Days In Blood Research Articles

Impact of Heat Stress on the Balance between Oxidative Markers and the Antioxidant Defence System in the Plasma of Mid-Lactating Modicana Dairy Cows.

Animal health is affected during heat stress as a result of impaired immune responses, increased production of reactive oxygen species, and/or a deficiency of antioxidants. This leads to an imbalance between oxidants and antioxidants and results in oxidative stress. Heat stress is usually measured in dairy cattle via the temperature-humidity index (THI). In the present study, we aimed at assessing the influence of incremental THI on the balance between oxidative markers and the antioxidant defence system in the plasma of Modicana cows. Twenty-four multiparous, mid-lactating dairy cows were divided into two groups on the basis of different levels of mean THI reached in the period of the previous week up until the day of blood and milk sampling (April THI1:55, May THI2:68, June THI3:71, July THI4:80). The blood samples were collected to measure reactive oxygen metabolites (ROM) and antioxidant defense markers (ferric reducing ability of plasma (FRAP), paraoxonase (PON), advanced oxidation protein products (AOPP), plasma thiol groups (SHp), as well as lipid-soluble antioxidant pro-vitamin (β-carotene) and vitamins (tocopherol and retinol). Milk characteristics, haematological values, and plasma biochemical metabolites were also evaluated. Results showed a significant increase in ROM (p < 0.05) and a significant decrease in PON (p < 0.05), AOPP (p < 0.05), and β-carotene (p < 0.001). Incremental THI significantly decreased levels of milk fat content, red and white blood cells, plasma glucose, and non-esterified fatty acids, while significantly increasing monocytes and the concentrations of β-hydroxybutyrate and creatinine, but not fructosamine. The results of the study show that heat stress significantly affects reactive oxygen species production and antioxidant parameters. Carotenoid supplementation should be considered to alleviate the impact of these effects.

DELINEATION OF HEMATOLOGICAL CHANGES OCCURRING IN STORED CITRATE PHOSPHATE DEXTROSE ADENINE CPDA-1 BLOOD

Objectives: To study the changes in red cell indices and counts over a period of 42 days in blood stored in blood bags. Materials and Methods: This research was done in Blood Bank, Khyber Teaching Hospital, Peshawar in collaboration with the main laboratory, Khyber Teaching Hospital, Peshawar. About 450 ml of whole blood was taken from 200 donors into Citrate Phosphate Dextrose Adenine blood bags. The storage temperature for the blood bags was maintained between 2–8°C. Blood samples were collected from blood bags. At days 1 and 42, using a haematology analyser, parameters including haemoglobin, RBC count, WBC count, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and platelet counts were measured. Mean and standard deviation were used for quantitative variables. Frequency and percentages were used for qualitative variables. Results: Through days 1 to 42, Haemoglobin (Hb) level decreased from 13.4gm/dl to 12.78 gm/dl, white cell count (WBC) decreased from 6.03x109/l to 2.95 x109/l, platelets count fell from 207 x109/l to 121 x109/l, neutrophils decreased from 59.6% to 23.8% while lymphocyte count increased from 28.36% to 66.3%. Conclusion: There is a decrease in haemoglobin level and white cell count over a period of 42 days of storage of blood bags. Lymphocyte counts increased during the storage duration of blood bags.

The role of WTX212, an erythrocyte-anti-PD1 antibody conjugate, in anti-tumor activities in tumor models and cancer patients with acquired resistance to immunotherapy.

29 Background: Despite the success of immune-checkpoint blockade (ICB) treatments, a considerable proportion of patients cannot benefit from ICB therapy due to acquired resistance. Recent studies have revealed that successful immunotherapy drives new immune responses arising from peripheral immune system instead of reinvigorating the pre-existing responses in the tumor. Here we designed WTX212, autologous engineered red blood cells (RBCs) conjugated with anti-PD1 antibodies capable of activating the peripheral immune system. We also evaluated its anti-cancer activities in anti-PD1 non-responsive tumor models and a first-in-human (FIH) trial. Methods: The anti-tumor efficacy of WTX212 was evaluated in both PD1 responsive (MC38 colorectal cancer) and non-responsive (KP lung cancer and pB3 breast cancer) mouse tumor models (n=7 / group). Mice were monitored twice a week for clinical signs and tumor sizes. The mechanism of action was investigated in splenectomized mice and by analyzing the biodistribution of WTX212 using a radiolabeling method. The immune landscape was also profiled through cytof mass and scRNA-seq analyses in the spleen, tumor and peripheral blood. The FIH trial (NCT05707325) explores safety, pharmacokinetics and mechanism in cancer patients with acquired resistance to ICB. WTX212 was manufactured by covalently engineering anti-PD1 antibody with RBCs isolated from patients. By 11th April 2023, 3 patients have been enrolled and received at least one dose of WTX212 (20*1010 cells, IV, Q3W). Results: Treatment of WTX212 significantly suppressed tumor growth up to 100% in anti-PD1 responsive and non-responsive tumor models. Unlike antibodies, WTX212 primarily distributed to the spleen and vascular system. The anti-tumor effect of WTX212 was abrogated in corresponding splenectomized mice. Compared to anti-PD1 antibody, single-cell analysis identified the immune landscape remodeling in the spleen from WTX212-treated mice, with down-regulation of PD-L1+ myeloid-derived suppressor cells (MDSCs) and up-regulation of effector CD8+ T cells. In the FIH trial, 3 patients have been administered with total thirteen cycles of WTX212. No treatment-related AEs greater than Grade 2 were observed. WTX212 remained in the peripheral blood day 21 post transfusion. WTX212 rapidly activates peripheral immune systems with decreased MDSCs, Tregs and increased effector T cells in peripheral blood from all patients. The first patient who received six cycles of WTX212 treatment demonstrated stable disease with a 20% reduction at the target lesion. Conclusions: WTX212 demonstrated anti-tumor activities by activating the peripheral immune system to overcome resistance to immunotherapy in both preclinical models and human patients. WTX212 is well-tolerated supporting the future clinical trials. Clinical trial information: NCT05707325 .

