Murine Models of Familial Cytokine Storm Syndromes.

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disease caused by mutations in effectors and regulators of cytotoxicity in cytotoxic T cells (CTL) and natural killer (NK) cells. The complexity of the immune system means that in vivo models are needed to efficiently study diseases like HLH. Mice with defects in the genes known to cause primary HLH (pHLH) are available. However, these mice only develop the characteristic features of HLH after the induction of an immune response (typically through infection with lymphocytic choriomeningitis virus). Nevertheless, murine models have been invaluable for understanding the mechanisms that lead to HLH. For example, the cytotoxic machinery (e.g., the transport of cytotoxic vesicles and the release of granzymes and perforin after membrane fusion) was first characterized in the mouse. Experiments in murine models of pHLH have emphasized the importance of cytotoxic cells, antigen-presenting cells (APC), and cytokines in hyperinflammatory positive feedback loops (e.g., cytokine storms). This knowledge has facilitated the development of treatments for human HLH, some of which are now being tested in the clinic.

Genetic algorithms applied to translational strategy in metabolic-dysfunction associated steatohepatitis (MASH). Learning from mouse models.

BACKGROUND & AIMS: We previously identified subsets of patients with metabolic (dysfunction)-associated steatotic liver disease (MASLD) with different metabolic phenotypes. Here, we aimed to refine this classification based on genetic algorithms implemented in a Python package. The use of these genetic algorithms can help scientists to solve problems which cannot be solved with other methods. We present this package and its capabilities with specific problems. The name, PyGenMet, comes from its main goal, solving problems in Python with Genetic Algorithms and Metabolomics data. METHODS: We collected serum from methionine adenosyltransferase 1a knockout (Mat1a-KO) mice, which have chronically low level of hepatic S-adenosylmethionine (SAMe) and the metabolomes of all samples were determined. We also analyzed serum metabolomes of 541 patients with biopsy proven MASLD (182 with simple steatosis and 359 with metabolic (dysfunction)-associated steatohepatitis or MASH) and compared them with the serum metabolomes of this specific MASLD mouse model using Genetic Algorithms in order to select patients with a specific phenotype. RESULTS: By applying genetic algorithms, we have found a subgroup of patients with a lipid profile similar to that observed in the mouse model. When analyzing the two groups of patients, we have seen that patients with a lipid profile reflecting the mouse model characteristics show significant differences in lipoproteins, especially in LDL-4, LDL-5, and LDL-6 associated with atherogenic risk. CONCLUSION: The results show that the application of genetic algorithms to subclassify patients with MASLD (or other metabolic disease) give consistent results and are a good approximation for the treatment of large volumes of data such as those from omics sciences and patient classification.

SkinCom, a synthetic skin microbial community, enables reproducible investigations of the human skin microbiome.

Existing models of the human skin have aided our understanding of skin health and disease. However, they currently lack a microbial component, despite microbes' demonstrated connections to various skin diseases. Here, we present a robust, standardized model of the skin microbial community (SkinCom) to support in vitro and in vivo investigations. Our methods lead to the formation of an accurate, reproducible, and diverse community of aerobic and anaerobic bacteria. Subsequent testing of SkinCom on the dorsal skin of mice allowed for DNA and RNA recovery from both the applied SkinCom and the dorsal skin, highlighting its practicality for in vivo studies and -omics analyses. Furthermore, 66% of the responses to common cosmetic chemicals in vitro were in agreement with a human trial. Therefore, SkinCom represents a valuable, standardized tool for investigating microbe-metabolite interactions and facilitates the experimental design of in vivo studies targeting host-microbe relationships.

Age-dependent Powassan virus lethality is linked to glial cell activation and divergent neuroinflammatory cytokine responses in a murine model.

