Treatment Of Young Mice Research Articles

The prostaglandin D2 antagonist asapiprant ameliorates clinical severity in young hosts infected with invasive Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus) remains a serious cause of pulmonary and systemic infections globally, and host-directed therapies are lacking. The aim of this study was to test the therapeutic efficacy of asapiprant, an inhibitor of prostaglandin D2 signaling, against pneumococcal infection. Treatment of young mice with asapiprant after pulmonary infection with invasive pneumococci significantly reduced systemic spread, disease severity, and host death. Protection was specific against bacterial dissemination from the lung to the blood but had no effect on pulmonary bacterial burden. Asapiprant-treated mice had enhanced antimicrobial activity in circulating neutrophils, elevated levels of reactive oxygen species (ROS) in lung macrophages/monocytes, and improved pulmonary barrier integrity indicated by significantly reduced diffusion of fluorescein isothiocyanate (FITC)-dextran from lungs into the circulation. These findings suggest that asapiprant protects the host against pneumococcal dissemination by enhancing the antimicrobial activity of immune cells and maintaining epithelial/endothelial barrier integrity in the lungs.

Abstract 1337: Aged pancreatic fibroblasts enhance pancreatic cancer growth and progression

Abstract Advancing age is a major independent risk factor for the development of pancreatic ductal adenocarcinoma (PDAC). However, the ways in which aging alters cells that eventually comprise the PDAC tumor microenvironment (TME) are incompletely understood. Fibroblasts comprise a large part of PDAC tumors, can alter the behavior of cancer cells, and impact treatment responses. Our studies investigate how normal pancreatic fibroblasts acquire tumor-promoting properties during aging and alter the tumorigenic properties of PDAC both in vitro and in vivo. To determine if PDAC tumor growth is altered by an aged microenvironment, we performed orthotopic injection of KPC and Panc02 PDAC cells into the pancreata of aged (>64-week-old) and young (6-8-week-old) C57Bl/6J mice. Tumors in aged mice are significantly larger compared to those in young mice, and there is increased incidence of liver metastases in the aged mouse cohort. We next queried whether aged pancreatic fibroblasts may contribute to this increase in tumor growth. We generated a panel of aged (donors >55 years old) and young (donors <35 years old) normal human pancreatic fibroblasts to determine the effects of healthy aging fibroblasts on PDAC cell behavior. Conditioned media from or co-culture with aged human pancreatic fibroblasts increases the proliferation, migration, and invasion of PDAC cells compared to young fibroblasts. Aged fibroblast conditioned media activated AKT signaling and enhances features of EMT and stemness. Proteomic analysis of conditioned media reveals a significantly altered secretome in aged fibroblasts, with a number of known tumor-promoting proteins released at higher levels from aged compared to young fibroblasts. We examined the contribution of GDF-15, secreted by aged fibroblasts at relatively high levels, as a paracrine modulator of PDAC growth and progression. Addition of recombinant GDF-15 to young fibroblast conditioned media increases proliferation and invasion of PDAC cells to levels similar to aged fibroblast conditioned media. Treatment of young mice with recombinant GDF-15 increases tumor growth and AKT phosphorylation in vivo. Additionally, inhibition of AKT reduces tumor size in aged but not young mice, suggesting that the aged microenvironment at least in part exerts its pro-tumor effects through AKT. In conclusion, we show that aged, non-cancer-associated pancreatic fibroblasts have the potential to promote growth, migration, and invasiveness in multiple models of pancreatic ductal adenocarcinoma. Further studies examining fibroblast-secreted factors and other changes in the aged TME are ongoing. Citation Format: Daniel J. Zabransky, Yash Chhabra, Mitchell Fane, Emma Kartalia, James Leatherman, Jacquelyn Zimmerman, Elizabeth Jaffee, Ashani Weeraratna. Aged pancreatic fibroblasts enhance pancreatic cancer growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1337.

Differential Impact of Calcitriol and Its Analogs on Tumor Stroma in Young and Aged Ovariectomized Mice Bearing 4T1 Mammary Gland Cancer.