Nicotinamide enhances natural killer cell function and yields remissions in patients with non-Hodgkin lymphoma.

Allogeneic natural killer (NK) cell adoptive transfer has shown the potential to induce remissions in relapsed or refractory leukemias and lymphomas, but strategies to enhance NK cell survival and function are needed to improve clinical efficacy. Here, we demonstrated that NK cells cultured ex vivo with interleukin-15 (IL-15) and nicotinamide (NAM) exhibited stable induction of l-selectin (CD62L), a lymphocyte adhesion molecule important for lymph node homing. High frequencies of CD62L were associated with elevated transcription factor forkhead box O1 (FOXO1), and NAM promoted the stability of FOXO1 by preventing proteasomal degradation. NK cells cultured with NAM exhibited metabolic changes associated with elevated glucose flux and protection against oxidative stress. NK cells incubated with NAM also displayed enhanced cytotoxicity and inflammatory cytokine production and preferentially persisted in xenogeneic adoptive transfer experiments. We also conducted a first-in-human phase 1 clinical trial testing adoptive transfer of NK cells expanded ex vivo with IL-15 and NAM (GDA-201) combined with monoclonal antibodies in patients with relapsed or refractory non-Hodgkin lymphoma (NHL) and multiple myeloma (MM) (NCT03019666). Cellular therapy with GDA-201 and rituximab was well tolerated and yielded an overall response rate of 74% in 19 patients with advanced NHL. Thirteen patients had a complete response, and 1 patient had a partial response. GDA-201 cells were detected for up to 14 days in blood, bone marrow, and tumor tissues and maintained a favorable metabolic profile. The safety and efficacy of GDA-201 in this study support further development as a cancer therapy.

Comparison between liquid biopsy and tissue NGS in two cohorts of advanced NSCLC.

e21043 Background: Molecular testing is mandatory in advanced non-small cell lung cancer (NSCLC) due to prognostic and therapeutic implications. Correlation between blood and tissue next-generation sequencing (NGS) has recently been demonstrated. Methods: A retrospective observational study of two cohorts of NSCLC patients who underwent liquid biopsy at the time of advanced disease diagnosis was conducted. In cohort 1 (N = 129), liquid biopsy was performed using a 74 genes panel (Guardant360 CDx). In cohort 2 (N = 149), NGS was performed in peripheral blood using a 63 genes panel (FoundationOne Liquid CDx). For tissue NGS analysis, Oncomine Precision Assay and Oncomine Focus Assay, with 50 and 52 genes, respectively, were performed. Data were analyzed in both cohorts independently, and compared by statistical descriptive methods. Results: In cohort 1, 74/129 (57.4%) were men and 55/129 (42.6%) were non-smokers or had a ≤ 20-pack-year tobacco history. The most frequent histology was adenocarcinoma (99/129; 76.7%). In 14/129 (10.9%) patients, a second determination of NGS in peripheral blood was necessary and 1/129 (0.8%) had a total of three determinations. Tissue NGS was not performed in 32/129 (24.8%) because of insufficient sample. In cohort 2, 99/149 (66.4%) were men and 48/149 (32.2%) were non-smokers or had a ≤ 20-pack-year tobacco history. The most frequent histology was adenocarcinoma (99/149; 66.4%). Tissue NGS was not performed in 40/149 (26.8%) due to insufficient sample (26/40; 65%) or technical difficulties to perform a biopsy (14/40; 35%). In cohort 1, pathogenic mutations were detected in 119/129 (92.2%) and in 62/97 (63.9%) patients (p &lt; 0.001), with median time to obtain results of 11 (IQR 9-14) and 29 (IQR 22-39) days for blood and tissue NGS, respectively (p &lt; 0.001). Actionable mutations included alterations of levels I-II according to ESCAT and were found in 40/129 (31%) and 33/97 (34%) patients using blood and tissue NGS, respectively (p = 0.63). In cohort 2, circulating tumor DNA was not detected in 5/149 (3.5%) patients. Pathogenic mutations were found in 123/149 (82.6%) and in 71/109 (65.1%) patients (p = 0.001), with median time to obtain results of 15 (IQR 14-15) and 27 (IQR 16-27) days for blood and tissue NGS, respectively (p &lt; 0.001). Actionable mutations were detected in 41/149 (27.5%) and 41/109 (37.6%) using blood and tissue NGS, respectively (p = 0.08). Targeted therapy was given to 31/129 (24%) and 39/149 (26.2%) patients in cohort 1 and 2, respectively. Conclusions: To our knowledge, this is the first retrospective observational study where liquid biopsy and tissue NGS are compared in two different cohorts of patients with advanced NSCLC. Time to obtain results was shorter for blood NGS than tissue sequencing, and liquid biopsy provided molecular information in cases in which tissue NGS is not available. Further studies to identify patients in which liquid biopsy could replace tissue analysis are needed.

