux [Resistance to treatment in movement disorders].

Therapeutic strategies for essential tremor (ET) and Parkinson's disease (PD) can be divided into two successive steps, one based on oral medications and the other, more invasive, using pumps or functional neurosurgery. When ET becomes refractory to propranolol, primidone and other, second-choice compounds, deep brain stimulation of the VIM nucleus of the thalamus can be considered. When PD becomes resistant to dopamine replacement therapy using various combinations of dopaminergic agents, then three options can be discussed: first, a subcutaneous apomorphine mini-pump, second, a jejunal levodopa-delivery system by means of percutaneous gastrostomy, and third, bilateral deep brain stimulation of the subthalamic nucleus. The above interventions are successful in about 80% of cases.

Resistance to diet-induced obesity and associated metabolic perturbations in haploinsufficient monocarboxylate transporter 1 mice

The monocarboxylate transporter 1 (MCT1 or SLC16A1) is a carrier of short-chain fatty acids, ketone bodies, and lactate in several tissues. Genetically modified C57BL/6J mice were produced by targeted disruption of the mct1 gene in order to understand the role of this transporter in energy homeostasis. Null mutation was embryonically lethal, but MCT1 (+/-) mice developed normally. However, when fed high fat diet (HFD), MCT1 (+/-) mice displayed resistance to development of diet-induced obesity (24.8% lower body weight after 16 weeks of HFD), as well as less insulin resistance and no hepatic steatosis as compared to littermate MCT1 (+/+) mice used as controls. Body composition analysis revealed that reduced weight gain in MCT1 (+/-) mice was due to decreased fat accumulation (50.0% less after 9 months of HFD) notably in liver and white adipose tissue. This phenotype was associated with reduced food intake under HFD (12.3% less over 10 weeks) and decreased intestinal energy absorption (9.6% higher stool energy content). Indirect calorimetry measurements showed ∼ 15% increase in O2 consumption and CO2 production during the resting phase, without any changes in physical activity. Determination of plasma concentrations for various metabolites and hormones did not reveal significant changes in lactate and ketone bodies levels between the two genotypes, but both insulin and leptin levels, which were elevated in MCT1 (+/+) mice when fed HFD, were reduced in MCT1 (+/-) mice under HFD. Interestingly, the enhancement in expression of several genes involved in lipid metabolism in the liver of MCT1 (+/+) mice under high fat diet was prevented in the liver of MCT1 (+/-) mice under the same diet, thus likely contributing to the observed phenotype. These findings uncover the critical role of MCT1 in the regulation of energy balance when animals are exposed to an obesogenic diet.

Theileria parva-transformed T cells show enhanced resistance to Fas/Fas ligand-induced apoptosis.

Lymphocyte homeostasis is regulated by mechanisms that control lymphocyte proliferation and apoptosis. Activation-induced cell death is mediated by the expression of death ligands and receptors, which, when triggered, activate an apoptotic cascade. Bovine T cells transformed by the intracellular parasite Theileria parva proliferate in an uncontrolled manner and undergo clonal expansion. They constitutively express the death receptor Fas and its ligand, FasL but do not undergo apoptosis. Upon elimination of the parasite from the host cell by treatment with a theilericidal drug, cells become increasingly sensitive to Fas/FasL-induced apoptosis. In normal T cells, the sensitivity to death receptor killing is regulated by specific inhibitor proteins. We found that anti-apoptotic proteins such as cellular (c)-FLIP, which functions as a catalytically inactive form of caspase-8, and X-chromosome-linked inhibitor of apoptosis protein (IAP) as well as c-IAP, which can block downstream executioner caspases, are constitutively expressed in T. parva-transformed T cells. Expression of these proteins is rapidly down-regulated upon parasite elimination. Antiapoptotic proteins of the Bcl-2 family such as Bcl-2 and Bcl-x(L) are also expressed but, in contrast to c-FLIP, c-IAP, and X-chromosome-linked IAP, do not appear to be tightly regulated by the presence of the parasite. Finally, we show that, in contrast to the situation in tumor cells, the phosphoinositide 3-kinase/Akt pathway is not essential for c-FLIP expression. Our findings indicate that by inducing the expression of antiapoptotic proteins, T. parva allows the host cell to escape destruction by homeostatic mechanisms that would normally be activated to limit the continuous expansion of a T cell population.

A mutation in the gene encoding the vaccinia virus 37,000-M(r) protein confers resistance to an inhibitor of virus envelopment and release.

Plaque formation in vaccinia virus is inhibited by the compound N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (IMCBH). We have isolated a mutant virus that forms wild-type plaques in the presence of the drug. Comparison of wild-type and mutant virus showed that both viruses produced similar amounts of infectious intracellular naked virus in the presence of the drug. In contrast to the mutant, no extracellular enveloped virus was obtained from IMCBH-treated cells infected with wild-type virus. Marker rescue experiments were used to map the mutation conferring IMCBH resistance to the mutant virus. The map position coincided with that of the gene encoding the viral envelope antigen of M(r) 37,000. Sequence analysis of both wild-type and mutant genes showed a single nucleotide change (G to T) in the mutant gene. In the deduced amino acid sequence, the mutation changes the codon for an acidic Asp residue in the wild-type gene to one for a polar noncharged Tyr residue in the mutant.

Matrix metalloproteinase 9 (MMP-9/gelatinase B) proteolytically cleaves ICAM-1 and participates in tumor cell resistance to natural killer cell-mediated cytotoxicity.