Powassan virus (POWV) is an emergent tick-borne flavivirus that causes fatal encephalitis in the elderly and long-term neurologic sequelae in survivors. How age contributes to severe POWV encephalitis remains an enigma, and no animal models have assessed age-dependent POWV neuropathology. Inoculating C57BL/6 mice with a POWV strain (LI9) currently circulating in Ixodes ticks resulted in age-dependent POWV lethality 10-20 dpi. POWV infection of 50-week-old mice was 82% fatal with lethality sequentially reduced by age to 7.1% in 10-week-old mice. POWV LI9 was neuroinvasive in mice of all ages, causing acute spongiform CNS pathology and reactive gliosis 5-15 dpi that persisted in survivors 30 dpi. High CNS viral loads were found in all mice 10 dpi. However, by 15 dpi, viral loads decreased by 2-4 logs in 10- to 40-week-old mice, while remaining at high levels in 50-week-old mice. Age-dependent differences in CNS viral loads 15 dpi occurred concomitantly with striking changes in CNS cytokine responses. In the CNS of 50-week-old mice, POWV induced Th1-type cytokines (IFNγ, IL-2, IL-12, IL-4, TNFα, IL-6), suggesting a neurodegenerative pro-inflammatory M1 microglial program. By contrast, in 10-week-old mice, POWV-induced Th2-type cytokines (IL-10, TGFß, IL-4) were consistent with a neuroprotective M2 microglial phenotype. These findings correlate age-dependent CNS cytokine responses and viral loads with POWV lethality and suggest potential neuroinflammatory therapeutic targets. Our results establish the age-dependent lethality of POWV in a murine model that mirrors human POWV severity and long-term CNS pathology in the elderly. IMPORTANCE: Powassan virus is an emerging tick-borne flavivirus causing lethal encephalitis in aged individuals. We reveal an age-dependent POWV murine model that mirrors human POWV encephalitis and long-term CNS damage in the elderly. We found that POWV is neuroinvasive and directs reactive gliosis in all age mice, but at acute stages selectively induces pro-inflammatory Th1 cytokine responses in 50-week-old mice and neuroprotective Th2 cytokine responses in 10-week-old mice. Our findings associate CNS viral loads and divergent cytokine responses with age-dependent POWV lethality and survival outcomes. Responses of young mice suggest potential therapeutic targets and approaches for preventing severe POWV encephalitis that may be broadly applicable to other neurodegenerative diseases. Our age-dependent murine POWV model permits analysis of vaccines that prevent POWV lethality, and therapeutics that resolve severe POWV encephalitis.

Effects of pH on the Pathogenicity of Escherichia coli and Klebsiella pneumoniae on the Kidney: In Vitro and In Vivo Studies.

Urine pH reflects the functional integrity of the body and may influence the virulence of uropathogenic Escherichia coli and Klebsiella pneumoniae, the main causes of urinary tract infections (UTIs). This study evaluated the effects of acidic pH on the pathogenicity of uropathogenic E. coli and K. pneumoniae strains, in vitro and in vivo. Four uropathogenic E. coli and four K. pneumoniae strains were used. Biofilm formation, growth competition indices, motility, and adhesion and invasion of human renal cells were analyzed in media with acidic, neutral, and alkaline pH. A murine lower UTI model was used, with urine adjusted to acidic, neutral, or alkaline pH. At acidic pH, E. coli and K. pneumoniae exhibited higher bacterial concentrations in the kidneys and systemic symptoms, including bacteremia. Alkaline urine pH did not affect bacterial concentrations of any strain. In mice with UTIs caused by E. coli Nu14 and K. pneumoniae HUVR42 and acidic urine pH, histopathological studies of the kidneys showed acute inflammation affecting the urothelium and renal parenchyma, which are traits of acute pyelonephritis. These results indicate that acidic pH could increase the pathogenicity of E. coli and K. pneumoniae in murine models of lower UTI, promoting renal infection and acute inflammation.

Preclinical human and murine models of hepatocellular carcinoma (HCC).

Hepatocellular carcinoma (HCC) is the most frequent liver cancer, which account for more than 90 % of all liver cancer cases. It is the fifth leading cause of cancer globally and the second leading cause of cancer-related mortality in men. The availability of competent HCC preclinical models is fundamental to the success of mechanistic studies, molecular target identification, and drug testing. However, there are challenges associated with the use of these models. In this review, we provided updates on various cell lines, animals, and human HCC models, their specific preclinic use and associated potential challenges. Overall, the understanding of the merits and demerits of a particular HCC model will improve model selection for various preclinical studies.

Modeling the CD8＋ T cell immune response to influenza infection in adult and aged mice.

