Mercurochrome Research Articles

First Preclinical Report of the Efficacy and PD Results of KHK2823, a Non-Fucosylated Fully Human Monoclonal Antibody Against IL-3Rα

Human interleukin-3 receptor alpha (IL-3Ra, CD123), which promotes the proliferation and differentiation of hematopoietic cells, is highly expressed in myeloid malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).We newly generated KHK2823, a non-fucosylated fully human IgG1 monoclonal antibody against human IL-3Ra, by utilizing the POTELLIGENT® technology. Here, we describe the in vitro and in vivo preclinical efficacy and safety of KHK2823, as well as its pharmacodynamic (PD) profile.At first, we explored that KHK2823 bound to various hematological malignant cells and leukemic stem cells. The cells from AML and MDS bone marrows were found to be bound by KHK2823. A significant part of bone marrow cells derived from B-cell acute lymphoblastic leukemia (B-ALL) patients was also bound by KHK2823.KHK2823 bound to soluble human IL-3Ra protein with a sub-nanomolar dissociation constant (KD), and recognized CD34+ CD38+ (leukemic blast) and/or CD34+ CD38- (leukemic stem cell) cells in patients with AML/MDS, as well as AML cell lines, thereby obtaining a high antibody-dependent cellular cytotoxic activity without complement-dependent cytotoxicity. Interestingly, KHK2823 did not interfere with the binding of IL-3 to IL-3R. The lack of a receptor-ligand interaction may conserve the IL-3 signal, which plays an important role in normal hematopoiesis. In a tumor model xenografting the human AML cell line MOLM-13 on nude rats, KHK2823 significantly suppressed the tumor growth at doses of 0.1 and 1 mg/kg (Figure 1).The PD and toxicity profiles of KHK2823 were assessed in cynomolgus monkeys administered at doses ranging from 0.1 to 100 mg/kg by i.v. infusion, once weekly for 4 weeks. KHK2823 was generally well tolerated in monkeys, even at 100 mg/kg. The number of IL-3Ra-positive cells in the peripheral blood of cynomolgus monkeys decreased in all groups receiving KHK2823, which suggest KHK2823 could exert its depletion activity of IL-3Ra-positive cells in human (Figure 2). Currently, the safety and tolerability of KHK2823 is being investigated in patients with AML or MDS in a Phase 1 study (NCT02181699, https://clinicaltrials.gov/ct2/show/NCT02181699). This is the first non-randomized, open-label, dose escalation clinical study to investigate the safety, PK, immunogenicity and PD of repeated doses of KHK2823.In summary, KHK2823 was confirmed to bind to AML, MDS and B-ALL cells as the IL-3Ra in accordance with other publications. KHK2823 was also found to eliminate AML cells in vitro and also suppressed the AML tumor growth in the in vivo model. In addition, the number of IL-3Ra-positive cells in cynomolgus monkeys decreased following i.v. infusion of 0.1mg/kg KHK2823 with a tolerable safety profile, even at a dose of 100 mg/kg. Taken together, KHK2823 may therefore be a promising anti-IL-3Ra therapeutic drug for the treatment of AML. [Display omitted] [Display omitted] DisclosuresAkiyama:Kyowa Hakko Kirin Co., Ltd.: Employment. Takayanagi:Kyowa Hakko Kirin Co., Ltd.: Employment. Maekawa:Kyowa Hakko Kirin Co., Ltd.: Employment. Shimabe:Kyowa Hakko Kirin Co., Ltd.: Employment. Nishikawa:Kyowa Hakko Kirin Co., Ltd.: Employment. Yamawaki:Kyowa Hakko Kirin Co., Ltd: Employment. Iijima:Kyowa Hakko Kirin Co., Ltd: Employment. Hiura:Kyowa Hakko Kirin Co., Ltd.: Employment. Takahashi:Kyowa Hakko Kirin Co., Ltd.: Employment. Akashi:Asahi Kasei: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau. Tawara:Kyowa Hakko Kirin Co., Ltd: Employment.

An expanded search for simian foamy viruses (SFV) in Brazilian New World primates identifies novel SFV lineages and host age-related infections.

