Innate Immune Zonation in the Liver: NF-κB (p50) Activation and C-Reactive Protein Expression in Response to Endotoxemia Are Zone Specific

Journal of immunology (Baltimore, Md. : 1950)

2023 Mar 22

McCarthy, WC;Sherlock, LG;Grayck, MR;Zheng, L;Lacayo, OA;Solar, M;Orlicky, DJ;Dobrinskikh, E;Wright, CJ;
PMID: 36946778 | DOI: 10.4049/jimmunol.2200900

Hepatic innate immune function plays an important role in the pathogenesis of many diseases. Importantly, a growing body of literature has firmly established the spatial heterogeneity of hepatocyte metabolic function; however, whether innate immune function is zonated remains unknown. To test this question, we exposed adult C57BL/6 mice to endotoxemia, and hepatic tissue was assessed for the acute phase response (APR). The zone-specific APR was evaluated in periportal and pericentral/centrilobular hepatocytes isolated using digitonin perfusion and on hepatic tissue using RNAscope and immunohistochemistry. Western blot, EMSA, chromatin immunoprecipitation, and immunohistochemistry were used to determine the role of the transcription factor NF-κB in mediating hepatic C-reactive protein (CRP) expression. Finally, the ability of mice lacking the NF-κB subunit p50 (p50-/-) to raise a hepatic APR was evaluated. We found that endotoxemia induces a hepatocyte transcriptional APR in both male and female mice, with Crp, Apcs, Fga, Hp, and Lbp expression being enriched in pericentral/centrilobular hepatocytes. Focusing our work on CRP expression, we determined that NF-κB transcription factor subunit p50 binds to consensus sequence elements present in the murine CRP promoter. Furthermore, pericentral/centrilobular hepatocyte p50 nuclear translocation is temporally associated with zone-specific APR during endotoxemia. Lastly, the APR and CRP expression is blunted in endotoxemic p50-/- mice. These results demonstrate that the murine hepatocyte innate immune response to endotoxemia includes zone-specific activation of transcription factors and target gene expression. These results support further study of zone-specific hepatocyte innate immunity and its role in the development of various disease states.

Angiotensin II and AT1a Receptors in the Proximal Tubules of the Kidney: New Roles in Blood Pressure Control and Hypertension

International journal of molecular sciences

2022 Feb 22

Leite, APO;Li, XC;Nwia, SM;Hassan, R;Zhuo, JL;
PMID: 35269547 | DOI: 10.3390/ijms23052402

Contrary to public perception, hypertension remains one of the most important public health problems in the United States, affecting 46% of adults with increased risk for heart attack, stroke, and kidney diseases. The mechanisms underlying poorly controlled hypertension remain incompletely understood. Recent development in the Cre/LoxP approach to study gain or loss of function of a particular gene has significantly helped advance our new insights into the role of proximal tubule angiotensin II (Ang II) and its AT1 (AT1a) receptors in basal blood pressure control and the development of Ang II-induced hypertension. This novel approach has provided us and others with an important tool to generate novel mouse models with proximal tubule-specific loss (deletion) or gain of the function (overexpression). The objective of this invited review article is to review and discuss recent findings using novel genetically modifying proximal tubule-specific mouse models. These new studies have consistently demonstrated that deletion of AT1 (AT1a) receptors or its direct downstream target Na+/H+ exchanger 3 (NHE3) selectively in the proximal tubules of the kidney lowers basal blood pressure, increases the pressure-natriuresis response, and induces natriuretic responses, whereas overexpression of an intracellular Ang II fusion protein or AT1 (AT1a) receptors selectively in the proximal tubules increases proximal tubule Na+ reabsorption, impairs the pressure-natriuresis response, and elevates blood pressure. Furthermore, the development of Ang II-induced hypertension by systemic Ang II infusion or by proximal tubule-specific overexpression of an intracellular Ang II fusion protein was attenuated in mutant mice with proximal tubule-specific deletion of AT1 (AT1a) receptors or NHE3. Thus, these recent studies provide evidence for and new insights into the important roles of intratubular Ang II via AT1 (AT1a) receptors and NHE3 in the proximal tubules in maintaining basal blood pressure homeostasis and the development of Ang II-induced hypertension.

