Jonathan C. Howard

A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics.

Novel chemotherapeutics with marked and selective antitumor activity are essential to develop, particularly those that can overcome resistance to established therapies. Iron (Fe) is critical for cell-cycle progression and DNA synthesis and potentially represents a novel molecular target for the design of new anticancer agents. The aim of this study was to evaluate the antitumor activity and Fe chelation efficacy of a new class of Fe chelators using human tumors. In this investigation, the ligands showed broad antitumor activity and could overcome resistance to established antitumor agents. The in vivo efficacy of the most effective chelator identified, di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT), was assessed by using a panel of human xenografts in nude mice. After 7 weeks, net growth of a melanoma xenograft in Dp44mT-treated mice was only 8% of that in mice treated with vehicle. In addition, no differences in these latter animals were found in hematological indices between Dp44mT-treated mice and controls. No marked systemic Fe depletion was observed comparing Dp44mT- and vehicle-treated mice, probably because of the very low doses required to induce anticancer activity. Dp44mT caused up-regulation of the Fe-responsive tumor growth and metastasis suppressor Ndrg1 in the tumor but not in the liver, indicating a potential mechanism of selective anticancer activity. These results indicate that the novel Fe chelators have potent and broad antitumor activity and can overcome resistance to established chemotherapeutics because of their unique mechanism of action.

Disruption of Toxoplasma gondii Parasitophorous Vacuoles by the Mouse p47-Resistance GTPases

The p47 GTPases are essential for interferon-γ-induced cell-autonomous immunity against the protozoan parasite, Toxoplasma gondii, in mice, but the mechanism of resistance is poorly understood. We show that the p47 GTPases, including IIGP1, accumulate at vacuoles containing T. gondii. The accumulation is GTP-dependent and requires live parasites. Vacuolar IIGP1 accumulations undergo a maturation-like process accompanied by vesiculation of the parasitophorous vacuole membrane. This culminates in disruption of the parasitophorous vacuole and finally of the parasite itself. Over-expression of IIGP1 leads to accelerated vacuolar disruption whereas a dominant negative form of IIGP1 interferes with interferon-γ-mediated killing of intracellular parasites. Targeted deletion of the IIGP1 gene results in partial loss of the IFN-γ-mediated T. gondii growth restriction in mouse astrocytes.

The interferon-inducible p47 (IRG) GTPases in vertebrates: loss of the cell autonomous resistance mechanism in the human lineage

BackgroundMembers of the p47 (immunity-related GTPases (IRG) family) GTPases are essential, interferon-inducible resistance factors in mice that are active against a broad spectrum of important intracellular pathogens. Surprisingly, there are no reports of p47 function in humans.ResultsHere we show that the p47 GTPases are represented by 23 genes in the mouse, whereas humans have only a single full-length p47 GTPase and an expressed, truncated presumed pseudo-gene. The human full-length gene is orthologous to an isolated mouse p47 GTPase that carries no interferon-inducible elements in the promoter of either species and is expressed constitutively in the mature testis of both species. Thus, there is no evidence for a p47 GTPase-based resistance system in humans. Dogs have several interferon-inducible p47s, and so the primate lineage that led to humans appears to have lost an ancient function. Multiple p47 GTPases are also present in the zebrafish, but there is only a tandem p47 gene pair in pufferfish.ConclusionMice and humans must deploy their immune resources against vacuolar pathogens in radically different ways. This carries significant implications for the use of the mouse as a model of human infectious disease. The absence of the p47 resistance system in humans suggests that possession of this resistance system carries significant costs that, in the primate lineage that led to humans, are not outweighed by the benefits. The origin of the vertebrate p47 system is obscure.

Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii

GTPases of the mouse IRG protein family, mediators of resistance against Toxoplasma gondii in the mouse, are inactivated by a polymorphic kinase of the parasite, resulting in enhanced parasite virulence.

Disruption of the Toxoplasma gondii Parasitophorous Vacuole by IFNγ-Inducible Immunity-Related GTPases (IRG Proteins) Triggers Necrotic Cell Death