WTX212, an erythrocyte-anti-PD1 antibody conjugate, to demonstrate anti-tumor activities in tumor models and patients with cancer with acquired resistance to immunotherapy.

e14529 Background: Despite the success of immune-checkpoint blockade (ICB) treatments, a considerable proportion of patients cannot benefit from ICB therapy due to acquired resistance. One major challenge is the systemic suppression of immune cells induced by the tumor. Recent studies have revealed that successful immunotherapy drives new immune responses arising from peripheral immune system instead of reinvigorating the pre-existing responses in the tumor. Here we designed WTX212, autologous engineered red blood cells (RBCs) conjugated with anti-PD1 antibodies capable of activating the peripheral immune system. We also evaluated its anti-cancer activities in anti-PD1 non-responsive tumor models and a first-in-human (FIH) trial. Methods: The anti-tumor efficacy of WTX212 was evaluated in both PD1 responsive (MC38 colorectal cancer) and non-responsive (KP lung cancer and pB3 breast cancer) mouse tumor models (n=7 / group). Mice were monitored twice a week for clinical signs and tumor sizes. The mechanism of action was investigated in splenectomized mice and by analyzing the biodistribution of WTX212 using a radiolabeling method. The immune landscape was also profiled through cytof mass and scRNA-seq analyses in the spleen, tumor and peripheral blood. The FIH trial (NCT05707325) explores safety, pharmacokinetics and mechanism in cancer patients with acquired resistance to ICB. WTX212 was manufactured by covalently engineering anti-PD1 antibody with RBCs isolated from patients. By 6th Feb 2023, 3 patients have been enrolled and received at least one dose of WTX212 (20*1010 cells, IV, Q3W). Results: Treatment of WTX212 significantly suppressed tumor growth up to 100% in anti-PD1 responsive and non-responsive tumor models and activated tumor microenvironment with increased infiltrated T cells. Unlike antibodies, WTX212 primarily distributed to the spleen and vascular system. The anti-tumor effect of WTX212 was abrogated in corresponding splenectomized mice. Compared to anti-PD1 antibody, single-cell analysis identified the immune landscape remodeling in the spleen from WTX212-treated mice, with down-regulation of PD-L1+ myeloid-derived suppressor cells (MDSCs) and up-regulation of effector CD8+ T cells. In the FIH trial, 3 patients have been administered with total eight cycles of WTX212. No treatment-related AEs greater than Grade 2 were observed. WTX212 remained in the peripheral blood day 21 post transfusion. WTX212 rapidly activates peripheral immune systems with decreased MDSCs, Tregs and increased effector T cells in peripheral blood from all patients. Conclusions: WTX212 demonstrated anti-tumor activities by activating the peripheral immune system to overcome resistance to immunotherapy in both preclinical models and human patients. WTX212 is well-tolerated supporting the future clinical trials.

Viral dynamics in patients with monkeypox infection: a prospective cohort study in Spain.