The CD8＋ T cell response is the main determinant of viral clearance during influenza infection. However, influenza viral dynamics and the respective immune responses are affected by the host's age. To investigate age-related differences in the CD8＋ T cell immune response dynamics, we propose 16 ordinary differential equation models of existing experimental data. These data consist of viral titer and CD8＋ T cell counts collected periodically over a period of 19 days from adult and aged mice infected with influenza A/Puerto Rico/8/34 (H1N1). We use the corrected Akaike Information Criterion to identify the models which best represent the considered data. Our model selection process indicates differences in mechanisms which reduce the CD8＋ T cell response: linear downregulation is favored for adult mice, while baseline exponential decay is favored for aged mice. Parameter fitting of the top ranked models suggests that the aged population has reduced CD8＋ T cell proliferation compared to the adult population. More experimental work is needed to determine the specific immunological features through which age might cause these differences. A better understanding of the immunological mechanisms by which aging leads to discrepant CD8＋ T cell dynamics may inform future treatment strategies.

Adherence and metal-ion acquisition gene expression increases during infection with Treponema phagedenis strains from bovine digital dermatitis.

Digital dermatitis (DD) is an ulcerative foot lesion on the heel bulbs of dairy cattle. DD is a polymicrobial disease with no precise etiology, although Treponema spirochetes are found disproportionally abundant in diseased tissue. Within Treponema, several different species are found in DD; however, the species Treponema phagedenis is uniformly found in copious quantities and deep within the skin layers of the active, ulcerative stages of disease. The pathogenic mechanisms these bacteria use to persist in the skin and the precise role they play in the pathology of DD are widely unknown. To explore the pathogenesis and virulence of Treponema phagedenis, newly isolated strains of this species were investigated in a subcutaneous murine abscess model. In the first trial, a dosage study was conducted to compare the pathogenicity of different strains across three different treponemes per inoculum (TPI) doses based on abscess volumes. In the second trial, the expression levels of 11 putative virulence genes were obtained to gain insight into their involvement in pathogenesis. During the RT-qPCR analysis, it was determined that genes encoding for two metal-ion import lipoproteins and two adherence genes were found highly upregulated during infection. Conversely, two genes involved in motility and chemotaxis were found to not be significantly upregulated or utilized during infection. These results were supported by gene expression data from natural M2 lesions of dairy cattle. This gene expression analysis could highlight the preference in strategy for T. phagedenis to persist and adhere in the host rather than engage in motility and disseminate.

The Oxidative Stress Markers' Protective Influence of Sea Buckthorn and Grape Extracts in Atorvastatin-Treated Hyperlipidemic Rats.

Free radicals and reactive oxygen species initiate when the oxidative stress arises. (1) Background: The effect of natural molecules on oxidative stress in hyperlipidemic rats, taking statins, was observed. (2) Methods: One hundred and twelve white Wistar rats, males and females, were divided into seven: Group I received 20 mg of atorvastatin while groups II and III received a combination of 20 mg of atorvastatin and 100 mg of Sea buckthorn and grape extract. Groups IV and V received 100 mg of Sea buckthorn and grape extract, while groups VI and VII received only high-fat diet (HFD) and normal rodents' fodder. After two and six months, rats were euthanized, and blood was gathered to measure the main paraclinical values and total antioxidant capacity (TAC). Also, the liver and kidney were stored for the organs' cytoarchitecture. For statistics, two-way analysis of variance (ANOVA), was performed. (3) Results: HFD produced hyperlipidemia, accompanied by augmented serum and hepatic oxidative stress markers, in addition to a reduction in antioxidant enzyme activities and glutathione levels. Polyphenolic substances proven efficient against HFD caused oxidative stress. (4) Conclusions: Atorvastatin heightened the histological injuries caused by the fatty diet, but these were diminished by taking atorvastatin in combination with 100 mg/kg of plant extracts.

The impact of high-IgE levels on metabolome and microbiome in experimental allergic enteritis.