Toxoplasma gondii is a natural intracellular protozoal pathogen of mice and other small mammals. After infection, the parasite replicates freely in many cell types (tachyzoite stage) before undergoing a phase transition and encysting in brain and muscle (bradyzoite stage). In the mouse, early immune resistance to the tachyzoite stage is mediated by the family of interferon-inducible immunity-related GTPases (IRG proteins), but little is known of the nature of this resistance. We reported earlier that IRG proteins accumulate on intracellular vacuoles containing the pathogen, and that the vacuolar membrane subsequently ruptures. In this report, live-cell imaging microscopy has been used to follow this process and its consequences in real time. We show that the rupture of the vacuole is inevitably followed by death of the intracellular parasite, shown by its permeability to cytosolic protein markers. Death of the parasite is followed by the death of the infected cell. The death of the cell has features of pyronecrosis, including membrane permeabilisation and release of the inflammatory protein, HMGB1, but caspase-1 cleavage is not detected. This sequence of events occurs on a large scale only following infection of IFNγ-induced cells with an avirulent strain of T. gondii, and is reduced by expression of a dominant negative mutant IRG protein. Cells infected by virulent strains rarely undergo necrosis. We did not find autophagy to play any role in the key steps leading to the death of the parasite. We conclude that IRG proteins resist infection by avirulent T. gondii by a novel mechanism involving disruption of the vacuolar membrane, which in turn ultimately leads to the necrotic death of the infected cell.

The gene conversion hypothesis of MHC evolution: a review.

Abstract Gene conversion is often invoked to explain the evolution of sequence patterns observed in major histocompatibility complex (MHC) genes and their alleles. This is the gene conversion hypothesis of MHC sequence evolution. These observations and their interpretation probably belong in a larger theoretical framework, namely the evolution of systems of resistance to rapidly evolving pathogens. This review looks critically at the evidence in favor of the gene conversion hypothesis in this context. We conclude that the case for the existence of an adaptive mechanism in the MHC favoring gene conversion mutations is not proven.

Comparative Genomics of the Apicomplexan Parasites Toxoplasma gondii and Neospora caninum: Coccidia Differing in Host Range and Transmission Strategy

Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species.

Monoclonal Antibodies as Tools to Analyze the Serological and Genetic Complexities of Major Transplantation Antigens

The rat MHC resembles that of other species in displaying extensive polymorphism for a variety of MHC-characteristic functions: antigens detected by serological assays, antigens detected by cellular assays such as the MLR, GVH and CML, and immune response genes for a variety of cellular and non-cellular antigens (Gunther & Stark 1977, Gasser 1977). Nevertheless very little is known about the genetic structure of the region because of the shortage of laboratory recombinants. The present study grew out of the realisation that the high resolving power of monoclonal antibodies against complex polymorphic antigens could compensate for lack of resolution at the genetic level. Granted suitable monoclonal alloantibodies, the number and antigenic structure of the polymorphic molecules specified by the MHC can in principle be examined with greater precision than is possible by analysis of recombinants using planned immunizations and absorptions of conventional sera. This review describes the preparation of monoclonal antibodies by fusion of spleen cells from alloimmunized rats with mouse plasmacytoma cells and some results of an analysis of the properties of these antibodies. Some preliminary data have been published elsewhere (Galfre et al. 1977, Howard et al. 1978).

The rat cim effect: TAP allele-dependent changes in a class I MHC anchor motif and evidence against C-terminal trimming of peptides in the ER.

Functional polymorphism in the rat peptide transporter associated with antigen processing (TAP) changes the peptide pool available for binding and presentation by a class I MHC allele, RT1.Aa. The peptide binding motif for RT1.Aa, determined by stabilization with synthetic peptides, included a strong preference for arginine at the peptide C terminus. Analysis of natural peptides bound to RT1.Aa by both pool sequencing and anhydrotrypsin chromatography revealed that TAP polymorphism determined the presence or absence of arginine as the peptide C-terminal residue. This result highlights the in vivo impact of TAP-peptide selectivity, and provides evidence against a high rate of generation of new C termini by protease activity in the endoplasmic reticulum.

Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole

The immunity‐related GTPases (IRGs) constitute an interferon‐induced intracellular resistance mechanism in mice against Toxoplasma gondii. IRG proteins accumulate on the parasitophorous vacuole membrane (PVM), leading to its disruption and to death of the parasite. How IRGs target the PVM is unknown. We show that accumulation of IRGs on the PVM begins minutes after parasite invasion and increases for about 1 h. Targeting occurs independently of several signalling pathways and the microtubule network, suggesting that IRG transport is diffusion‐driven. The intensity of IRG accumulation on the PVM, however, is reduced in absence of the autophagy regulator, Atg5. In wild‐type cells IRG proteins accumulate cooperatively on PVMs in a definite order reflecting a temporal hierarchy, with Irgb6 and Irgb10 apparently acting as pioneers. Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected. The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A–D, are not individually responsible for this effect. Thus IRG proteins protect mice against avirulent strains of Toxoplasma but fail against virulent strains. The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.