Monkeypox DNA has been detected in skin lesions, saliva, oropharynx, urine, semen, and stool of patients infected during the 2022 clade IIb outbreak; however, the viral dynamics within these compartments remain unknown. We aimed to characterise the viral load kinetics over time in various parts of the body. This was an observational, prospective, multicentre study of outpatients diagnosed with monkeypox in two hospitals and two sexual health clinics in Spain between June 28, 2022, and Sept 22, 2022. Men and women aged over 18 years were eligible if they reported having symptom onset within the previous 10 days of presentation, and were ineligible if disease was severe enough to be admitted to hospital. Samples were collected from five body locations (skin lesions, oropharynx, rectum, semen or vagina, and a dried blood spot) at six time points up to 57 days after the screening visit. Samples were analysed by quantitative PCR and a subset by cell culture. The primary endpoint was time from symptom onset to viral DNA clearance. Overall, 1663 samples were collected from 77 study participants. 75 (97%) participants were men, the median age was 35·0 years (IQR 29·0-46·0), and 39 (51%) participants were living with HIV. The median time from symptom onset to viral clearance was 25 days (95% CI 23-28) in the skin lesions, 16 days (13-19) in the oropharynx, 16 days (13-23) in the rectum, 13 days in semen (9-18), and 1 day in blood (0-5). The time from symptom onset to viral clearance for 90% of cases was 41 days (95% CI 34-47) in skin lesions and 39 days (27-56) in semen. The median viral load in skin lesions was 7·3 log10 copies per mL (IQR 6·5-8·2) at baseline, compared with 4·6 log10 copies per mL (2·9-5·8) in oropharyngeal samples, 5·0 log10 copies per mL (2·9-7·5) in rectal samples, 3·5 log10 copies per mL (2·9-4·7) in semen samples, and 4·0 log10 copies per mL (4·0-4·0) in blood specimens. Replication-competent viruses were isolated in samples with high DNA levels (>6·5 log10 copies per mL). In immunocompetent patients with mild monkeypox disease, PCR data alone would suggest a contact isolation period of 3 to 6 weeks but, based on detection of replication-competent virus, this time could be reduced. Based on findings from this cohort of patients, semen testing and prolonged use of condoms after recovery from monkeypox might not be necessary. University Hospital Germans Trias i Pujol and the YoMeCorono. For the Spanish translation of the abstract see Supplementary Materials section.

Site of infusion of a commercially available direct-fed microbial on performance and digestibility in lactating Holstein cows

The objective of this research was to evalu ate site of infusion of a commercially available direct-fed microbial (DFM) containing 109 cfu/g of Lactobacillus acidophilus and 109 cfu/g Propionibacterium freudenreichii on DMI, rumen kinetics, ruminal VFA, digestibility, milk production, milk components, and blood metabolites in lactating dairy cows. Four Holstein cows equipped with ruminal cannulas were used in a Latin square design experiment with 4 periods. Each 37-d period consisted of 14 d of no treatment to prevent crossover contamination, 14 d of adaptation to treatments, 8 d of sampling, and 1 d for ruminal evacuations. Within each period, cows were assigned to 1 of 4 treatments: (1) cows were fed a TMR formulated to meet or exceed nutrient requirements plus 5 g of lactose twice daily without the addition of DFM (control); (2) cows were fed the TMR with a daily dose of DFM top dressed on the feed twice daily (TD); (3) cows were fed the TMR with ruminal infusion of the DFM administered twice daily (RuI); or (4) cows were fed the TMR plus abomasal infusion of the DFM twice daily (AbI). During the sampling period within each period, DMI and milk production were measured daily with set days for blood and rumen fluid collection. Data were analyzed using the MIXED procedure of SAS with animal within period as a random effect. Dry matter intake was not different among treatments. No differences were detected in rumen kinetics, pH, individual VFA or VFA ratios, ammonia, or digestibility. There were no differences in kilograms of milk production (P > 0.87); SCC (P > 0.54); or percentage of butter fat (P > 0.21), milk protein (P > 0.83), lactose (P > 0.91), SNF (P > 0.88), and MUN (P > 0.49). No difference existed in most of the milk fatty acids except for 8:0, which had a greater concentration (P > 0.01) in AbI versus control, TD, and RuI. Route of administering DFM overall had no effects on DMI, rumen kinetics, ruminal VFA, digestibility, milk production, or milk components in the present experiment.

RAAV expressing recombinant antibody for emergency prevention and long-term prophylaxis of COVID-19.

Numerous agents for prophylaxis of SARS-CoV-2-induced diseases are currently registered for the clinical use. Formation of the immunity happens within several weeks following vaccine administration which is their key disadvantage. In contrast, drugs based on monoclonal antibodies, enable rapid passive immunization and therefore can be used for emergency pre- and post-exposure prophylaxis of COVID-19. However rapid elimination of antibody-based drugs from the circulation limits their usage for prolonged pre-exposure prophylaxis. In current work we developed a recombinant adeno-associated viral vector (rAAV), expressing a SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibody P2C5 fused with a human IgG1 Fc fragment (P2C5-Fc) using methods of molecular biotechnology and bioprocessing. A P2C5-Fc antibody expressed by a proposed rAAV (rAAV-P2C5-Fc) was shown to circulate within more than 300 days in blood of transduced mice and protect animals from lethal SARS-CoV-2 virus (B.1.1.1 and Omicron BA.5 variants) lethal dose of 105 TCID50. In addition, rAAV-P2C5-Fc demonstrated 100% protective activity as emergency prevention and long-term prophylaxis, respectively. It was also demonstrated that high titers of neutralizing antibodies to the SARS-CoV-2 virus were detected in the blood serum of animals that received rAAV-P2C5-Fc for more than 10 months from the moment of administration.Our data therefore indicate applicability of an rAAV for passive immunization and induction of a rapid long-term protection against various SARS-CoV-2 variants.