BACKGROUND: The pathological mechanism of the gastrointestinal forms of food allergies is less understood in comparison to other clinical phenotypes, such as asthma and anaphylaxis Importantly, high-IgE levels are a poor prognostic factor in gastrointestinal allergies. METHODS: This study investigated how high-IgE levels influence the development of intestinal inflammation and the metabolome in allergic enteritis (AE), using IgE knock-in (IgEki) mice expressing high levels of IgE. In addition, correlation of the altered metabolome with gut microbiome was analysed. RESULTS: Ovalbumin-sensitized and egg-white diet-fed (OVA/EW) BALB/c WT mice developed moderate AE, whereas OVA/EW IgEki mice induced more aggravated intestinal inflammation with enhanced eosinophil accumulation. Untargeted metabolomics detected the increased levels of N-tau-methylhistamine and 2,3-butanediol, and reduced levels of butyric acid in faeces and/or sera of OVA/EW IgEki mice, which was accompanied with reduced Clostridium and increased Lactobacillus at the genus level. Non-sensitized and egg-white diet-fed (NC/EW) WT mice did not exhibit any signs of AE, whereas NC/EW IgEki mice developed marginal degrees of AE. Compared to NC/EW WT mice, enhanced levels of lysophospholipids, sphinganine and sphingosine were detected in serum and faecal samples of NC/EW IgEki mice. In addition, several associations of altered metabolome with gut microbiome-for example Akkermansia with lysophosphatidylserine-were detected. CONCLUSIONS: Our results suggest that high-IgE levels alter intestinal and systemic levels of endogenous and microbiota-associated metabolites in experimental AE. This study contributes to deepening the knowledge of molecular mechanisms for the development of AE and provides clues to advance diagnostic and therapeutic strategies of allergic diseases.

α7 nicotinic acetylcholine receptor agonist attenuates allergen-induced immediate nasal response in murine model of allergic rhinitis.

The expression of nicotinic acetylcholine receptor (nAChR) subunits on various immune cells suggests their involvement in allergic rhinitis. However, how exactly they contribute to this pathogenesis is not yet confirmed. Our present study examined the therapeutic potential of GTS-21, an α7 nAChR agonist, for treating allergic rhinitis by employing its mouse models. GTS-21 treatment reduced allergen-induced immediate nasal response in ovalbumin (OVA)-sensitized model. However, nasal hyperresponsiveness or eosinophil infiltration elicited in either the OVA-sensitized or T helper 2 cell-transplanted model was not affected by GTS-21. GTS-21 did not alter allergen-induced passive cutaneous anaphylaxis response in anti-dinitrophenyl IgE-sensitized mice. This evidence implies GTS-21's potential to alleviate allergic rhinitis without perturbing T cells or mast cells.

Developing a Novel Murine Meningococcal Meningitis Model Using a Capsule-Null Bacterial Strain.

BACKGROUND: Neisseria meningitidis (meningococcus) is a Gram-negative bacterium that colonises the nasopharynx of about 10% of the healthy human population. Under certain conditions, it spreads into the body, causing infections with high morbidity and mortality rates. Although the capsule is the key virulence factor, unencapsulated strains have proved to possess significant clinical implications as well. Meningococcal meningitis is a primarily human infection, with limited animal models that are dependent on a variety of parameters such as bacterial virulence and mouse strain. In this study, we aimed to develop a murine Neisseria meningitidis meningitis model to be used in the study of various antimicrobial compounds. METHOD: We used a capsule-deficient Neisseria meningitidis strain that was thoroughly analysed through various methods. The bacterial strain was incubated for 48 h in brain-heart infusion (BHI) broth before being concentrated and injected intracisternally to bypass the blood-brain barrier in CD-1 mice. This prolonged incubation time was a key factor in increasing the virulence of the bacterial strain. A total of three more differently prepared inoculums were tested to further solidify the importance of the protocol (a 24-h incubated inoculum, a diluted inoculum, and an inactivated inoculum). Antibiotic treatment groups were also established. The clinical parameters and number of deaths were recorded over a period of 5 days, and comatose mice with no chance of recovery were euthanised. RESULTS: The bacterial strain was confirmed to have no capsule but was found to harbour a total of 56 genes coding virulence factors, and its antibiotic susceptibility was established. Meningitis was confirmed through positive tissue culture and histological evaluation, where specific lesions were observed, such as perivascular sheaths with inflammatory infiltrate. In the treatment groups, survival rates were significantly higher (up to 81.25% in one of the treatment groups compared to 18.75% in the control group). CONCLUSION: We managed to successfully develop a cost-efficient murine (using simple CD-1 mice instead of expensive transgenic mice) meningococcal meningitis model using an unencapsulated strain with a novel method of preparation.

AAV mediated repression of Neat1 lncRNA combined with F8 gene augmentation mitigates pathological mediators of joint disease in haemophilia.

Haemophilic arthropathy (HA), a common comorbidity in haemophilic patients leads to joint pain, deformity and reduced quality of life. We have recently demonstrated that a long non-coding RNA, Neat1 as a primary regulator of matrix metalloproteinase (MMP) 3 and MMP13 activity, and its induction in the target joint has a deteriorating effect on articular cartilage. In the present study, we administered an Adeno-associated virus (AAV) 5 vector carrying an short hairpin (sh)RNA to Neat1 via intra-articular injection alone or in conjunction with systemic administration of a capsid-modified AAV8 (K31Q) vector carrying F8 gene (F8-BDD-V3) to study its impact on HA. AAV8K31Q-F8 vector administration at low dose, led to an increase in FVIII activity (16%-28%) in treated mice. We further observed a significant knockdown of Neat1 (~40 fold vs. untreated injured joint, p = 0.005) in joint tissue of treated mice and a downregulation of chondrodegenerative enzymes, MMP3, MMP13 and the inflammatory mediator- cPLA2, in mice receiving combination therapy. These data demonstrate that AAV mediated Neat1 knockdown in combination with F8 gene augmentation can potentially impact mediators of haemophilic joint disease.

Usefulness of a murine model of hepatic cystic echinococcosis for preclinical drug trials: Efficacy of albendazole vs albendazole nanocrystals.

Cystic echinococcosis is a zoonotic infection caused by the larval stage of Echinococcus granulosus sensu lato. The disease is characterized by the long-term growth of cysts, most commonly in the liver and lungs. Although an ideal model of cystic echinococcosis should induce the development of cysts in the liver and imitate the natural infection route, the murine model of intraperitoneal is still widely used in the field of experimental theraphy. The aim of the present work was to evaluate the usefulness of the murine model of hepatic CE for preclinical drug trials. The effectiveness of albendazole could also be assessed by measuring the diameter of the hepatic cyst. The albendazole significantly reduced the size of the cysts. The ultrastructural alterations of the germinal layer of hepatic cysts provoked by albendazole coincided with those observed in the intraperitoneal model. Similar results were obtained with both albendazole doses. Therefore, the efficacy of albendazole nanocrystals in the murine model of hepatic cystic echinococcosis was carried out at albendazole doses of 25 mg/kg. The abdominal ultrasound allows us to assess the response of cysts to drugs only in a qualitative manner. Although the size of cysts in the albendazole nanocrystal group was not significantly lower than that observed with albendazole, at the ultrastructural level, a greater extent of damage was observed. The murine model of hepatic cystic echinococcosis can be effectively used for assessing the effect of novel formulations or compounds. The main advantage of this model is that cysts are located in the orthotopic organ, which resembles the location most commonly found in human cases. In future studies, the usefulness of the model for pharmacokinetics studies in hepatic cysts will be evaluated.

Establishment of a Posttraumatic Osteoarthritis Model in Mice Induced by Noninvasive Anterior Cruciate Ligament Tear.

BACKGROUND: Animal models that use open surgical transection of the anterior cruciate ligament (ACL) do not accurately simulate the clinical condition regarding the pivot-shift mechanism and the associated inflammatory response that occurs before reconstruction. PURPOSE/HYPOTHESIS: The purpose was to characterize a reproducible manual, nonsurgical method to mimic an isolated ACL tear in a clinically relevant model and to evaluate the development of progressive posttraumatic osteoarthritis due to ACL injury. It was hypothesized that the ACL could be reproducibly torn with minimal damage to other ligaments and that there would be progressive development of degenerative joint disease after ACL injury. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 37 mice (strain C57BL/6) were used to compare the manual procedure with sham surgery (sham group; n = 10) and with the established surgical ACL transection (ACLT) procedure (surgical group; n = 27). In the sham group, a closed manual procedure was performed on the right knee and sham surgery on the left knee. In the surgical group, the closed manual procedure was performed on the right knee and surgical ACLT on the left knee. Dissection using India ink, histological assessment with safranin O and hematoxylin-eosin staining, radiological evaluation through radiographs and microfocus computed tomography scans, and gait analyses were performed to assess cartilage/ligament status. Osteoarthritis Research Society International (OARSI) and synovitis scores, anterior tibial translation, range of motion, bone microstructure, osteophyte volume, and pain were assessed at 2, 4, and 8 weeks postoperatively. RESULTS: The manual procedure successfully resulted in an ACL rupture and associated meniscal injury. The posterior cruciate, lateral collateral, and medial collateral ligaments were intact in all dissected knees. Two weeks after ACL tear, the surgical group showed a significantly higher synovitis score, whereas 8 weeks after ACL tear, the manual group showed a significantly higher volume of osteophytes. No significant differences were found between the groups in terms of OARSI score, anterior tibial translation, range of motion, bone microstructure computed tomography values, and stride distance/irregularity. CONCLUSION: This procedure can be used to create an ACL tear model without causing grossly evident injuries to other ligaments and avoiding the risk of cartilage damage from surgical instruments. CLINICAL RELEVANCE: This procedure offers a more clinically relevant ACL tear model and facilitates simple, inexpensive, and reproducible development of posttraumatic osteoarthritis.

KR158 Spheres Harboring Slow-Cycling Cells Recapitulate High-Grade Glioma Features in an Immunocompetent System.

Glioblastoma (GBM) poses a significant challenge in clinical oncology due to its aggressive nature, heterogeneity, and resistance to therapies. Cancer stem cells (CSCs) play a critical role in GBM, particularly in treatment resistance and tumor relapse, emphasizing the need to comprehend the mechanisms regulating these cells. Also, their multifaceted contributions to the tumor microenvironment (TME) underline their significance, driven by their unique properties. This study aimed to characterize glioblastoma stem cells (GSCs), specifically slow-cycling cells (SCCs), in an immunocompetent murine GBM model to explore their similarities with their human counterparts. Using the KR158 mouse model, we confirmed that SCCs isolated from this model exhibited key traits and functional properties akin to human SCCs. KR158 murine SCCs, expanded in the gliomasphere assay, demonstrated sphere forming ability, self-renewing capacity, positive tumorigenicity, enhanced stemness and resistance to chemotherapy. Together, our findings validate the KR158 murine model as a framework to investigate GSCs and SCCs in GBM pathology, and explore specifically the SCC-immune system communications, understand their role in disease progression, and evaluate the effect of therapeutic strategies targeting these specific connections.

A pathological study on the efficacy of Syk inhibitors in a Candida albicans-induced aortic root vasculitis murine model.

BACKGROUND: The activation of innate immunity may be involved in the development of Candida albicans-induced murine vasculitis, which resembles Kawasaki disease (KD) vasculitis. This study aimed to histologically clarify the time course of the development of vasculitis in this model in detail and to estimate the potential role of spleen tyrosine kinase (Syk) inhibitors in KD vasculitis. METHODS AND RESULTS: DBA/2 male mice were intraperitoneally injected with a vasculitis-inducing substance and treated with a Syk inhibitor (R788 or GS-9973). Systemic vasculitis, especially in the aortic annulus area, was histologically evaluated. Regarding lesions in the aortic annulus area, some mice in the untreated control group already showed initiation of vasculitis 1 day after the final injection of a vasculitis-inducing substance. The vasculitis expanded over time. Inflammation occurred more frequently at the aortic root than at the coronary artery. The distribution of inflammatory cells was limited to the intima, intima plus adventitia, or all layers. In the Syk inhibitor-treated groups, only one mouse had vasculitis at all observation periods. The severity and area of the vasculitis were reduced by both Syk inhibitors. CONCLUSION: Candida albicans-induced murine vasculitis may occur within 1 day after the injection of a vasculitis-inducing substance. Additionally, Syk inhibitors suppress murine vasculitis.

Sexually dimorphic effects of prenatal alcohol exposure on the murine skeleton.