(1) Background: Vitamin D compounds (VDC) are extensively studied in the field of anticancer properties, including breast cancer. Previously, we showed that calcitriol and its analogs (PRI-2191 and PRI-2205) stimulate metastasis in 4T1 murine mammary gland cancer models in young mice, whereas the reverse effect was observed in aged ovariectomized (OVX) mice; (2) Methods: We determined the phenotype of monocytes/macrophages using FACS and examined the expression of selected genes and proteins by Real-Time PCR and ELISA; (3) Results: Activities of VDC are accompanied by an increase in the percentage of Ly6Clow anti-inflammatory monocytes in the spleen of young and a decrease in aged OVX mice. Treatment of young mice with VDC resulted in an increase of CCL2 plasma and tumor concentration and Arg1 in tumor. In later stage of tumor progression the expression of genes related to metastasis in lung tissue was decreased or increased, in old OVX or young mice, respectively; (4) Conclusions: Pro- or anti-metastatic effects of calcitriol and its analogs in young or aged OVX mice, respectively, can be attributed to the differences in the effects of VDC on the tumor microenvironment, as a consequence of differences in the immunity status of young and aged mice.

Plasma cells are obligate enhancers of age-associated myeloid skewing

Abstract In humans and mice, aging results in significantly elevated myelopoiesis and the increased production of inflammatory cytokines with age is thought to underlie this phenomenon. The observation that treatment of young mice with factors such as interleukin (IL)-1 can skew hematopoiesis towards myeloid development is consistent with this hypothesis. However, the cellular source(s) of inflammatory cytokines in the aging bone marrow has not been fully defined. We observed that plasma cells (PCs) accumulate in the bone marrow with age and acquire an inflammatory gene signature. Furthermore, we observed that their depletion in vivo using anti-CD138 antibodies resulted in a significant decline in myelopoiesis as demonstrated by a reduction in the number of myeloid biased hematopoietic stem cells, myeloid progenitors and mature myeloid cells. We also found that anti-CD138 treated mice exhibited reduced expression of genes encoding inflammatory cytokines in bone marrow stromal cells, indicating that plasma cells can regulate inflammatory cytokine production by other microenvironmental components. We tested whether this inflammatory network was necessary to drive age-associated myeloid skewing by treating old mice with Anakinra or Enbrel to abrogate IL-1 and tumor necrosis factor alpha (TNF-α) signaling, respectively. Concurrent blockade of both signaling pathways in old mice resulted in the significant reduction of bone marrow myelopoiesis, which was not observed upon inhibition of either pathway alone. These data demonstrate a previously unappreciated ability of inflammatory PCs to stimulate myelopoiesis and implicate them at the center of an inflammatory regulatory network in the aging bone marrow.

Long-lived plasma cells regulate patterns of hematopoiesis in aging.

Abstract In both humans and mice, aging results in significant alterations to the patterns of bone marrow hematopoiesis. This is highlighted by a precipitous reduction in B lymphopoiesis while myelopoiesis is enhanced. Treatment of young mice with factors such as GM-CSF or IL-1 can skew hematopoiesis towards myeloid development. This suggests that imbalances in the relative concentrations of various cytokines such as inflammatory mediators, whose production is thought to increase with age, could contribute to suppressed lymphoid and enhanced myeloid development in aging. In this case, the resulting stimulation of myelopoiesis would dominate the limited number of niches in the bone marrow environment at the expense of lymphopoiesis. However, whether the age-related decline in lymphopoiesis and increase in myelopoiesis are linked or represent distinct processes is not known. We observed that long-lived plasma cells from old mice acquire an inflammatory gene signature and accumulate in the bone marrow. Addition of long-lived plasma cells to long-term in vitro cultures demonstrated the ability of these cells to promote granulopoiesis from hematopoietic stem cells as well as myeloid progenitors. However, while depletion of long-lived plasma cells in old mice by injection of anti-CD138 antibodies resulted in a significant reduction in myeloid development, B lymphopoiesis remained suppressed. These data demonstrate a previously unappreciated ability of inflammatory plasma cells to stimulate myelopoiesis and provide evidence that, in the context of aging, enhanced myelopoiesis and depressed lymphopoiesis are distinct, independently regulated processes.

Oral treatment with enrofloxacin early in life promotes Th2-mediated immune response in mice.