BackgroundWhile simian foamy viruses have co-evolved with their primate hosts for millennia, most scientific studies have focused on understanding infection in Old World primates with little knowledge available on the epidemiology and natural history of SFV infection in New World primates (NWPs). To better understand the geographic and species distribution and evolutionary history of SFV in NWPs we extend our previous studies in Brazil by screening 15 genera consisting of 29 NWP species (140 monkeys total), including five genera (Brachyteles, Cacajao, Callimico, Mico, and Pithecia) not previously analyzed. Monkey blood specimens were tested using a combination of both serology and PCR to more accurately estimate prevalence and investigate transmission patterns. Sequences were phylogenetically analyzed to infer SFV and host evolutionary histories.ResultsThe overall serologic and molecular prevalences were 42.8 and 33.6 %, respectively, with a combined assay prevalence of 55.8 %. Discordant serology and PCR results were observed for 28.5 % of the samples, indicating that both methods are currently necessary for estimating NWP SFV prevalence. SFV prevalence in sexually mature NWPs with a positive result in any of the WB or PCR assays was 51/107 (47.7 %) compared to 20/33 (61 %) for immature animals. Epidemiological analyses revealed an increase in SFV prevalence with age in captive Cebus monkeys. Phylogenetic analysis identified novel SFVs in Cacajao,Leontopithecus, and Chiropotes species that had 6–37 % nucleotide divergence to other NWP SFV. Comparison of host and SFV phylogenies showed an overall cospeciation evolutionary history with rare ancient and contemporaneous host-switching for Saimiri and Leontopithecus and Cebus xanthosternos, respectively.ConclusionsWe identified novel SFV in four neotropical monkey genera in Brazil and demonstrate that SFV prevalence increases with age in Cebus monkeys. Importantly, our test results suggest that both molecular and serological screening are currently required to accurately determine infection with NWP SFV. Our study significantly expands knowledge of the epidemiology and natural history of NWP SFVs. The tools and information provided in our study will facilitate further investigation of SFV in NWPs and the potential for zoonotic infection with these viruses.

Large Hernia of Umbilical Cord Misdiagnosed as Omphalocele.

We read with interest case series of “Hernia of umbilical cord: Report of three unusual cases” [1] and want to mention our own case where a misdiagnosis led to inadequate management resulting in loss of 30cm of small bowel. A 12hour-old male baby delivered via Caesarian section due to prenatal diagnosis of omphalocele. He was attended by a surgical resident and labelled as omphalocele major and advised mercurochrome paint. Next day, the neonate became lethargic. Abdominal examination showed protrusion of bowel through the umbilicus into an omphalocele like sac with dark maroon discoloration on one side (Fig. 1). There was not abdominal wall defect and umbilicus was normally sited. At the base there was a skin collar of about 2 cm. The patient was immediately prepared for laparotomy with a suspicion of bowel necrosis. On opening the sac, serosanguineous fluid was drained. The part of ileum was necrotic while most of jejunum and ileum were edematous. The incision extended cranially in midline and small bowel was delivered. Grossly necrotic ileum measuring 30cm was excised and end to end anastomosis was done. Malrotation was also present and corrected. Primary closure easily achieved due to bowel resection. The postoperative recovery was uneventful with a follow-up of 6 months. Our case is unique since large sac with hemorrhage was giving appearance of herniated liver causing the condition to be misdiagnosed as omphalocele major. It is mandatory to look carefully in a case of omphalocele for presence of any true abdominal wall defect, umbilical ring, and the base for presence of skin collar so that the condition is not misdiagnosed.

Pharmacokinetics and disposition of CS-0777, a sphingosine 1-phosphate receptor modulator, in rats and monkeys

1. Disposition and metabolism of CS-0777 (1-{5-[(3R)-3-amino-4-hydroxy-3- methylbutyl]-1-methyl-1H-pyrrol-2-yl}-4-(4-methylphenyl) butan-1-one), a selective sphingosine 1-phosphate receptor-1 modulator under development for autoimmune conditions was investigated following oral and/or i.v. bolus administration to rats and monkeys.2. After oral administration of [14C]CS-0777, CS-0777 was well absorbed in rats and monkeys with total recoveries of over 90% of the dose, majorly in feces. CS-0777 and phosphorylated pharmacologically active metabolite of CS-0777 (M1) were highly bound to plasma proteins among rats, monkeys and humans (>93%).3. The structures of 12 metabolites were identified and phosphorylation and two hydroxylation pathways were proposed as primary metabolism. In the blood of rats and monkeys, the major metabolite was M1 and a few phosphorylated metabolites were also detected. Meanwhile, in urine and feces of rats and monkeys, not phosphorylated, but oxidized CS-0777 metabolites and/or those various conjugated metabolites were observed. This suggests that CS-0777 and its oxidized metabolites would be phosphorylated in the body, but their phosphorylated metabolites would revert back to their dephosphorylated form again then be further metabolized and finally eliminated from the body.4. Pharmacokinetic analysis using a reversible metabolism model revealed that the clearance of phosphorylation was larger than the clearance of dephosphorylation and elimination.