“A Day of Blood and Valor”: terrorism and social tensions in 1970s Israel

ABSTRACT On June 13, 1974, four Palestinian militants penetrated kibbutz Shamir in northern Israel, killing three women before being killed by an ad-hoc force of kibbutz members. The attack on Shamir generated a new discourse in Israel about civilian reaction to terrorism and the concept of the citizen-soldier. But this discourse was intertwined with contemporaneous social and ethnic conflicts, as it included comparisons between the events in Shamir and in other communities struck by Palestinian terrorism around the same period. Analyzing the public reactions to the raids on Shamir and other Israeli communities – mainly the towns of Kiryat Shmona and Ma’alot – this article argues that, in contrast to received opinion, security threats did not serve as a social glue keeping together a divided Israeli society, but rather accentuated ethnic and social tensions plaguing Israel in the 1970s.

Precise quantitative evaluation of pharmacokinetics of cisplatin using a radio-platinum tracer in tumor-bearing mice.

ObjectiveThe platinum-based antineoplastic drug cisplatin is commonly used for chemotherapy in clinics. This work aims to demonstrate a radio-platinum tracer is useful for precisely quantifying small amounts of platinum in pharmacokinetics studies.MethodsA cisplatin radiotracer (radio-cisplatin) was synthesized, and a comprehensive evaluation of cisplatin over 7 days after its intravenous injection into nude mice bearing a subcutaneous lung tumor (H460) was conducted.ResultsA biphasic retention curve in the whole body and blood was observed [T1/2(α) = 1.14 h, T1/2(β) = 5.33 days for the whole body, and T1/2(α) = 23.9 min, T1/2(β) = 4.72 days for blood]. The blood concentration decreased within 1 day after injection. Most of the intact cisplatin was excreted via the kidneys in the early time points, and a small part was distributed in tissues including tumors. The plasma protein binding rate of cisplatin increased rapidly after injection, and the protein-bound cisplatin remained in the blood longer than intact cisplatin. The peak uptake in H460 tumors was 4.7% injected dose per gram at 15 min after injection, and the area under the curve (AUC0–7 days) was approximately one-half to one-third of the AUC0–7 days in the kidneys, liver, and bone, where some toxicity is observed in humans.ConclusionThe radio-platinum tracer revealed the highly quantitative biodistribution of cisplatin, providing insights into the properties of cisplatin, including its adverse effects. The tracer enables a precise evaluation of pharmacokinetics for platinum-based drugs with high sensitivity.

Acute serum protein and cytokine response of single dose of prednisone in adult volunteers

Pharmacological glucocorticoids are the most prescribed anti-inflammatory medications, and are chemical variants of cortisol, the circadian and stress hormone. Both endogenous and pharmacological glucocorticoids bind the glucocorticoid receptor (NR3C1) with high affinity, and both then bind downstream gene promoter elements (GRE) to drive positive gene transcription of many proteins. Glucocorticoid/GR complexes also bind distinct negative gene promoter elements (nGRE) to inhibit expression of genes involved in NF-κB innate immunity signaling. We sought to define the acute response of a single dose of prednisone (0.2 mg/kg) in young adult volunteers, with blood samples taken at baseline, 2, 3, 4 and 6 h post-oral dose. To control for circadian morning cortisol hitting the same molecular pathways, a day of blood draws was done without oral prednisone (same time of day), one day prior to drug day. Serum samples were processed for steroid hormone profiles (mass spectrometry; 9 steroidal hormones), proteomics (SOMAscan aptamer panels, 1,305 proteins), and inflammatory markers (Meso Scale Discovery; 10 pro-inflammatory cytokines). The pharmacological effect of the prednisone dose was shown by significant declines of adrenal steroids by 3 h after dosing. IL-10 showed drug-related increase to 4 hrs, then decrease to 6 hrs. IL-8 showed drug-related decrease in serum by 4 h, consistent with direct negative action of GR/ligand on IL-8 gene promoter. Proteomics data showed beta-2 microglobulin, TNFSF15, TSH, CST3, NBL1 to show time-related decreases with prednisone, while CXCL13 showed increases, although these require validation. In summary, a single low dose of prednisone leads to broad suppression of the adrenal axis within 3 h, and down-regulation of inflammatory serum proteins by 6 h.

Excretion and Distribution of the 60-kDa PEG Moiety Used in BAY 94-9027 in Male Wistar Rats

