Public-private partnerships influencing the initiation and duration of clinical trials for neglected tropical diseases.

Public-private partnerships (PPPs) for neglected tropical diseases (NTDs) are often studied as an organizational form that facilitates the management and control of the huge costs of drug research and development. Especially the later stages of drug development, including clinical trials, become very expensive. This present study investigates whether and how the type of PPPs influences the initiation and duration of NTD clinical trials. Using the ClinicalTrials.gov database, a dataset of 1175 NTD clinical studies that started between 2000 and 2021 is analyzed based on affiliation information and project duration. For the NTD clinical trials that resulted from PPPs, the collaborating types were determined and analyzed, including the public sector-, private sector-, governmental sector-, and nongovernmental organization-led collaborations. The determinants for the discontinuation of all stopped clinical trials were categorized into scientific-, funding-, political-, and logistic dimensions. The results reveal that public sector-led PPPs were the most common collaborative types, and logistic and scientific issues were the most frequent determinants of stopped clinical trials. Trial registration: ClinicalTrials.gov.

Collaborative university-industry R&D practices supporting the pharmaceutical innovation process: Insights from a bibliometric review.

University-industry collaborative research and development (UIC R&D) is generally seen as a driver of the pharmaceutical innovation process. Here, we perform a bibliometric review of UIC R&D practices over the past 30 years (1991-2020) by analyzing 800+ publications. At the strategic level of organizational cooperation patterns, the analysis shows that pharmaceutical UIC R&D mainly aims at strategic alliance formation, which gears toward universities and companies collaboratively exploring and commercializing technological breakthroughs. At the structural level of universities and companies investing in cooperation and aligning their activities, analytical results indicate that universities and companies organize themselves as interdependent entities in an open innovation ecosystem. At the cultural level of generally accepted collaboration norms and habits, analytical results show that university-company partnerships are becoming a rule rather than an exception. This study delves into a 30-year history of UIC R&D practices that support the pharmaceutical innovation process. It provides academics and practitioners with an insight into possible strategies for UIC R&D in the future and presents avenues for science, business and innovation research.

Neutralization of the Plasmodium-encoded MIF ortholog confers protective immunity against malaria infection.

Plasmodium species produce an ortholog of the cytokine macrophage migration inhibitory factor, PMIF, which modulates the host inflammatory response to malaria. Using a novel RNA replicon-based vaccine, we show the impact of PMIF immunoneutralization on the host response and observed improved control of liver and blood-stage Plasmodium infection, and complete protection from re-infection. Vaccination against PMIF delayed blood-stage patency after sporozoite infection, reduced the expression of the Th1-associated inflammatory markers TNF-α, IL-12, and IFN-γ during blood-stage infection, augmented Tfh cell and germinal center responses, increased anti-Plasmodium antibody titers, and enhanced the differentiation of antigen-experienced memory CD4 T cells and liver-resident CD8 T cells. Protection from re-infection was recapitulated by the adoptive transfer of CD8 or CD4 T cells from PMIF RNA immunized hosts. Parasite MIF inhibition may be a useful approach to promote immunity to Plasmodium and potentially other parasite genera that produce MIF orthologous proteins.

Identification of novel translational urinary biomarkers for acetaminophen-induced acute liver injury using proteomic profiling in mice.

Drug-induced liver injury (DILI) is the leading cause of acute liver failure. Currently, no adequate predictive biomarkers for DILI are available. This study describes a translational approach using proteomic profiling for the identification of urinary proteins related to acute liver injury induced by acetaminophen (APAP). Mice were given a single intraperitoneal dose of APAP (0-350 mg/kg bw) followed by 24 h urine collection. Doses of ≥275 mg/kg bw APAP resulted in hepatic centrilobular necrosis and significantly elevated plasma alanine aminotransferase (ALT) values (p<0.0001). Proteomic profiling resulted in the identification of 12 differentially excreted proteins in urine of mice with acute liver injury (p<0.001), including superoxide dismutase 1 (SOD1), carbonic anhydrase 3 (CA3) and calmodulin (CaM), as novel biomarkers for APAP-induced liver injury. Urinary levels of SOD1 and CA3 increased with rising plasma ALT levels, but urinary CaM was already present in mice treated with high dose of APAP without elevated plasma ALT levels. Importantly, we showed in human urine after APAP intoxication the presence of SOD1 and CA3, whereas both proteins were absent in control urine samples. Urinary concentrations of CaM were significantly increased and correlated well with plasma APAP concentrations (r = 0.97; p<0.0001) in human APAP intoxicants, who did not present with elevated plasma ALT levels. In conclusion, using this urinary proteomics approach we demonstrate CA3, SOD1 and, most importantly, CaM as potential human biomarkers for APAP-induced liver injury.