Immune analysis of lymph nodes in relation to the presence or absence of tumor infiltrating lymphocytes in triple-negative breast cancer

European journal of cancer (Oxford, England : 1990)

2021 Mar 17

Quintana, Á;Peg, V;Prat, A;Moliné, T;Villacampa, G;Paré, L;Galván, P;Dientsmann, R;Schmid, P;Curigliano, G;Muñoz-Couselo, E;Perez-García, J;Marti, M;Blanco-Heredia, J;Anjos, CD;Vazquez, M;De Mattos-Arruda, L;Cortés, J;
PMID: 33743482 | DOI: 10.1016/j.ejca.2021.01.037

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with unmet medical needs. Several studies have proved that high levels of tumor infiltrating lymphocytes (TILs) at diagnosis of TNBC confer better prognosis and patients respond better to specific chemotherapies. Nonetheless, current evidence suggests that only 15% of TNBC patients have very high levels of TILs, and another 15% lacks TILs. One possible reason to explain why patients have low TILs at diagnosis is that lymphocytes might be deactivated by an immune checkpoint in local lymph nodes, provoking their retention in there as they are unresponsive to other immune stimuli. We have identified 15 high TILs (≥50%) and 20 low TILs (≤5%) TNBC patients with localised tumour (T1c-T2N0M0) and compared the protein expression of five immune checkpoints in lymph nodes. We have also performed a customised 50-immune gene NanoString expression panel, the NanoString 360 Breast Cancer panel, and whole exome sequencing for mutation and neoantigen load analyses. In low TILs, we observed higher expression of CTLA-4 in local lymph nodes, which could explain why lymphocytes get retained in there and do not migrate to tumour. These patients have also higher neoantigen load and higher expression of B7.H3 and B7.H4 in the tumour. In high TILs, we observed more PD-L1+ tumour cells and more expanded humoral response. These results could provide a strategy to revert low tumour immune infiltration at diagnosis of TNBC, improving their prognosis.

Spatial Distribution of Neurons Expressing Single, Double, and Triple Molecular Characteristics of Glutamatergic, Dopaminergic, or GABAergic Neurons in the Mouse Ventral Tegmental Area

Journal of molecular neuroscience : MN

2023 May 27

Ma, S;Zhong, H;Liu, X;Wang, L;
PMID: 37243808 | DOI: 10.1007/s12031-023-02121-2

The ventral tegmental area (VTA) is a heterogeneous midbrain area that plays a significant role in diverse neural processes such as reward, aversion, and motivation. The VTA contains three main neuronal populations, namely, dopamine (DA), γ-aminobutyric acid (GABA), and glutamate neurons, but some neurons exhibit combinatorial molecular characteristics of dopaminergic, GABAergic, or glutamatergic neurons. However, little information is available regarding detailed distribution of neurons with single, double, and triple molecular characteristics of glutamatergic, dopaminergic, or GABAergic neurons in mice. We present a topographical distribution map of three main neuronal populations expressing a single molecular characteristic of dopaminergic, GABAergic, or glutamatergic neurons, and four neuronal populations co-expressing double or triple molecular characteristics in combinatorial manners, in the mouse VTA, following analysis of triple fluorescent in situ hybridization for the simultaneous detection of tyrosine hydroxylase (TH, marker for dopaminergic neurons), vesicular glutamate transporter 2 (VGLUT2, marker for glutamatergic neurons), and glutamic acid decarboxylase 2 (GAD2, marker for GABAergic neurons) mRNA. We found that the vast majority of neurons expressed a single type of mRNA, and these neurons were intermingled with neurons co-expressing double or triple combinations of VGLUT2, TH, or GAD2 in the VTA. These seven neuronal populations were differentially distributed in the VTA sub-nuclei across the rostro-caudal and latero-medial axes. This histochemical study will lead to a deeper understanding of the complexity of neuronal molecular characteristics in different VTA sub-nuclei, and potentially facilitate clarification of diverse functions of the VTA.