Introduction: PEGylation is a widely used method to prolong the half-life of therapeutic proteins. Long-acting factor VIII (FVIII) products developed through PEGylation may potentially reduce the burden associated with prophylaxis in patients with hemophilia A. BAY 94-9027 is a prolonged-half-life FVIII product modified through site-specific addition of a 60-kDa (2 × 30-kDa branched) PEG via a maleimide linker to a cysteine amino acid. BAY 1025662 is the 60-kDa PEG moiety used for PEGylation including linker and cysteine (PEG-60-Mal-Cys). In toxicology studies, BAY 94-9027 and BAY 1025662 were not associated with any adverse events or PEG-related cellular vacuolation (Ivens et al. Haemophilia. 2013;19:11-20). The rate-limiting clearance of the PEG part of PEGylated proteins after metabolism is expected to be similar to that when the corresponding PEG is administered alone (Baumann et al. Drug Discov Today . 2014;19:1623-1631). The study objective was to investigate the pharmacokinetics and mass balance of total radioactivity in male Wistar rats after a single intravenous (IV) administration of [14C]-labeled BAY 1025662, including size exclusion chromatography profiles of urine at selected time points.Methods: Radiolabeled BAY 1025662 ([prop-14C]BAY 1025662) was synthesized using a 2-step process resulting in a maleimide linker bearing the [14C] label at the propionic acid moiety. Radioactivity following a single IV dose of 11 mg/kg (the estimated maximal human PEG dose over ~30 years' treatment with BAY 94-9027) was determined in body fluids, organs and tissues, and in excreta by liquid scintillation counting after appropriate sample preparation and by quantitative whole-body autoradiography during and at the end of the study.Results: Single-dose mass balance studies using [prop-14C]BAY 1025662 showed that 30.4% of administered radioactivity was excreted within 1 week (23.7% in urine and 6.3% in feces); cumulative excretion at day 168 via urine and feces was 82.6% of the administered dose (68.4% via urine, 13.8% via feces). High-performance liquid chromatography profiling analysis of rat urine suggested that BAY 1025662 is excreted nearly unchanged (74% of total radioactivity, corresponding to 17.6% of dose, present unchanged in urine in the 0- to 168-hour interval). Radioactivity was continuously, albeit slowly, eliminated throughout the study; elimination half-life was 23 and 24 days in blood and plasma, respectively. The corresponding elimination half-lives of radioactivity were 33, 31, 39, 54, and 68 days in the liver, skin, carcass, testes, and kidneys, respectively. Elimination was almost complete at the end of experiment, with a total recovery of radioactivity (cumulative excretion plus residues in animals) of 89% on day 168. Following [prop-14C]BAY 1025662 administration, radioactivity was slowly distributed from blood to peripheral organs, reaching maximum concentration 2 hours postdose; highest maximum concentration was observed in blood, followed by lung and liver. At day 168, only ~4% of the administered radioactive dose was still present in the animal body, partially localized in endocrine/exocrine glands and lymphatic organs. No residual radioactivity was observed in the brain, indicating no crossing of the blood-brain barrier.Conclusions: Following single-dose administration of [prop-14C]BAY 1025662, elimination was almost complete by the end of the experiment on day 168. These results suggest that there are excretion processes in place for high-molecular-weight PEGs such as the PEG 60 moiety used in BAY 94-9027. Two clearance processes are proposed for large PEGs (>30 kDa), as indicated by a faster initial excretion over the first 1-2 weeks, followed by a nonspecific uptake into cells by pinocytosis leading to a longer elimination half-life. The PEG moiety can be recycled from inside the cells back to the plasma, possibly by exocytosis, to then undergo further (mainly renal) excretion (Baumann et al. Drug Discov Today . 2014;19:1623-1631). DisclosuresBaumann:Bayer AG: Employment. Schwarz:Bayer AG: Employment. Hucke:Bayer AG: Employment. Piel:Bayer AG: Employment. Sandmann:Bayer AG: Employment.

IgG modulation in male mice with reproductive failure after prenatal inflammation.

Sexual performance in adult male rats is highly sensitive to prenatal stress which can affect the functionality of the reproductive system and various brain structures involved in modulating sexual behavior. The immunomodulatory effect of mouse IgG on reproductive maturity in male offspring after LPS exposure in vivo and in vitro was studied. Prenatal IgG injection (20 µg/mouse) had a positive impact on the puberty of male mice whose mothers were exposed to LPS (100 µg/kg) on the 12th day of pregnancy. The numbers of Sertoli cells were increased, whereas the body weight and the number of symplastic spermatids were decreased in offspring as compared to LPS-treated animals. Besides, IgG had a positive effect on altered hormone levels: reduced estradiol level on the 5th and 14th postnatal days and increased testosterone level on the 30th postnatal day in blood that led to an increased number of mounting attempts in sexually mature males. The cAMP-dependent pathway may be involved in the regulation of the LPS-induced inflammation. IgG reduced the increased level of cAMP in mouse peritoneal macrophages activated by LPS in vitro. IgG is able to modulate inflammation processes but its exposure time is important.

Development and Validation of a Bioanalytical LC-MS/MS Method for Simultaneous Determination of Sirolimus in Porcine Whole Blood and Lung Tissue and Pharmacokinetic Application with Coronary Stents.