Receptor occupancy and blocking of STAT5 signaling by an anti-IL-7 receptor α antibody in cynomolgus monkeys.

Interleukin-7 receptor α (IL-7Rα) is associated with autoimmune disease. Blocking its activation by interleukin-7 (IL-7) with a therapeutic monoclonal antibody may reduce pathogenic T cells and effectively control the autoimmune response in these disorders. Two flow cytometry-based assays were developed and implemented to evaluate the interaction between cell surface IL-7Rα and an anti-IL-7Rα monoclonal antibody (Ab1). The receptor occupancy assay utilized competing and noncompeting commercial detection antibodies for "free" and "total" IL-7Rα, respectively. STAT5 phosphorylation (pSTAT5) was measured as a proximal biomarker of IL-7Rα inhibition by Ab1. Monkeys administered Ab1 had no free IL-7Rα detectable on the CD3+ T cell surface at 0.25 hours postdose through day 4, in all treatment groups. Ab1 treatment resulted in a significant reduction in total IL-7Rα, dropping to 53%, 44%, and 55% on day 4 at 0.3, 3, and 30 mg/kg, respectively, compared to predose levels. There were treatment-related decreases in the ability of IL-7 to induce STAT5 phosphorylation in both CD4+ and CD8+ T cells in monkey blood samples from all treated animals from 0.25 hours through Day 4 postdose. The nonclinical receptor occupancy assay was developed and applied to detect free and total IL-7Rα on the surface of CD3+ T cells in cynomolgus monkeys treated with Ab1. The results showed good correlation with the phosphorylation of STAT5 and serum concentration of Ab1. The approach for IL-7Rα occupancy and pSTAT5 measurements established in monkeys can be utilized in clinical trials for pharmacokinetic/pharmacodynamic evaluation of Ab1 effect in humans.

Sequential Monitoring and Stability of Ex Vivo–Expanded Autologous and Nonautologous Regulatory T Cells Following Infusion in Nonhuman Primates

Ex vivo-expanded cynomolgus monkey CD4(+)CD25(+)CD127(-) regulatory T cells (Treg) maintained Foxp3 demethylation status at the Treg-specific demethylation region, and potently suppressed T cell proliferation through three rounds of expansion. When carboxyfluorescein succinimidyl ester- or violet proliferation dye 450-labeled autologous (auto) and nonautologous (non-auto)-expanded Treg were infused into monkeys, the number of labeled auto-Treg in peripheral blood declined rapidly during the first week, but persisted at low levels in both normal and anti-thymocyte globulin plus rapamycin-treated (immunosuppressed; IS) animals for at least 3 weeks. By contrast, MHC-mismatched non-auto-Treg could not be detected in normal monkey blood or in blood of two out of the three IS monkeys by day 6 postinfusion. They were also more difficult to detect than auto-Treg in peripheral lymphoid tissue. Both auto- and non-auto-Treg maintained Ki67 expression early after infusion. Sequential monitoring revealed that adoptively transferred auto-Treg maintained similarly high levels of Foxp3 and CD25 and low CD127 compared with endogenous Treg, although Foxp3 staining diminished over time in these nontransplanted recipients. Thus, infused ex vivo-expanded auto-Treg persist longer than MHC-mismatched non-auto-Treg in blood of nonhuman primates and can be detected in secondary lymphoid tissue. Host lymphodepletion and rapamycin administration did not consistently prolong the persistence of non-auto-Treg in these sites.