Gut Microbiota And Metabolites Drive Persistent Pain In Sickle Cell Disease

The Journal of Pain

2023 Apr 01

Sadler, K;Atkinson, S;Ehlers, V;Waltz, T;Hayward, M;Rodriguez-Garcia, D;Salzman, N;Stucky, C;Brandow, A;
| DOI: 10.1016/j.jpain.2023.02.104

Severe debilitating pain is the most common complication and reason for hospitalization for individuals with sickle cell disease (SCD), a genetic blood disorder that affects 100,000 people in the US and over 3 million worldwide. Despite this, the biological basis of chronic SCD pain is not fully understood. Using transgenic SCD mice and fecal material transplant paradigms, we determined that gastrointestinal tract contents drive persistent SCD pain. Mechanical allodynia was temporarily alleviated in SCD mice following fecal transplant from wildtype animals. In contrast, wildtype mice developed mechanical and cold allodynia following fecal transplant from SCD animals. To identify gut bacterial species and metabolites responsible for SCD pain, we completed 16s rRNA sequencing and metabolomic screening respectively on transplant recipient feces. Bilirubin, a product of heme breakdown, was significantly elevated in the feces of SCD mice and mice that received SCD fecal transplants, as well as in the plasma of individuals with SCD. Oral administration of bilirubin induced mechanical allodynia in wildtype mice that depended on vagus nerve signaling. Using whole cell patch clamp recordings, we demonstrated that bilirubin directly activates vagal afferents and increases afferent excitability. Ongoing experiments are investigating the specific receptors through which bilirubin alters neuronal activity as drugs targeting these proteins may prove effective analgesics for SCD pain. In summary, these experiments are the first to demonstrate that sickle cell gut contents drive chronic widespread pain in this disease, and furthermore, are the first to identify gut metabolites that should be targeted for chronic SCD pain management. National Institutes of Health: K99HL155791(KS), R01HL142657(AB), R01NS070711 CS).

Skin Resident Memory T Cell Dysfunction In The Tibia Fracture Model Of Complex Regional Pain Syndrome

The Journal of Pain

2023 Apr 01

Wickman, J;Shenoda, B;Van Duyne, R;Kline, Z;Ajit, S;
| DOI: 10.1016/j.jpain.2023.02.060

Complex regional pain syndrome (CRPS) is a debilitating chronic pain disorder that with no effective treatments. Several microRNA (miRNA) are commonly dysregulated in CRPS patient and tibia fracture model of CRPS (TFM) mice, including miR-25 which is associated with positive treatment outcomes in patients. Interestingly, these miRNAs are predicted to target several genes critical to resident memory T cell (Trm) function. We hypothesize that miRNA dysregulation contributes to the pathology of CRPS through regulation of skin Trm development and maintenance. Therapeutic strategies blocking Trm development or maintenance may be beneficial in treating this disease. Whole blood samples were obtained from CRPS patients or healthy controls. miRNA and gene expression changes in blood and T cells were assessed by qPCR. Animals were treated with therapeutic agents after development of TFM and monitored for behavioral outcomes and T cell populations of collected tissues were analyzed at different time points by flow cytometry. There was an inverse correlation of miR-25 and CD69 in blood samples from CRPS patients compared to controls. TFM hindlimb skin shows increased epidermal CD8+ and CD4+ Trm, dermal CD4+ Trm. Epidermal CD8+ Trm, dermal CD4+ Trm are marked by increases in CD103+CD49a+ populations, and along with splenic CD8+ Tem show increased CD122+ cells. Therapeutic studies are ongoing. miRNA signatures in CRPS patients and TFM mice show common alterations which are capable of regulating CD69, a core Trm marker. TFM hindlimb skin shows increased pathological Trm populations and treatments targeting Trm development and maintenance may be beneficial in treating CRPS. 1RF1NS130481-01.