Sirolimus is a hydrophobic macrolide compound that has been used for long-term immunosuppressive therapy, prevention of restenosis, and treatment of lymphangioleiomyomatosis. In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous determination of sirolimus in both porcine whole blood and lung tissue. Blood and lung tissue homogenates were deproteinized with acetonitrile and injected into the LC-MS/MS system for analysis using the positive electrospray ionization mode. The drug was separated on a C18 reversed phase column with a gradient mobile phase (ammonium formate buffer (5 mM) with 0.1% formic acid and acetonitrile) at 0.2 mL/min. The selected reaction monitoring transitions of m/z 931.5 → 864.4 and m/z 809.5 → 756.5 were applied for sirolimus and ascomycin (the internal standard, IS), respectively. The method was selective and linear over a concentration range of 0.5–50 ng/mL. The method was validated for sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability in porcine whole blood and lung tissue homogenates, and all values were within acceptable ranges. The method was applied to a pharmacokinetic study to quantitate sirolimus levels in porcine blood and its distribution in lung tissue following the application of stents in the porcine coronary arteries. It enabled the quantification of sirolimus concentration until 2 and 14 days in blood and in lung tissue, respectively. This method would be appropriate for both routine porcine pharmacokinetic and bio-distribution studies of sirolimus formulations.

Rites of the Day of Blood (dies sanguinis) in the Graeco-Roman Cult of Cybele and Attis

The cult of Cybele and Attis was an ancient cult disseminated throughout the entire Roman Empire. Among the rites held by its followers, there were the so-called Day of Blood (dies sanguinis) which, according to the Calendar (or Chronography) of Philocalus (354 CE), was celebrated on 24 March. On this day the worshipers and priests (galli) of Cybele/Attis flagellated themselves until they bled profusely, and with their blood they sprinkled Cybele’s effigy as well as the altars of the temple, while the initiates castrated themselves and offered their testes to the goddess as a real-life imitation of what happened mythologically to the goddess’ consort Attis. The present contribution offers a preliminary systemization of this glaringly maladaptive and quite puzzling belief-behaviour complex in the anthropological and neurocognitive frame of the so-called extreme rituals, highlighting the specific in-group benefit reaped by worshipers and initiates (e.g., community cohesion through costly signalling and credibilityenhancing displays).

Evaluating the use of hypoxia sensitive markers for body fluid stain age prediction

To augment DNA profiling and body fluid identification techniques efforts are being made to increase the amount of information available from a crime scene stain, which includes efforts to identify externally visible characteristics through phenotypic analysis. A key question surrounding crime scene stains is the length of time between deposition of the stain and its subsequent recovery, in that is the stain recovered related to the incident in question or from a previously deposited stain number of weeks earlier? The inability to answer this fundamental question has a detrimental effect upon the successful completion of a criminal investigation. Once a body fluid leaves the body, the oxygen concentration in the environment changes; therefore, it may be that this change could cause a change in the expression of hypoxia-sensitive biomarkers. Here, a range of bloodstains, liquid saliva and liquid semen samples were collected at 0 days, 7 days, 14 days, 21 days and 28 days of degrading at room temperature (19–22 °C), before undergoing total RNA extraction and cDNA synthesis. Blood was recovered from filter paper with 3 mm2, with saliva and semen being left in their tubes and swabbed at the appropriate times. All samples then underwent quantitative PCR targeting Vascular Endothelial Growth Factor A (VEGFA) and Hypoxia-Inducible Factor 1 Alpha (HIF1A), with B-Actin (ACTB) as a reference gene. A range of linear and quadratic correlation values was obtained from the qPCR data and used to develop a predictive model with a mean absolute deviation (MAD) of 4.2, 2.1, and 5 days for blood, saliva, and semen respectively. Blind testing indicated that a stain age prediction model based upon VEGFA with ACTB as a reference gene could be used on samples up to four weeks old with a margin of error ranging from 2 days through to 5 days. While a sizeable potential time frame exists using this model; this represents a significant step towards the target of having an accurate stain age prediction model.

SAT-LB134 Aging Related Changes in Sex Hormone-Binding Globulin in Men With HIV

Background: Sex hormone-binding globulin (SHBG) is a glycoprotein that regulates the bioavailability of sex hormones, may directly affect glucose metabolism, and increases with age in the general population. SHBG concentrations are higher in people with HIV, a population in whom accelerated aging has been hypothesized. It is unclear whether age-related trajectories of SHBG differ by HIV serostatus. Methods: SHBG was measured in 182 men with HIV (MWH) and 267 men without HIV (MW/oH) from the Multicenter AIDS Cohort Study (MACS) in four U.S. cities, who had ≥2 samples over a 10 year period. Outcome measure: SHBG, log2SHBG. Multivariable linear mixed-effects regression models were used to evaluate whether SHBG (log2-transformed) and its rate of change differed by HIV serostatus after adjustment for covariates: age, race, BMI, smoking, education, hepatitis C virus infection, total testosterone concentrations, time of day of blood drawn and comorbidities (history of diabetes, kidney disease, liver disease, cancer, depression, hypertension) Results: At baseline, mean age among MWH was similar to MW/oH (51±5 vs 49±6 years). However, SHBG values were higher in MWH compared to MWo/H (65.6±48.8 nmol/L vs. 45.4±22 nmol/L, p<0.001). The rate of SHBG change with age was (1.021 (95% CI:1.010-1.028) or 2.1% per year in MWH and (1.014 nmol/L/year (95% CI:1.010-1.019) or 1.4% per year in MW/oH, p=0.05 for difference in slopes. Among MWH, higher SHBG levels were significantly associated with lower CD4+ T cell count (0.99 (5%CI:0.98-1.00), p<0.05), fewer cumulative years on zidovudine (0.98 nmol/L (95%CI:0.96,1.00), p<0.001) and greater cumulative years on non-nucleoside reverse transcriptase inhibitors drugs (1.02 (95%CI:1.00,1.04), p<0.05). Conclusion: Aging-related increases in SHBG were greater in magnitude among men with HIV and were related to poorer immunologic status and antiretroviral factors. The mechanisms and consequences of these findings require further investigation.