MGD011, Humanized CD19 x CD3 DART® Protein with Enhanced Pharmacokinetic Properties, Demonstrates Potent T-Cell Mediated Anti-Tumor Activity in Preclinical Models and Durable B-Cell Depletion in Cynomolgus Monkeys Following Once-a-Week Dosing

Abstract CD19, a lymphocyte-specific marker expressed from early B-lymphocyte development through mature memory B cells, is highly represented in B-cell malignancies, making it attractive for targeted interventions. MGD011 is a Dual-Affinity Re-Targeting (DART®) protein designed to redirect T-cells to eliminate CD19-expressing cells through co-engagement of CD19 and CD3 in a monovalent binding fashion. MGD011 was engineered with a modified Fc domain that is inactivated for binding to FcγRs but retains binding to the neonatal Fc receptor (FcRn) of the immunoglobulin salvage pathway, allowing for extended pharmacokinetic (PK) properties and convenient dosing at a once-a-week or longer interval. To enable preclinical toxicokinetics and dose optimization, MGD00011 was also designed to cross-react with non-human primates. MGD011 demonstrates bispecific binding to B and T cells isolated from human and cynomolgus monkey blood. MGD011 mediates potent in vitro redirected T-cell killing of B-cell lymphoma cell lines and effective autologous B-cell depletion of human or cynomolgus monkey peripheral blood mononuclear cells (PBMCs). MGD011-mediated killing of CD19 target cells was accompanied by target-dependent T-cell expansion and activation, cytokine release and upregulation of perforin and granzyme B, consistent with the redirected T-cell mediated killing mechanism of action endowed in its design. Two tumor models in NOD/SCID/IL2 receptor gamma chain-deficient (NSG) or NSG/beta2-microglobulin-deficient (NSG/Bm2-/-) mice were used to ascertain in vivo activity of MGD011. The growth of newly-implanted tumor xenografts of HBL-2 cells, a mantle cell lymphoma line, or Raji cells, a Burkitt’s lymphoma line, was efficiently inhibited by intravenous (IV) administration of MGD011 following the subcutaneous (SC) implantation of the target cells co-mixed with activated human T cells in Winn’s tumor models. Furthermore, in human PBMC-reconstituted mice bearing pre-established intradermal (ID) HBL-2 xenografts, MGD011 demonstrated potent anti-tumor activity with high complete response rates and no evidence of relapse over the study duration. To assess the safety and pharmacodynamic activity of MGD011, a dose-escalation study of MGD011 administered once weekly by 2-hour IV infusion was conducted in cynomolgus monkeys. MGD011 was well tolerated at doses up to 100 µg/kg, the highest dose tested, with no drug-related adverse findings and modest elevations in serum cytokines. MGD011-related pharmacological effects included marked decrease in circulating B cells at doses as low as 0.5 µg/kg, profound reduction of CD20+ cells in lymphoid organs and only modest recovery 4 weeks after the last dose. As expected, MGD011 displays prolonged PK properties, consistent with that of a human IgG1 in monkeys. In summary, the safety, activity and PK profiles of MGD011 support further investigation as a therapeutic candidate for treatment of B-cell malignancies. Disclosures Liu: Macrogenics Inc.: Employment, Equity Ownership. Lam:Macrogenics Inc.: Employment, Equity Ownership. Alderson:Macrogenics Inc: Employment, Equity Ownership. Yang:Macrogenics Inc: Employment, Equity Ownership. Li:Macrogenics Inc: Employment, Equity Ownership. Long:Macrogenics Inc: Employment, Equity Ownership. Gorlatov:Macrogenics Inc: Employment, Equity Ownership. Burke:Macrogenics Inc: Employment, Equity Ownership. Ciccarone:Macrogenics Inc: Employment, Equity Ownership. Nordstrom:Macrogenics Inc: Employment, Equity Ownership. Johnson:Macrogenics Inc: Employment, Equity Ownership. Moore:Macrogenics Inc: Employment, Equity Ownership. Bonvini:Macrogenics Inc: Employment, Equity Ownership.

Enhancing the Acute Hemostatic Efficacy in Cynomolgus Monkeys By Targeting Activated Coagulation Factor VII to Platelets