Mfge8 is expressed by pericytes in human gastric antrum submucosa from obese patients

American journal of physiology. Cell physiology

2023 Mar 20

Perrino, BA;Malogan, J;Cobine, CA;Sasse, KC;
PMID: 36939201 | DOI: 10.1152/ajpcell.00043.2023

The main function of the stomach is to digest ingested food. Gastric antrum muscular contractions mix ingested food with digestive enzymes and stomach acid and propel the chyme through the pyloric sphincter at a rate in which the small intestine can process the chyme for optimal nutrient absorption. Mfge8 binding to α8β1 integrins helps regulate gastric emptying by reducing the force of antral smooth muscle contractions. The source of Mfge8 within gastric muscles is unclear. Since Mfge8 is a secreted protein, Mfge8 could be delivered via the circulation, or be locally secreted by cells within the muscle layers. In this study we identify a source of Mfge8 within human gastric antrum muscles using spatial transcriptomic analysis. We show that Mfge8 is expressed in subpopulations of Mef2c+ perivascular cells within the submucosa layer of the gastric antrum. Mef2c is expressed in subpopulations of NG2+ and PDGFRB+ pericytes. Mfge8 is expressed in NG2+/Mef2c+ pericytes, but not in NG2+/Mef2c-, PDGFRB+/Mef2c-, or PDGFRB+/Mef2c+ pericytes. Mfge8 is absent from CD34+ endothelial cells but is expressed in a small population of perivascular ACTA2+ cells. We also show that α8 integrin is not expressed by ICC, supporting the findings that Mfge8 attenuates gastric antrum smooth muscle contractions by binding to α8β1 integrins on enteric smooth muscle cells. These findings suggest a novel, supplementary mechanism of regulation of gastric antrum motility by cellular regulators of capillary blood flow, in addition to the regulation of gastric antrum motility by the enteric nervous system and the SIP syncytium.

The infertile individual analysis based on whole-exome sequencing in chinese multi-ethnic groups

Genes & genomics

2022 Sep 17

Zhao, Q;Li, Y;Liang, Q;Zhao, J;Kang, K;Hou, M;Zhang, X;Du, R;Kong, L;Liang, B;Huang, W;
PMID: 36115009 | DOI: 10.1007/s13258-022-01307-z

Infertility is a common and rapidly growing health issue around the world. The genetic analysis based on the infertile population is crucial for intervention and treatment.To find candidate gene locus led to azoospermia in Chinese multi-ethnic groups and provide theoretical guidance for the diagnosis of genetic diseases to progressively aggravated infertility patients and sterile offspring with ART.The study based on whole-exome sequencing (WES) was presented for genetic characteristic analysis of multi-ethnics and identification of variants related to infertility in Xinjiang area of China.The frequency of pathogenic variants showed significant ethnic differences among four main ethnics in Xinjiang. The population structure analysis confirmed that the Hui was close to the Han population, the Kazak was close to the Uygur population, and there are three ancestry components in the four ethnics. In addition, ten candidate variants potentially regulated azoospermia were detected, and KNTC1 (rs7968222: G > T) was chosen to validate the association. Through the analysis in the valid group, the frequency of rs7968222 (G > T) has a significant difference in the azoospermia population (11.76%, 8/68) and normospermia population (4.63%, 35/756) (P < 0.001). Interestingly, the proportion of people with abnormal follicle-stimulating hormone (FSH) level in the group carrying rs7968222 (G > T) was significantly higher than non-carriers (P < 0.05). Therefore, rs7968222 may regulate spermatogenesis through affecting hormone level.Our study establishes the genetics analysis of Northwest China and finds a candidate gene locus KNTC1 (rs7968222: G > T), which is one of the genetic susceptibility factors for male azoospermia.