Shedding of intercellular adhesion molecule 1 (ICAM-1) is believed to play a role in tumor cell resistance to cell-mediated cytotoxicity. However, the mechanism whereby ICAM-1 is shed from the surface of tumor cells remains unclear. In this study, we have addressed the possibility that matrix metalloproteinases are implicated in ICAM-1 shedding. Our observations suggest a functional relationship between ICAM-1 and matrix metalloproteinase 9 (MMP-9) whereby ICAM-1 provides a cell surface docking mechanism for proMMP-9, which, upon activation, proteolytically cleaves the extracellular domain of ICAM-1 leading to its release from the cell surface. MMP-9-dependent shedding of ICAM-1 is found to augment tumor cell resistance to natural killer (NK) cell-mediated cytotoxicity. Taken together, our observations propose a mechanism for ICAM-1 shedding from the cell surface and provide support for MMP involvement in tumor cell evasion of immune surveillance.

Lentivirus-mediated Bcl-2 expression in betaTC-tet cells improves resistance to hypoxia and cytokine-induced apoptosis while preserving in vitro and in vivo control of insulin secretion.

betaTC-tet cells are conditionally immortalized pancreatic beta cells which can confer long-term correction of hyperglycemia when transplanted in syngeneic streptozocin diabetic mice. The use of these cells for control of type I diabetes in humans will require their encapsulation and transplantation in non-native sites where relative hypoxia and cytokines may threaten their survival. In this study we genetically engineered betaTC-tet cells with the anti-apoptotic gene Bcl-2 using new lentiviral vectors and showed that it protected this cell line against apoptosis induced by hypoxia, staurosporine and a mixture of cytokines (IL-1beta, IFN-gamma and TNF-alpha). We further demonstrated that Bcl-2 expression permitted growth at higher cell density and with shorter doubling time. Expression of Bcl-2, however, did not inter- fere either with the intrinsic mechanism of growth arrest present in the betaTC-tet cells or with their normal glucose dose-dependent insulin secretory activity. Furthermore, Bcl-2 expressing betaTC-tet cells retained their capacity to secrete insulin under mild hypoxia. Finally, transplantation of these cells under the kidney capsule of streptozocin diabetic C3H mice corrected hyperglycemia for several months. These results demonstrate that the murine betaTC-tet cell line can be genetically modified to improve its resistance against different stress-induced apoptosis while preserving its normal physiological function. These modified cells represent an improved source for cell transplantation therapy of type I diabetes.

Expression of the NH(2)-terminal fragment of RasGAP in pancreatic beta-cells increases their resistance to stresses and protects mice from diabetes.

OBJECTIVE: Our laboratory has previously established in vitro that a caspase-generated RasGAP NH(2)-terminal moiety, called fragment N, potently protects cells, including insulinomas, from apoptotic stress. We aimed to determine whether fragment N can increase the resistance of pancreatic beta-cells in a physiological setting. RESEARCH DESIGN AND METHODS: A mouse line, called rat insulin promoter (RIP)-N, was generated that bears a transgene containing the rat insulin promoter followed by the cDNA-encoding fragment N. The histology, functionality, and resistance to stress of RIP-N islets were then assessed. RESULTS: Pancreatic beta-cells of RIP-N mice express fragment N, activate Akt, and block nuclear factor kappaB activity without affecting islet cell proliferation or the morphology and cellular composition of islets. Intraperitoneal glucose tolerance tests revealed that RIP-N mice control their glycemia similarly as wild-type mice throughout their lifespan. Moreover, islets isolated from RIP-N mice showed normal glucose-induced insulin secretory capacities. They, however, displayed increased resistance to apoptosis induced by a series of stresses including inflammatory cytokines, fatty acids, and hyperglycemia. RIP-N mice were also protected from multiple low-dose streptozotocin-induced diabetes, and this was associated with reduced in vivo beta-cell apoptosis. CONCLUSIONS: Fragment N efficiently increases the overall resistance of beta-cells to noxious stimuli without interfering with the physiological functions of the cells. Fragment N and the pathway it regulates represent, therefore, a potential target for the development of antidiabetes tools.

In vivo over-expression of interleukin-10 increases resistance to focal brain ischemia in mice.

Early studies showed that the administration of the anti-inflammatory cytokine interleukin-10 (IL10) protects against permanent middle cerebral artery occlusion (MCAO) in mice. In this study, transgenic mice expressing murine IL10 (IL10T) directed by the major histocompatibility complex Ea promoter were produced and used to explore the effect of chronically increased IL10 levels on MCAO-related molecular mechanisms. IL10 was over-expressed in astrocytes, microglia, and endothelial brain cells in IL10T compared with wild type mice. Four days following MCAO, IL10T mice showed a 40% reduction in infarct size which was associated to significantly reduced levels of active caspase 3 compared with wild type mice. Under basal conditions, anti-inflammatory factors such as nerve growth factor and GSH were up-regulated and the pro-inflammatory cytokine IL1beta was down-regulated in the brain of IL10T animals. In addition, these mice displayed increased basal GSH levels in microglial and endothelial cells as well as a marked increase in manganese superoxide dismutase in endothelial lining blood vessels. Following ischemia, IL10T mice showed a marked reduction in pro-inflammatory cytokines, including tumor necrosis factor-alpha, interferon-gamma, and IL1beta. Our data indicate that constitutive IL10 over-expression is associated with a striking resistance to cerebral ischemia that may be attributed to changes in the basal redox properties of glial/endothelial cells.