Recombinant activated factor VII (rFVIIa) is used for controlling bleeds in hemophilia A and B patients with inhibitory antibodies against factor VIII or factor IX. The hemostatic effect of rFVIIa at pharmacological doses is believed to be mediated by activating the tenase complex on platelets directly at the site of injury to promote thrombin generation. To increase the hemostatic activity of rFVIIa, we sought to target rFVIIa to platelets by fusing a single chain variable region of a monoclonal antibody against human platelet receptor αIIbβ3. The resulting fusion protein, FVII-189, displays higher affinity to human platelets and about 50-fold higher hemostatic activity than that of rFVIIa in whole blood from hemophilia A patients by rotational thromboelastometry (ROTEM). Here we investigated the safety, pharmacokinetics, and pharmacodynamics of FVII-189 in cynomolgus monkeys.First, we confirmed that FVII-189 cross-reacted to platelets from cynomolgus monkeys. When spiked in the monkey whole blood, FVII-189 could bind platelets in a dose-dependent manner, as measured by flow cytometry using fluorescently labeled antibodies against FVII, whereas platelet-binding by rFVIIa was not detectable in the same assay. More importantly, increased hemostatic activity of FVII-189 was observed by ROTEM in acquired hemophilic monkey blood where factor VIII activity was neutralized by the inhibitory antibodies.We then evaluated FVII-189 in cynomolgus monkeys following a bolus intravenous administration (n=3) at an equal molar dose to the pharmacological dose of rFVIIa (~2 nmol/kg). rFVIIa was included as a comparator. Both FVII-189 and rFVIIa appeared to be well tolerated with no abnormal clinical observations that could be attributed to the dosing. No apparent effects on platelet count in whole blood, as well as other hematology and coagulation parameters except for a temporary decrease in prothrombin time over a few hours, which was an expected pharmacological effect of rFVIIa and FVII-189. Compared to rFVIIa, FVII-189 cleared more rapidly in plasma, and distributed more to the platelets. The half-life of FVII-189 antigen on platelets was found to be several fold longer than that of rFVIIa antigen in plasma, as measured by ELISA of the platelet-rich plasma, or by flow cytometry of platelet-associated FVII-189 antigen. Despite the lower initial recovery and faster clearance of FVII-189 in plasma, directing FVII-189 to platelets resulted in much higher hemostatic activity of the whole blood 5 minute and 1 hour after dosing, as measured by ex vivo ROTEM assay.In conclusion, these results indicate that in cynomolgus monkeys, 1) FVII-189 is safe and does not affect the platelet clearance, 2) FVII-189 protein is targeted to platelets with fast clearance in plasma and prolonged half-life on platelets and 3) FVII-189 could be more efficacious in controlling acute bleeds than rFVIIa. DisclosuresTan:Biogen Idec: Employment, Equity Ownership. Salas:Biogen Idec: Employment, Equity Ownership. Chen:Biogen Idec: Employment, Equity Ownership. Ashworth:Biogen Idec: Employment, Equity Ownership. Smith:Biogen Idec: Employment, Equity Ownership. Kistanova:Biogen Idec: Employment, Equity Ownership. Moore:Biogen Idec: Employment, Equity Ownership. Acosta:Biogen Idec: Employment, Equity Ownership. Kole:Biogen Idec: Employment, Equity Ownership. Light:Biogen Idec: Employment, Equity Ownership. Peters:Biogen Idec: Employment. Jiang:Biogen Idec: Employment, Equity Ownership.

Frequencies of fatal cellular and cell-free DNA in maternal blood of pregnant cynomolgus monkeys (Macaca fascicularis)

Frequencies of fatal cellular and cell-free DNA in maternal blood of pregnant cynomolgus monkeys (Macaca fascicularis)

MORC1 exhibits cross-species differential methylation in association with early life stress as well as genome-wide association with MDD.

Early life stress (ELS) is associated with increased vulnerability for diseases in later life, including psychiatric disorders. Animal models and human studies suggest that this effect is mediated by epigenetic mechanisms. In humans, epigenetic studies to investigate the influence of ELS on psychiatric phenotypes are limited by the inaccessibility of living brain tissue. Due to the tissue-specific nature of epigenetic signatures, it is impossible to determine whether ELS induced epigenetic changes in accessible peripheral cells, for example, blood lymphocytes, reflect epigenetic changes in the brain. To overcome these limitations, we applied a cross-species approach involving: (i) the analysis of CD34+ cells from human cord blood; (ii) the examination of blood-derived CD3+ T cells of newborn and adolescent nonhuman primates (Macaca mulatta); and (iii) the investigation of the prefrontal cortex of adult rats. Several regions in MORC1 (MORC family CW-type zinc finger 1; previously known as: microrchidia (mouse) homolog) were differentially methylated in response to ELS in CD34+ cells and CD3+ T cells derived from the blood of human and monkey neonates, as well as in CD3+ T cells derived from the blood of adolescent monkeys and in the prefrontal cortex of adult rats. MORC1 is thus the first identified epigenetic marker of ELS to be present in blood cell progenitors at birth and in the brain in adulthood. Interestingly, a gene-set-based analysis of data from a genome-wide association study of major depressive disorder (MDD) revealed an association of MORC1 with MDD.