A Plasmodium-encoded cytokine suppresses T-cell immunity during malaria.

The inability to acquire protective immunity against Plasmodia is the chief obstacle to malaria control, and inadequate T-cell responses may facilitate persistent blood-stage infection. Malaria is characterized by a highly inflammatory cytokine milieu, and the lack of effective protection against infection suggests that memory T cells are not adequately formed or maintained. Using a genetically targeted strain of Plasmodium berghei, we observed that the Plasmodium ortholog of macrophage migration inhibitory factor enhanced inflammatory cytokine production and also induced antigen-experienced CD4 T cells to develop into short-lived effector cells rather than memory precursor cells. The short-lived effector CD4 T cells were more susceptible to Bcl-2-associated apoptosis, resulting in decreased CD4 T-cell recall responses against challenge infections. These findings indicate that Plasmodia actively interfere with the development of immunological memory and may account for the evolutionary conservation of parasite macrophage migration inhibitory factor orthologs.

Plasmodium Cysteine Repeat Modular Proteins 3 and 4 are essential for malaria parasite transmission from the mosquito to the host.

BACKGROUND: The Plasmodium Cysteine Repeat Modular Proteins (PCRMP) are a family of four conserved proteins of malaria parasites, that contain a number of motifs implicated in host-parasite interactions. Analysis of mutants of the rodent parasite Plasmodium berghei lacking expression of PCRMP1 or 2 showed that these proteins are essential for targeting of P. berghei sporozoites to the mosquito salivary gland and, hence, for transmission from the mosquito to the mouse. METHODS: In this work, the role of the remaining PCRMP family members, PCRMP3 and 4, has been investigated throughout the Plasmodium life cycle by generation and analysis of P. berghei gene deletion mutants, Δpcrmp3 and Δpcrmp4. The role of PCRMP members during the transmission and hepatic stages of the Plasmodium lifecycle has been evaluated by light- and electron microscopy and by analysis of liver stage development in HEPG2 cells in vitro and by infecting mice with mutant sporozoites. In addition, mice were immunized with live Δpcrmp3 and Δpcrmp4 sporozoites to evaluate their immunization potential as a genetically-attenuated parasite-based vaccine. RESULTS: Disruption of pcrmp3 and pcrmp4 in P. berghei revealed that they are also essential for transmission of the parasite through the mosquito vector, although acting in a distinct way to pbcrmp1 and 2. Mutants lacking expression of PCRMP3 or PCRMP4 show normal blood stage development and oocyst formation in the mosquito and develop into morphologically normal sporozoites, but these have a defect in egress from oocysts and do not enter the salivary glands. Sporozoites extracted from oocysts perform gliding motility and invade and infect hepatocytes but do not undergo further development and proliferation. Furthermore, the study shows that immunization with Δcrmp3 and Δcrmp4 sporozoites does not confer protective immunity upon subsequent challenge. CONCLUSIONS: PCRMP3 and 4 play multiple roles during the Plasmodium life cycle; they are essential for the establishment of sporozoite infection in the mosquito salivary gland, and subsequently for development in hepatocytes. However, although Δpcrmp3 and Δpcrmp4 parasites are completely growth-impaired in the liver, immunization with live sporozoites does not induce the protective immune responses that have been shown for other genetically-attenuated parasites.

The crystal structures of macrophage migration inhibitory factor from Plasmodium falciparum and Plasmodium berghei.

Malaria, caused by Plasmodium falciparum and related parasites, is responsible for millions of deaths each year, mainly from complications arising from the blood stages of its life cycle. Macrophage migration inhibitory factor (MIF), a protein expressed by the parasite during these stages, has been characterized in mammals as a cytokine involved in a broad spectrum of immune responses. It also possesses two catalytic activities, a tautomerase and an oxidoreductase, though the physiological significance of neither reaction is known. Here, we have determined the crystal structure of MIF from two malaria parasites, Plasmodium falciparum and Plasmodium berghei at 2.2 A and 1.8 A, respectively. The structures have an alpha/beta fold and each reveals a trimer, in agreement with the results of analytical ultracentrifugation. We observed open and closed active sites, these being distinguished by movements of proline-1, the catalytic base in the tautomerase reaction. These states correlate with the covalent modification of cysteine 2 to form a mercaptoethanol adduct, an observation confirmed by mass spectrometry. The Plasmodium MIFs have a different pattern of conserved cysteine residues to the mammalian MIFs and the side chain of Cys58, which is implicated in the oxidoreductase activity, is buried. This observation and the evident redox reactivity of Cys2 suggest quite different oxidoreductase characteristics. Finally, we show in pull-down assays that Plasmodium MIF binds to the cell surface receptor CD74, a known mammalian MIF receptor implying that parasite MIF has the ability to interfere with, or modulate, host MIF activity through a competitive binding mechanism.