Th2 lymphocytes play a crucial role in the development of allergy. These pathologies are caused by coordinated production of the cytokines IL-4, IL-5 and IL-13 that regulate the activity of eosinophils, basophils and B cells. According to the 'hygiene hypothesis', the reduced exposure to microorganisms favors allergy occurrence. The advances in medicine in the field of infection therapy promoted an increasing application of antibiotics which, apart from eliminating pathogens, also partially eliminate the microbiota. Epicutaneous (EC) immunization with ovalbumin (OVA) followed by OVA challenge was used to study the influence of partial gut flora depletion by oral treatment with enrofloxacin on type-2 immune response. Current work describes the influence of enrofloxacin application on anti-OVA antibody production and cytokine synthesis in young and adult mice. Immune response in adult mice is less sensitive to modification of natural gut flora. We observed that enrofloxacin treatment of adult mice leads to significant decrease of anti-OVA IgG2a production while synthesis of anti-OVA IgE was not changed. The production of type-1 (IFN-γ), type-2 (IL-4, IL-5, IL-10, IL-13) and Th17-associated (IL-17A) cytokines was inhibited. On the other hand, treatment of young mice with enrofloxacin significantly upregulates the production of anti-OVA IgE and inhibits the secretion of anti-OVA IgG2a antibodies. Additionally, treatment with enrofloxacin early in life prior to OVA immunization results in increased production of type-2 (IL-4, IL-10 and IL-13) cytokines. Our results clearly indicate that the immune system is more vulnerable to decreased bacterial exposure early in life that may promote development of allergy.

Abstract C110: Abstract B: Hedgehog pathway inhibition in young mice results in severe bone defects

Abstract Current treatment for the most common pediatric brain tumor, medulloblastoma (MB) compromises neurological and endocrine development, particularly in younger patients. Mutations in Patched (PTCH1), the receptor for Hedgehog (Hh) proteins, cause MB in approximately 30% of patients. Loss of PTCH1 leads to constitutive activation of Smoothened (SMO) and increased expression of downstream target genes such as GLI1. Previously, we investigated the use of the SMO inhibitor, HhAntag, in a mouse model of MB. Treatment of Ptch1+/−p53−/− mice with HhAntag completely eliminates spontaneous and transplanted tumors. However, treatment of young mice with HhAntag results in permanent defects in bone structure. Histological analyses of long bones from these mice showed a significant reduction in proliferating chondrocytes and an increase in the number of hypertrophic chondrocytes. In our current study, we have further characterized the bone toxicities caused by HhAntag treatment and are testing potential molecules that might lessen the severity of these toxicities. We treated P10-old mice for 4 or 6 days and analyzed their limbs at 0, 2, or 4 days after HhAntag treatment. We found that the severity of bone defects increases with the length of time the mice are treated with HhAntag. MicroCT and MRI analysis showed that exposure to HhAntag for as little as 4 days caused premature growth plate fusion and an increase in bone mineral density. In an effort to protect proliferating chondrocytes from the toxicities caused by HhAntag, we have identified several candidate molecules that have been shown to alter developing bone. One candidate, Parathyroid Hormone-Related Protein (PTHrP), is a downstream effector of the Hh pathway. Hh signaling activates the expression of PTHrP, which then signals to proliferating chondrocytes, preventing them from differentiating. Previous studies show that overexpression of PTHrP in developing bones delays chondrocyte hypertrophy. Therefore, exogenous PTHrP may offset the premature differentiation evident in HhAntag treated animals. We have identified a physiologically relevant dose of PTHrP that is sufficient to mediate a change in bone morphology. We are currently treating mice simultaneously with PTHrP and HhAntag, in an effort to reduce the HhAntag associated bone toxicities. Another candidate is the proteasome inhibitor, bortezomib. Bortezomib has been shown to alter Hh pathway activity and therefore may also reduce the toxicities associated with HhAntag treatment. We are currently establishing the proper dosing strategy appropriate for bortezomib treatment of young mice. We have shown that treatment of young mice with HhAntag leads to severe bone defects and are currently employing a multi-drug approach to lessen the severity of these defects. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C110.