Genetic diversity, haplotypes and allele groups of Duffy binding protein (PkDBPαII) of Plasmodium knowlesi clinical isolates from Peninsular Malaysia

BackgroundThe monkey malaria parasite Plasmodium knowlesi is now recognized as the fifth species of Plasmodium that can cause human malaria. Like the region II of the Duffy binding protein of P. vivax (PvDBPII), the region II of the P. knowlesi Duffy binding protein (PkDBPαII) plays an essential role in the parasite’s invasion into the host’s erythrocyte. Numerous polymorphism studies have been carried out on PvDBPII, but none has been reported on PkDBPαII. In this study, the genetic diversity, haplotyes and allele groups of PkDBPαII of P. knowlesi clinical isolates from Peninsular Malaysia were investigated.MethodsBlood samples from 20 knowlesi malaria patients and 2 wild monkeys (Macaca fascicularis) were used. These samples were collected between 2010 and 2012. The PkDBPαII region of the isolates was amplified by PCR, cloned into Escherichia coli, and sequenced. The genetic diversity, natural selection and haplotypes of PkDBPαII were analysed using MEGA5 and DnaSP ver. 5.10.00 programmes.ResultsFifty-three PkDBPαII sequences from human infections and 6 from monkeys were obtained. Comparison at the nucleotide level against P. knowlesi strain H as reference sequence showed 52 synonymous and 76 nonsynonymous mutations. Analysis on the rate of these mutations indicated that PkDBPαII was under purifying (negative) selection. At the amino acid level, 36 different PkDBPαII haplotypes were identified. Twelve of the 20 human and 1 monkey blood samples had mixed haplotype infections. These haplotypes were clustered into 2 distinct allele groups. The majority of the haplotypes clustered into the large dominant group.ConclusionsOur present study is the first to report the genetic diversity and natural selection of PkDBPαII. Hence, the haplotypes described in this report can be considered as novel. Although a high level of genetic diversity was observed, the PkDBPαII appeared to be under purifying selection. The distribution of the haplotypes was skewed, with one dominant major and one minor group. Future study should investigate PkDBPαII of P. knowlesi from Borneo, which hitherto has recorded the highest number of human knowlesi malaria.

Th1-skewed tissue responses to a mycolyl glycolipid in mycobacteria-infected rhesus macaques

Trehalose 6,6′-dimycolate (TDM) is a major glycolipid of the cell wall of mycobacteria with remarkable adjuvant functions. To avoid detection by the host innate immune system, invading mycobacteria down-regulate the expression of TDM by utilizing host-derived glucose as a competitive substrate for their mycolyltransferases; however, this enzymatic reaction results in the concomitant biosynthesis of glucose monomycolate (GMM) which is recognized by the acquired immune system. GMM-specific, CD1-restricted T cell responses have been detected in the peripheral blood of infected human subjects and monkeys as well as in secondary lymphoid organs of small animals, such as guinea pigs and human CD1-transgenic mice. Nevertheless, it remains to be determined how tissues respond at the site where GMM is produced. Here we found that rhesus macaques vaccinated with Mycobacterium bovis bacillus Calmette–Guerin mounted a chemokine response in GMM-challenged skin that was favorable for recruiting T helper (Th)1 T cells. Indeed, the expression of interferon-γ, but not Th2 or Th17 cytokines, was prominent in the GMM-injected tissue. The GMM-elicited tissue response was also associated with the expression of monocyte/macrophage-attracting CC chemokines, such as CCL2, CCL4 and CCL8. Furthermore, the skin response to GMM involved the up-regulated expression of granulysin and perforin. Given that GMM is produced primarily by pathogenic mycobacteria proliferating within the host, the Th1-skewed tissue response to GMM may function efficiently at the site of infection.

Biomarkers of oral exposure to 3-nitro-1,2,4-triazol-5-one (NTO) and 2,4-dinitroanisole (DNAN) in blood and urine of rhesus macaques (Macaca mulatta)

The U.S. Department of Defense is using the chemicals 2,4-dinitroanisole (DNAN) and 3-nitro-1, 2,4-triazol-5-one (NTO) in new munitions development. In a screen for biomarkers of exposure, these compounds were measured in urine and blood of male rhesus monkeys after oral doses. NTO peaked at 4 h, with urinary concentrations at least 100-fold higher than that of blood or serum while 4-dinitrophenol (DNP), a metabolite of DNAN, appeared in blood at concentrations 10- to 20-fold higher than the parent compound. For human exposure monitoring, urine is optimal for NTO while the metabolite DNP in blood is best for DNAN.