Role of Ecogenomics in Conservation and Management

Plant Ecogenomics

2022 Aug 19

Islam, S;Mangral, Z;Tariq, L;Bhat, B;Mehraj, H;Islam, S;Dar, T;
| DOI: 10.1201/9781003282006-9

Ecogenomics is the scientific approach to understand the relationship between structural and functional aspects of genomes with biotic/abiotic environmental factors. The classification of ecology depends upon the overall complexity (behavioral and population ecology) of an organism (plant and animal ecology) and system under investigation (soil and forest ecology). The molecular techniques adopted by these various ecology branches result in a new field known as ecological genomics or ecogenomics that focuses on an organism’s overall development during the evolutionary period. It is an interdisciplinary research field covering ecological science, microbiology, 176environmental and molecular biology, toxicology, physiology, chemistry, etc. The study related to the ecology of plants has a direct relationship with the adaptation mechanism because plants have no alternative to cope with the environment in which they grow. The world is facing biodiversity loss at an alarming rate, with a loss of 90% crop varieties in a century from the field. It is essential to select the genes in biological pathways responsible for an organism’s stability in the ecological system. The challenge is to understand the basic phenomenon behind adaptation, migration, inbreeding mechanism of endangered or critically endangered species. in ecogenomics, we know that the genes are responsible for the effective management strategies from a conservation point of view. Therefore, in this chapter, we discuss various aspects of community shaping and visualization. The community’s function and structure need to be studied due to the availability of plenty of molecular data. Further, the role of ecogenomics and multiomic approaches in conservation and management will also be emphasized.

Quantitative Imaging Analysis of the Spatial Relationship between Antiretrovirals, Reverse Transcriptase Simian-Human Immunodeficiency Virus RNA, and Fibrosis in the Spleens of Nonhuman Primates

Antimicrobial agents and chemotherapy

2022 Jul 20

Devanathan, AS;White, NR;Desyaterik, Y;De la Cruz, G;Nekorchuk, M;Terry, M;Busman-Sahay, K;Adamson, L;Luciw, P;Fedoriw, Y;Estes, JD;Rosen, EP;Kashuba, ADM;
PMID: 35856680 | DOI: 10.1128/aac.00609-22

Although current antiretroviral therapy (ART) has increased life expectancy, a cure for human immunodeficiency virus (HIV) remains elusive due to the persistence of the virus in tissue reservoirs. In the present study, we sought to elucidate the relationship between antiretrovirals (ARVs) and viral expression in the spleen. We performed mass spectrometry imaging (MSI) of 6 different ARVs, RNAscope in situ hybridization of viral RNA, and immunohistochemistry of three different fibrosis markers in the spleens of 8 uninfected and 10 reverse transcriptase simian-human immunodeficiency virus (RT-SHIV)-infected rhesus macaques (infected for 6 weeks) that had been dosed for 10 days with combination ART. Using MATLAB, computational quantitative imaging analysis was performed to evaluate the spatial and pharmacological relationships between the 6 ARVs, viral RNA, and fibrotic deposition. In these spleens, >50% of the spleen tissue area was not covered by any detectable ARV response (any concentration above the limits of detection for individual ARVs). The median spatial ARV coverage across all tissues was driven by maraviroc followed by efavirenz. Yet >50% of RNA-positive cells were not exposed to any detectable ARV. Quantifiable maraviroc and efavirenz colocalization with RNA-positive cells was usually greater than the in vitro concentration inhibiting 50% replication (IC50). Fibrosis markers covered more than 50% of the spleen tissue area and had negative relationships with cumulative ARV coverages. Our findings suggest that a heterogeneous ARV spatial distribution must be considered when evaluating viral persistence in lymphoid tissue reservoirs.