The nude mouse as a model for the study of human pancreatic cancer

The purpose of this study was to characterize an in vivo model of human pancreatic cancer suitable for chemotherapy and immunotherapy studies. In this study we report a 2-year experience in growing the MIA PaCa-2 (CRL 1420) human pancreatic cancer cell line in 92 adult (8 weeks old) and 256 young (3–6 weeks old) nude mice. Ten million tumor cells were transplanted into orthotopic (duodenal lobe of the pancreas) and/or heterotopic positions (hepatic and subcutaneous) and data on operative mortality, effect of total body irradiation (TBI), tumor growth kinetics, and survival are presented comparing the two age groups. Operative mortality was due to anesthetic intolerance which was higher in the young mouse population (13.4% versus 5.7%). Adult mice withstood TBI (500 rad) without mortality but young mice were highly sensitive to radiation damage and their maximum tolerated dose (LD 50) was 425–450 rad. Subcutaneous tumors grew significantly more often in young compared to adult animals (97.9% versus 69%) and this finding was not affected by TBI (96.9% versus 75%), though tumors did appear more quickly after TBI. An average of 14.7 ± 2.8 days was required for the subcutaneous tumors to become macroscopically apparent in the adult population compared with 3.1 ± 0.8 days in the young mice. The largest subcutaneous tumor diameter 28 days following tumor implant averaged 9.3 ± 0.6 mm in the young animals and 5.5 ± 1.7 mm in the adult population ( P < 0.01). Treatment of young mice with human recombinant interleukin-2 (IL-2) (10,000 Units twice a day for 28 days) produced a 27% decrease in tumor growth. This effect was abolished by prior irradiation of the young mice with 375 rad TBI. Pancreatic tumor growth also occurred more consistently in young than in adult animals (91.2% versus 64.3%) and irradiation did not affect pancreatic tumor take in either group. Occasionally intrapancreatic tumor growth was associated with liver metastases in animals that were killed after 28 days (17.8% in young and 22.2% in adult animals). However, when more than 45 days elapsed before sacrificing the animals, the incidence of hepatic metastases increased to 57.1%. This was slightly less than the incidence of hepatic lesions found after direct injection of cancer cells into the liver by portal vein injection (71.4%). Direct extension of tumor into surrounding tissues was common with frequent involvement of the duodenum (83.7%), kidneys (30.6%), and other intraabdominal organs (43.9%). Survival was significantly longer in adult compared to young mice. All young mice that grew intrapancreatic tumors died within 3 months from the injection of tumor cells, while about 50% of the adult animals with documented pancreatic tumors survived up to 5 months. The model is an efficient means for the in vivo study of human pancreatic cancer and the autologous anti-tumor response can be controlled by the use of young and irradiated nude mice by methods described in this paper.

Increased immunological efficiency in young mice by short-term treatment with L-thyroxine.

Treatment of young mice with L-thyroxine increases their capacity to form hemolytic plaques after sheep erythrocyte immunization. Such an increment is independent from the quantity of antigenic challenge, from sex and strains variations and from the temporal relationship between timing of hormonal treatment and antigenic challenge. The increment seems to depend on an increased number of both T and B reactive cells, rather than on an augmented proliferation rate of antigen triggered cells. The thyroxine induced increase of T reactive cells requires the presence of a functional thymus. These data suggest that the maximal immune response obtained under physiological conditions does not correspond to the maximal potentiality which can be expressed through appropriate manipulation of the internal microenvironment and further confirm the relevance that thyroxine may have as an immunostimulant agent.

Suppression of activity of mouse natural killer (NK) cells by activated macrophages from mice treated with pyran copolymer.

Treatment of young mice with pyran copolymer caused a substantial decrease in natural killer (NK) cell activity at 7 days. The decrease in cytotoxicity was associated with the presence of splenic suppressor cells, capable of inhibiting in vitro the NK activity of spleen cells from normal mice. The suppressor cells appeared to be macrophages, being plastic-adherent, phagocytic and radioresistant, and lacking demonstrable Thy 1.2 antigen. Sonicates or culture supernatants of adherent spleen cells from pyran-treated mice were also able to inhibit NK activity, suggesting that suppressor cells act by release of soluble factors.

The effect of immunopharmacological agents on mouse natural cell-mediated cytotoxicity and on its augmentation by poly I:C

Treatment of young mice with lethal X-irradiation (900 R), cyclophosphamide, or hydrocortisone significantly depressed their spontaneous NK activity. The same treatments, however, did not inhibit the augmentation of NK function by poly I:C, suggesting the existence of a treatment resistant pre-NK cell. Both the spontaneous activity and its augmentation were readily inhibited by macrophage-toxic agents such as silica and carrageenan in vivo. Since the NK cells themselves were not macrophages, as shown by the inability of silica or carrageenan to block in vitro cytolysis of target cells, we postulated that macrophages were required to maintain NK activity in vivo and that they were essential accessory cells in the augmentation. The induction of interferon by poly I:C was also inhibited by silica and carrageenan. The augmentation of NK activity induced by poly I:C was consistently accompanied by the rise in serum IF levels of the treated mice, and its inhibition by the macrophage-toxic agents was followed by decreased production of interferon. These observations support our hypothesis that macrophages, in response to poly I:C, produced interferon which in turn activated NK cells to become cytolytic.