144: Receptor occupancy and internalization of an anti-IL-7 receptor antibody

In vitro (or ex vivo) quantification of cellular parameters is often employed to inform systems models that define the relationship between drug exposure and response. We developed two flow cytometry based assays to evaluate the interaction between the IL-7 receptor (IL-7R) and an anti-IL-7R monoclonal antibody (Ab1). IL-7R occupancy and rate of internalization in the presence of Ab1 was determined using flow cytometry in monkeys and human whole blood. Two commercially available antibodies were used to detect IL-7R. One detected “free” and the other determined “total” receptor. Receptor internalization was measured ex vivo at various time points at 37 °C. The receptor occupancy assay was used in vivo following administration of Ab1 into cynomolgus monkeys at 0.3, 3, and 30 mg/kg. The amount of Ab1 bound to receptor and the total amount of receptor was quantified. The ex vivo receptor occupancy mean IC50 values in monkey and human whole blood were 5.64 ± 2.38 and 9.39 ± 1.54 ng/ml, respectively. The ex vivo rate of internalization of Ab1-IL-7R complex was characterized by a slope of 384 ± 24 and 2482 ± 319, for monkey and human, respectively. In the in vivo monkey study, no free IL-7R was detectable on the CD3 T cell surface at 0.25 h post-dose in all treatment groups. Free receptor levels returned to 78% on day 8 at 0.3 mg/kg, 55% on day 15 at 3 mg/kg, and 12% on day 15 at 30 mg/kg, compared to pre-dose levels. By Day 22, free IL-7R levels in all dose groups returned to their baseline. Ab1 treatment resulted in a significant reduction in total IL-7R. Effective biomeasures provide a link between species and the ex vivo and in vivo systems. Integration of these values into systems models reduces the number of model assumptions and increases the likelihood of success.

Telemetering system and its application for fruit cultivation in greenhouses.

Circuit of the temperature measurement equipment was improved in order to develop temperature telemetering system for cultivating fruit facilities. The traditional temperature measuring equipments used in monitoring temperature changes in greenhouse include mercury column, merbromine column, kerosene column glass rod thermometer and mechanical inductive thermometer. Compared with the traditional method of temperature measurement the new system reduces the labor requirement and promotes precision monitoring and control of the temperature. In addition, the temperature in the greenhouse was very accurate especially at night or in rainy and snowy weather. This new temperature telemetering system will promote the development of the fruit protection cultivation.

Natural Plasmodium infection in monkeys in the state of Rondônia (Brazilian Western Amazon)

BackgroundSimian malaria is still an open question concerning the species of Plasmodium parasites and species of New World monkeys susceptible to the parasites. In addition, the lingering question as to whether these animals are reservoirs for human malaria might become important especially in a scenario of eradication of the disease. To aid in the answers to these questions, monkeys were surveyed for malaria parasite natural infection in the Amazonian state of Rondônia, Brazil, a state with intense environmental alterations due to human activities, which facilitated sampling of the animals.MethodsParasites were detected and identified in DNA from blood of monkeys, by PCR with primers for the 18S rRNA, CSP and MSP1 genes and sequencing of the amplified fragments. Multiplex PCR primers for the 18S rRNA genes were designed for the parasite species Plasmodium falciparum and Plasmodium vivax, Plasmodium malariae/Plasmodium brasilianum and Plasmodium simium.ResultsAn overall infection rate of 10.9% was observed or 20 out 184 monkey specimens surveyed, mostly by P. brasilianum. However, four specimens of monkeys were found infected with P. falciparum, two of them doubly infected with P. brasilianum and P. falciparum. In addition, a species of monkey of the family Aotidae, Aotus nigriceps, is firstly reported here naturally infected with P. brasilianum. None of the monkeys surveyed was found infected with P. simium/P. vivax.ConclusionThe rate of natural Plasmodium infection in monkeys in the Brazilian state of Rondônia is in line with previous surveys of simian malaria in the Amazon region. The fact that a monkey species was found that had not previously been described to harbour malaria parasites indicates that the list of monkey species susceptible to Plasmodium infection is yet to be completed. Furthermore, finding monkeys in the region infected with P. falciparum clearly indicates parasite transfer from humans to the animals. Whether this parasite can be transferred back to humans and how persistent the parasite is in monkeys in the wild so to be efficient reservoirs of the disease, is yet to be evaluated. Finding different species of monkeys infected with this parasite species suggests indeed that these animals can act as reservoirs of human malaria.