Performance evaluation of the MedMira reveal G4 LAB S/P and POC HIV antibody rapid screening tests using plasma and whole blood specimens

BackgroundThe Reveal G4 antibody rapid test is FDA-approved for HIV-1 detection using the versions LAB S/P and POC in CLIA-moderate complexity settings with serum/plasma and whole blood, respectively. The same Reveal tests are CE-marked for HIV-1 and HIV-2 detection in laboratory and point-of-care (POC) settings. ObjectiveWe compared the performance of G4 LAB S/P with plasma and POC with whole blood (blood) for detecting early and established HIV-1/HIV-2 infections. Study designMatched well-characterized plasma and simulated blood were used to evaluate: sensitivity in 104 HIV-1 and 55 HIV-2 established infections, specificity in 49 HIV-negative, and reactivity in early HIV-1 infection in a performance panel (n=38) and 18 plasma panels from seroconverters (SCs, n=183). Median number of days after first RNA-positive was calculated for 13 SCs. Impact of viral suppression (VS) was evaluated in 3 SCs receiving early antiretroviral therapy (ART). ResultsSensitivity was 100 % for HIV-1 and 98.18 % for HIV-2, while specificity was 100 %. All 38 plasma and blood become reactive by Fiebig stage V. Of 18 SCs, 10 had similar reactivity in plasma/blood, 7 showed delayed reactivity in blood, and 1 was nonreactive in plasma/blood. The median days for a G4-reactive after first RNApositive was 13 for plasma and 14 for blood. Long-term VS had no impact on G4 reactivity. ConclusionsOverall reactivity in early HIV-1 infections is delayed by one day in blood compared to plasma. If FDA-approved for POC settings, the G4 POC is a fast sensitive screening tool for HIV-1/HIV-2-specific IgG even during VS.

Abstract WP127: Intravenous Administration of Microrna-122 Mimic for Treatment of Ischemic and Hemorrhagic Strokes in Rats

MicroRNAs(miRs)are very promising next generation drug targets due to their unique miR-target binding that is different from the traditional ligand-receptor. A single miR binds to complementary bases in the 3’ untranslated regions(3’UTR) of hundreds of target genes and down-regulates these genes. MiR therapeutics have been developed for treatment of various diseases, with several miR drugs(e.g., miR-122/miR-155 inhibitors, miR-16/29/34 mimics) being advanced into human trials in the last decade. Our previous studies showed that intravenous (i.v.) miR-122 mimic (2.4mg/kg, wrapped in PEG-liposomes) improves outcomes after suture middle cerebral artery occlusion(MCAO)-induced ischemic stroke (IS) with a 6 hour time window. In pilot whole genome miR expression studies, we demonstrated that microRNA-122 (miR-122) is the most significantly decreased miR in blood after intraventricularautologousfresh blood-induced intracerebral hemorrhage (ICH) in rats. As compared to sham controls, miR-122 decreased 28 fold at 3 hrs, 34 fold at 24 hrs, 31 fold at 7 days and 19 fold at 14 days in blood after ICH in rats. These miRNA expression data suggest that elevating miR-122 in blood has great potential to treat ICH in addition to IS. Therefore, we hypothesized that i.v. miR-122 mimic improves outcomes after ICH, in addition to IS. Our miR-122 targetome studies show that a set of miR-122 target genes (Pla2g2a, Vcam1, Nos2, Rhbdf1, Olig1, Nrep) are responsible for the therapeutic efficacy of miR-122 mimic on ischemic stroke, while another set of genes (Ywhaq, Klrk1, Tpst1, Vars2)account for efficacy in ICH. Using3’UTR luciferase reporter assays and anti-sense Morpholino Oligos (MOs), we show that miR-122 binds to 3’UTR of its target genes Pla2g2a and Vcam1, rather than Nos2. In addition, in vivo MO-miR-122-Pla2g2a blocks miR-122 mimic treatment-induced decrease of Pla2g2a in blood cells after ischemic stroke. In summary, our data suggest miR-122 mimic can treat IS and ICH. Therefore, the miR-122 mimic treatment for IS and ICH could likely be performed without brain imaging (e.g. in the ambulance, and/or emergency room) since it is broadly efficacious for both IS and ICH.