Detection accuracy of the Cobas HPV assay for high-risk HPV in head and neck FNA biopsy specimens

Cancer cytopathology

2022 Feb 22

Guo, M;Khanna, A;Tinnirello, AA;Hwang, J;Zhang, P;Xu, L;Li, G;Dahlstrom, KR;Sturgis, EM;Stewart, J;
PMID: 35192231 | DOI: 10.1002/cncy.22563

This study evaluated the detection accuracy of the Cobas human papillomavirus (HPV) assay for high-risk human papillomavirus (hrHPV) and HPV-16 in head and neck fine-needle aspiration (FNA) specimens with squamous cell carcinoma.Head and neck FNA biopsy specimens from 2012 to 2020 were retrospectively collected. Cobas HPV testing was performed on 90 FNA specimens with valid Cervista HPV testing results. Results of Cobas HPV and Cervista HPV assays were compared. A Linear Array or SPF10-LiPA25 HPV genotyping assay resolved cases with discrepant results. The κ value and accuracy of Cobas HPV testing were calculated. The accuracy of the Cobas HPV assay was also determined in 42 FNA needle-rinse specimens.Cobas HPV was positive in 82% of the FNA specimens (74 of 90). The concordance between Cobas HPV and Cervista HPV test results was 88.9% (80 of 90) with substantial agreement (κ = 0.669; 95% CI, 0.481-0.856). With HPV genotyping confirmation in cases with discrepant results between the 2 HPV assays, Cobas HPV showed 100% sensitivity and specificity for hrHPV. HPV-16 was detected in 88% of HPV-positive cases (65 of 74). HPV genotyping confirmed 1 false-negative HPV-16 result and 1 false-positive HPV-16 result. Overall, the accuracy of Cobas HPV for HPV-16 was 97.8%. The accuracy of Cobas HPV in FNA needle-rinse specimens was 100%.The Cobas HPV assay is highly accurate for determining the HPV status in head and neck FNA specimens. FNA needle rinse is valid for Cobas HPV testing in patients with squamous cell carcinoma.

Spatial distribution of beta-klotho mRNA in the mouse hypothalamus, hippocampal region, subiculum, and amygdala

The Journal of comparative neurology

2022 Feb 10

Bono, BS;Koziel Ly, NK;Miller, PA;Williams-Ikhenoba, J;Dumiaty, Y;Chee, MJ;
PMID: 35143049 | DOI: 10.1002/cne.25306

Beta-klotho (KLB) is a coreceptor required for endocrine fibroblast growth factor (FGF) 15/19 and FGF21 signaling in the brain. Klb is prominent within the hypothalamus, which is consistent with its metabolic functions, but diverse roles for Klb are now emerging. Central Klb expression is low but discrete and may govern FGF-targeted sites. However, given its low expression, it is unclear if Klb mRNA is more widespread. We performed in situ hybridization to label Klb mRNA to generate spatial maps capturing the distribution and levels of Klb within the mouse hypothalamus, hippocampal region, subiculum, and amygdala. Semiquantitative analysis revealed that Klb-labeled cells may express low, medium, or high levels of Klb mRNA. Hypothalamic Klb hybridization was heterogeneous and varied rostrocaudally within the same region. Most Klb-labeled cells were found in the lateral hypothalamic zone, but the periventricular hypothalamic region, including the suprachiasmatic nucleus, contained the greatest proportion of cells expressing medium or high Klb levels. We also found heterogeneous Klb hybridization in the amygdala and subiculum, where Klb was especially distinct within the central amygdalar nucleus and ventral subiculum, respectively. By contrast, Klb-labeled cells in the hippocampal region only expressed low levels of Klb and were typically found in the pyramidal layer of Ammon's horn or dentate gyrus. The Klb-labeled regions identified in this study are consistent with reported roles of Klb in metabolism, taste preference, and neuroprotection. However, additional identified sites, including within the hypothalamus and amygdala, may suggest novel roles for FGF15/19 or FGF21 signaling.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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