Comparison of the in vitro and in vivo metabolism of Cladribine (Leustatin, Movectro) in animals and human

1. New insight into the in vitro and in vivo metabolism of Cladribine (2-chloro-2′-deoxyadenosine, [2-CdA]) are presented.2. Following incubation of [14C]-2-CdA in mouse, rat, rabbit, dog, monkey and human hepatocyte cultures, variable turnover was observed with oxidations and direct glucuronidation pathways. The oxidative cleavage to 2-chloroadenine (2-CA, M1) was only observed in rabbit and rat.3. Following incubation of [14C]-2-CdA in whole blood from mouse, monkey and human, a significant turnover was observed. The main metabolites in monkey and human were 2-chlorodeoxyinosine (M11, 16% of total radioactivity) and 2-chlorodeoxyinosine (M12, 43%). In mouse, 2-CA was the major metabolite (2-CA; M1, 73%).4. After single intravenous and oral administration of [14C]-2-CdA to mice, 2-chlorodeoxyinosine (M11) was confirmed in plasma, while 2-chlorohypoxanthine (M12) and 2-CA (M1) were found in urine.5. Overall, the use of [14C]-2-CdA both in vitro (incubations in mouse, monkey and human whole blood) and in vivo (mouse) has confirmed the existence of an additional metabolism pathway leading to the formation of 2-chlorodeoxyinosine (M11) and 2-chlorohypoxanthine (M12). Formation of these two metabolites demonstrates that Cladribine as free form is not fully resistant to adenosine deaminase as suggested earlier, an enzyme involved in its mode of action.

Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.

Synthesis and characterization in monkey of [11C]SP203 as a radioligand for imaging brain metabotropic glutamate 5 receptors

[(18)F]SP203 (3-fluoro-5-(2-(2-([(18)F]fluoromethyl)-thiazol-4-yl)ethynyl)benzonitrile) is an effective high-affinity and selective radioligand for imaging metabotropic 5 receptors (mGluR5) in human brain with PET. To provide a radioligand that may be used for more than one scanning session in the same subject in a single day, we set out to label SP203 with shorter-lived (11)C (t (1/2) = 20.4 min) and to characterize its behavior as a radioligand with PET in the monkey. Iodo and bromo precursors were obtained by cross-coupling 2-fluoromethyl-4-((trimethylsilyl)ethynyl)-1,3-thiazole with 3,5-diiodofluorobenzene and 3,5-dibromofluorobenzene, respectively. Treatment of either precursor with [(11)C]cyanide ion rapidly gave [(11)C]SP203, which was purified with high-performance liquid chromatography. PET was used to measure the uptake of radioactivity in brain regions after injecting [(11)C]SP203 intravenously into rhesus monkeys at baseline and under conditions in which mGluR5 were blocked with 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP). The emergence of radiometabolites in monkey blood in vitro and in vivo was assessed with radio-HPLC. The stability of [(11)C]SP203 in human blood in vitro was also measured. The iodo precursor gave [(11)C]SP203 in higher radiochemical yield (>98 %) than the bromo precursor (20-52 %). After intravenous administration of [(11)C]SP203 into three rhesus monkeys, radioactivity peaked early in brain (average 12.5 min) with a regional distribution in rank order of expected mGluR5 density. Peak uptake was followed by a steady decline. No radioactivity accumulated in the skull. In monkeys pretreated with MTEP before [(11)C]SP203 administration, radioactivity uptake in brain was again high but then declined more rapidly than in the baseline scan to a common low level. [(11)C]SP203 was unstable in monkey blood in vitro and in vivo, and gave predominantly less lipophilic radiometabolites. By contrast, [(11)C]SP203 was stable in human blood in vitro. [(11)C]SP203 emulates [(18)F]SP203 with regard to providing a sizeable mGluR5-specific signal in monkey brain, and advantageously avoids troublesome accumulation of radioactivity in bone. Although [(11)C]SP203 is unsuitable for mGluR5 quantification in monkey brain, its evaluation as a PET radioligand for studying human brain mGluR5 is nevertheless warranted.

Expression and biological characterization of an anti-CD20 biosimilar candidate antibody

The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials.