In vitro human helper T-cell assay to screen antibody drug candidates for immunogenicity.

Monoclonal antibody (mAb) drugs offer a number of valuable treatments. Many newly developed mAb drugs include artificial modification of amino acid sequences from human origin, which may cause higher immunogenicity to induce anti-drug antibodies (ADA). If the immunogenicity of a new candidate can be understood in the nonclinical phase, clinical studies will be safer and the success rate of development improved. Empirically, in vitro immunogenicity assays with human cells have proved to be sufficiently sensitive to nonhuman proteins, but not to human/humanized mAb. To detect the weaker immunogenicity of human-based mAb, a more sensitive biomarker for in vitro assays is needed. The in vitro study here developed a proliferation assay (TH cell assay) using flow cytometry analysis that can detect a slight increase in proliferating TH cells. Samples from 218 donors treated with a low-immunogenic drug (etanercept) were measured to determine a positive threshold level. With this threshold, positive donor percentages among PBMC after treatment with higher-immunogenicity mAb drugs were noted, that is, 39.5% with humanized anti-human A33 antibody (hA33), 27.3% with abciximab, 25.9% with adalimumab, and 14.8% with infliximab. Biotherapeutics with low immunogenicity yielded values of 0% for basiliximab and 3.7% for etanercept. These data showed a good comparability with previously reported incidences of clinical ADA with the evaluated drugs. Calculations based on the data here showed that a TH cell assay with 40 donors could provide statistically significant differences when comparing low- (etanercept) versus highly immunogenic mAb (except for infliximab). Based on the outcomes here, for screening purposes, a practical cutoff point of 3/20 positives with 20 donors was proposed to alert immunogenicity of mAb drug candidates.

Neural progenitor cell proliferation in the hypothalamus is involved in acquired heat tolerance in long-term heat-acclimated rats.

Constant exposure to moderate heat facilitates progenitor cell proliferation and neuronal differentiation in the hypothalamus of heat-acclimated (HA) rats. In this study, we investigated neural phenotype and responsiveness to heat in HA rats' hypothalamic newborn cells. Additionally, the effect of hypothalamic neurogenesis on heat acclimation in rats was evaluated. Male Wistar rats (5 weeks old) were housed at an ambient temperature (Ta) of 32°C for 6 days (STHA) or 40 days (LTHA), while control (CN) rats were kept at a Ta of 24°C for 6 days (STCN) or 40 days (LTCN). Bromodeoxyuridine (BrdU) was intraperitoneally injected daily for five consecutive days (50 mg/kg/day) after commencing heat exposure. The number of hypothalamic BrdU-immunopositive (BrdU+) cells in STHA and LTHA rats was determined immunohistochemically in brain samples and found to be significantly greater than those in respective CN groups. In LTHA rats, approximately 32.6% of BrdU+ cells in the preoptic area (POA) of the anterior hypothalamus were stained by GAD67, a GABAergic neuron marker, and 15.2% of BrdU+ cells were stained by the glutamate transporter, a glutamatergic neuron marker. In addition, 63.2% of BrdU+ cells in the POA were immunolabeled with c-Fos. Intracerebral administration of the mitosis inhibitor, cytosine arabinoside (AraC), interfered with the proliferation of neural progenitor cells and acquired heat tolerance in LTHA rats, whereas the selected ambient temperature was not changed. These results demonstrate that heat exposure generates heat responsive neurons in the POA, suggesting a pivotal role in autonomic thermoregulation in long-term heat-acclimated rats.

Oral intake of encapsulated dried ginger root powder hardly affects human thermoregulatory function, but appears to facilitate fat utilization.

The present study investigated the impact of a single oral ingestion of ginger on thermoregulatory function and fat oxidation in humans. Morning and afternoon oral intake of 1.0 g dried ginger root powder did not alter rectal temperature, skin blood flow, O2 consumption, CO2 production, and thermal sensation and comfort, or induce sweating at an ambient temperature of 28 °C. Ginger ingestion had no effect on threshold temperatures for skin blood flow or thermal sweating. Serum levels of free fatty acids were significantly elevated at 120 min after ginger ingestion in both the morning and afternoon. Morning ginger intake significantly reduced respiratory exchange ratios and elevated fat oxidation by 13.5 % at 120 min after ingestion. This was not the case in the afternoon. These results suggest that the effect of a single oral ginger administration on the peripheral and central thermoregulatory function is miniscule, but does facilitate fat utilization although the timing of the administration may be relevant.

Aging attenuates acquired heat tolerance and hypothalamic neurogenesis in rats.

This study investigated age-dependent changes in heat exposure-induced hypothalamic neurogenesis and acquired heat tolerance in rats. We previously reported that neuronal progenitor cell proliferation and neural differentiation are enhanced in the hypothalamus of long-term heat-acclimated (HA) rats. Male Wistar rats, 5 weeks (Young), 10-11 months (Adult), or 22-25 months (Old) old, were subjected to an ambient temperature of 32°C for 40-50 days (HA rats). Rats underwent a heat tolerance test. In HA rats, increases in abdominal temperature (Tab ) in the the Young, Adult, and Old groups were significantly smaller than those in their respective controls. However, the increase in Tab of HA rats became greater with advancing age. The number of hypothalamic bromodeoxyuridine (BrdU)-immunopositive cells double stained with a mature neuron marker, neuronal nuclei (NeuN), of HA rats was significantly higher in the Young group than that in the control group. In Young HA, BrdU/NeuN-immunopositive cells of the preoptic area/anterior hypothalamus appeared to be the highest among regions examined. Large numbers of newborn neurons were also located in the ventromedial and dorsomedial nuclei, as well as the posterior hypothalamic area, whereas heat exposure did not increase such numbers in the Adult and Old groups. Aging may interfere with heat exposure-induced hypothalamic neurogenesis and acquired heat tolerance in rats.

Proliferation of neuronal progenitor cells and neuronal differentiation in the hypothalamus are enhanced in heat-acclimated rats.

Male Wistar rats, initially maintained at an ambient temperature (T (a)) of 24 degrees C, were subjected to a constant high T (a) of 32 degrees C (HE) or were constantly kept at 24 degrees C (controls, CN). Bromodeoxyuridine (BrdU) was intraperitoneally injected daily for five consecutive days after commencing heat exposure. On the 6th, 13th, 23rd, 33rd, 43rd, and 53rd day of heat exposure, rats' brains were removed. Immunohistochemical analysis showed that the numbers of BrdU-positive cells in the hypothalamus of HE were significantly and consistently greater than those of CN. In HE, the number of BrdU-positive cells double-stained by a mature neuron marker increased abruptly after 33 days of heat exposure by about seven times. This was not the case in CN. The results suggest that heat exposure facilitates proliferation of neuronal progenitor cells in the hypothalamus and promotes differentiation to neurons, which might have certain relation to establishing long-term heat acclimation in rats.

Improvement of spatial cognition with dietary docosahexaenoic acid is associated with an increase in Fos expression in rat CA1 hippocampus.

Twenty 5-week-old male Wistar rats were divided into two groups: one group was fed a fish oil-deficient diet and the other group was fed the same diet supplemented with per orally administered docosahexaenoic acid (DHA) for 12 weeks. Six weeks after the start of the administration of DHA, rats were trained for 6 weeks to acquire a reward at the end of each of four arms of an eight-arm radial maze. On completion of the radial maze task, the Fos expression in the hippocampus was examined immunohistochemically. Chronic DHA administration significantly reduced the number of reference and working memory errors. The number of Fos-positive neurons in the CA1 hippocampus significantly increased in DHA-treated rats compared with control rats, demonstrating a statistically significant negative correlation with the number of reference memory errors. These results suggest that the DHA-induced improvement in spatial cognition is associated with increased Fos expression in the CA1 hippocampus.