Functional characterization of the Plasmodium falciparum and P. berghei homologues of macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a mammalian cytokine that participates in innate and adaptive immune responses. Homologues of mammalian MIF have been discovered in parasite species infecting mammalian hosts (nematodes and malaria parasites), which suggests that the parasites express MIF to modulate the host immune response upon infection. Here we report the first biochemical and genetic characterization of a Plasmodium MIF (PMIF). Like human MIF, histidine-tagged purified recombinant PMIF shows tautomerase and oxidoreductase activities (although the activities are reduced compared to those of histidine-tagged human MIF) and efficiently inhibits AP-1 activity in human embryonic kidney cells. Furthermore, we found that Plasmodium berghei MIF is expressed in both a mammalian host and a mosquito vector and that, in blood stages, it is secreted into the infected erythrocytes and released upon schizont rupture. Mutant P. berghei parasites lacking PMIF were able to complete the entire life cycle and exhibited no significant changes in growth characteristics or virulence features during blood stage infection. However, rodent hosts infected with knockout parasites had significantly higher numbers of circulating reticulocytes. Our results suggest that PMIF is produced by the parasite to influence host immune responses and the course of anemia upon infection.

Plasmodium berghei alpha-tubulin II: a role in both male gamete formation and asexual blood stages.

Plasmodium falciparum contains two genes encoding different isotypes of alpha-tubulin, alpha-tubulin I and alpha-tubulin II. alpha-Tubulin II is highly expressed in male gametocytes and forms part of the microtubules of the axoneme of male gametes. Here we present the characterization of Plasmodium berghei alpha-tubulin I and alpha-tubulin II that encode proteins of 453 and 450 amino acids, respectively. alpha-Tubulin II lacks the well-conserved three amino acid C-terminal extension including a terminal tyrosine residue present in alpha-tubulin I. Investigation of transcription by Northern analysis and RT-PCR and analysis of promoter activity by GFP tagging showed that alpha-tubulin I is expressed in all blood and mosquito stages. As expected, alpha-tubulin II was highly expressed in the male gametocytes, but transcription was also observed in the asexual blood stages, female gametocytes, ookinetes and oocysts. Gene disruption experiments using standard transfection technologies did not produce viable parasites indicating that both alpha-tubulin isotypes are essential for the asexual blood stages. Targeted modification of alpha-tubulin II by the addition of the three C-terminal amino acids of alpha-tubulin I did not affect either blood stage development nor male gamete formation. Attempts to modify the C-terminal region by adding a TAP tag to the endogenous alpha-tubulin II gene were not successful. Introduction of a transgene, expressing TAP-tagged alpha-tubulin II, next to the endogenous alpha-tubulin II gene, had no effect on the asexual blood stages but strongly impaired formation of male gametes. These results show that alpha-tubulin II not only plays an important role in the male gamete but is also expressed in and essential for asexual blood stage development.

Structural characterization of the PIT-1/ETS-1 interaction: PIT-1 phosphorylation regulates PIT-1/ETS-1 binding.

The POU-domain transcription factor Pit-1 and Ets-1, a member of the ETS family of transcription factors, can associate in solution and synergistically activate the prolactin promoter by binding to a composite response element in the prolactin promoter. We mapped the minimal region of Ets-1 required for the interaction with the Pit-1 POU-homeodomain. Here, we describe a detailed NMR study of the interaction between the POU-homeodomain of Pit-1 and the minimal interacting region of Ets-1. By using heteronuclear single quantum coherence titration experiments, we were able to map exact residues on the POU-homeodomain that are involved in the interaction with this minimal Ets-1 interaction domain. By using our NMR data, we generated point mutants in the POU-homeodomain and tested their effect on the interaction with Ets-1. Our results show that phosphorylation of Pit-1 can regulate the interaction with Ets-1.