BACKGROUND: Prenatal alcohol exposure (PAE) can result in lifelong disabilities known as foetal alcohol spectrum disorder (FASD) and is associated with childhood growth deficiencies and increased bone fracture risk. However, the effects of PAE on the adult skeleton remain unclear and any potential sexual dimorphism is undetermined. Therefore, we utilised a murine model to examine sex differences with PAE on in vitro bone formation, and in the juvenile and adult skeleton. METHODS: Pregnant C57BL/6J female mice received 5% ethanol in their drinking water during gestation. Primary calvarial osteoblasts were isolated from neonatal offspring and mineralised bone nodule formation and gene expression assessed. Skeletal phenotyping of 4- and 12-week-old male and female offspring was conducted by micro-computed tomography (µCT), 3-point bending, growth plate analyses, and histology. RESULTS: Osteoblasts from male and female PAE mice displayed reduced bone formation, compared to control (≤ 30%). Vegfa, Vegfb, Bmp6, Tgfbr1, Flt1 and Ahsg were downregulated in PAE male osteoblasts only, whilst Ahsg was upregulated in PAE females. In 12-week-old mice, µCT analysis revealed a sex and exposure interaction across several trabecular bone parameters. PAE was detrimental to the trabecular compartment in male mice compared to control, yet PAE females were unaffected. Both male and female mice had significant reductions in cortical parameters with PAE. Whilst male mice were negatively affected along the tibial length, females were only distally affected. Posterior cortical porosity was increased in PAE females only. Mechanical testing revealed PAE males had significantly reduced bone stiffness compared to controls; maximum load and yield were reduced in both sexes. PAE had no effect on total body weight or tibial bone length in either sex. However, total growth plate width in male PAE mice compared to control was reduced, whilst female PAE mice were unaffected. 4-week-old mice did not display the altered skeletal phenotype with PAE observed in 12-week-old animals. CONCLUSIONS: Evidence herein suggests, for the first time, that PAE exerts divergent sex effects on the skeleton, possibly influenced by underlying sex-specific transcriptional mechanisms of osteoblasts. Establishing these sex differences will support future policies and clinical management of FASD.

Prenatal alcohol exposure (PAE) can lead to a set of lifelong cognitive, behavioural, and physical disabilities known as foetal alcohol spectrum disorder (FASD). FASD is a significant burden on healthcare, justice and education systems, which is set to worsen with rising alcohol consumption rates. FASD children have an increased risk of long bone fracture and adolescents are smaller in stature. However, sex differences and the long-term effects of PAE on the skeleton have not been investigated and was the aim of this study. Using a mouse model of PAE, we examined the function and gene expression of bone-forming cells (osteoblasts). We then analysed the skeletons of male and female mice at 12-weeks-old (adult) and 4-weeks-old (juvenile). PAE reduced osteoblast bone formation in both sexes, compared to control. Differential gene expression was predominantly observed in PAE males and largely involved genes related to blood vessel formation. High resolution x-ray imaging (micro-CT) revealed PAE had a detrimental effect on the inner trabecular bone component in 12-week-old male mice only. Analysis of the outer cortical bone revealed that whilst both male and female PAE mice were negatively affected, anatomical variations were observed. Mechanical testing also revealed differences in bone strength in PAE mice, compared to control. Interestingly, 4-week-old mice did not possess these sex differences observed in our PAE model at 12 weeks of age. Our data suggest PAE has detrimental and yet sex-dependent effects on the skeleton. Establishing these sex differences will support future policies and clinical management of FASD.

In Vivo Modeling of Cryptococcus neoformans Infection and Collection of Murine Samples.

In vivo models provide advantages to study the progression of disease and to identify potential biomarkers to detect and monitor infections. For the human fungal pathogen Cryptococcus neoformans, murine intranasal models aim to recapitulate natural infection from inhalation of desiccated fungal cells from the environment and permit monitoring of disease over time. In this chapter, we describe the establishment of a murine model for cryptococcosis and the subsequent collection of organs, tissues, and fluids for sampling. These samples may support novel diagnostic strategies and opportunities to monitor dissemination of the fungal cells throughout the host and propose new treatment options to combat disease.

Proteomic Profiling of Samples Derived from a Murine Model Following Cryptococcus neoformans Infection.

Proteomic profiling provides in-depth information about the regulation of diverse biological processes, activation of and communication across signaling networks, and alterations to protein production, modifications, and interactions. For infectious disease research, mass spectrometry-based proteomics enables detection of host defenses against infection and mechanisms used by the pathogen to evade such responses. In this chapter, we outline protein extraction from organs, tissues, and fluids collected following intranasal inoculation of a murine model with the human fungal pathogen Cryptococcus neoformans. We describe sample preparation, followed by purification, processing on the mass spectrometer, and a robust bioinformatics analysis. The information gleaned from proteomic profiling of fungal infections supports the detection of novel biomarkers for diagnostic and prognostic purposes.