Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo.

Gut

2017 Apr 20

Allweiss L, Volz T, Giersch K, Kah J, Raffa G, Petersen J, Lohse AW, Beninati C, Pollicino T, Urban S, Lütgehetmann M, Dandri M.
PMID: 28428345 | DOI: 10.1136/gutjnl-2016-312162

Abstract

OBJECTIVE:

The stability of the covalently closed circular DNA (cccDNA) in nuclei of non-dividing hepatocytes represents a key determinant of HBV persistence. Contrarily, studies with animal hepadnaviruses indicated that hepatocyte turnover can reduce cccDNA loads but knowledge on the proliferative capacity of HBV-infected primary human hepatocytes (PHHs) in vivo and the fate of cccDNA in dividing PHHs is still lacking. This study aimed to determine the impact of human hepatocyte division on cccDNA stability in vivo.

METHODS:

PHH proliferation was triggered by serially transplanting hepatocytes from HBV-infected humanised mice into naïve recipients. Cell proliferation and virological changes were assessed by quantitative PCR, immunofluorescence and RNA in situ hybridisation. Viral integrations were analysed by gel separation and deep sequencing.

RESULTS:

PHH proliferation strongly reduced all infection markers, including cccDNA (median 2.4 log/PHH). Remarkably, cell division appeared to cause cccDNA dilution among daughter cells and intrahepatic cccDNA loss. Nevertheless, HBV survived in sporadic non-proliferating human hepatocytes, so that virological markers rebounded as hepatocyte expansion relented. This was due to reinfection of quiescent PHHs since treatment with the entry inhibitor myrcludex-B or nucleoside analogues blocked viral spread and intrahepatic cccDNA accumulation. Viral integrations were detected both in donors and recipient mice but did not appear to contribute to antigen production.

CONCLUSIONS:

We demonstrate that human hepatocyte division even without involvement of cytolytic mechanisms triggers substantial cccDNA loss. This process may be fundamental to resolve self-limiting acute infection and should be considered in future therapeutic interventions along with entry inhibition strategies.

Analysis of Cytokine Gene Expression using a Novel Chromogenic In-situ Hybridization Method in Pulmonary Granulomas of Cattle Infected Experimentally by Aerosolized Mycobacterium bovis.

J Comp Pathol. 2015 Jul 16.

Palmer MV, Thacker TC, Waters WR.
PMID: 26189773 | DOI: 10.1016/j.jcpa.2015.06.004.

Mycobacterium bovis is the cause of tuberculosis in most animal species including cattle and is a serious zoonotic pathogen. In man, M. bovis infection can result in disease clinically indistinguishable from that caused by Mycobacterium tuberculosis, the cause of most human tuberculosis. Regardless of host, the typical lesion induced by M. bovis or M. tuberculosis is the tuberculoid granuloma. Tuberculoid granulomas are dynamic structures reflecting the interface between host and pathogen and, therefore, pass through various morphological stages (I to IV). Using a novel in-situ hybridization assay, transcription of various cytokine and chemokine genes was examined qualitatively and quantitatively using image analysis. In experimentally infected cattle, pulmonary granulomas of all stages were examined 150 days after aerosol exposure to M. bovis. Expression of mRNA encoding tumour necrosis factor (TNF)-α, transforming growth factor-β, interferon (IFN)-γ, interleukin (IL)-17A, IL-16, IL-10, CXCL9 and CXCL10 did not differ significantly between granulomas of different stages. However, relative expression of the various cytokines was characteristic of a Th1 response, with high TNF-α and IFN-γ expression and low IL-10 expression. Expression of IL-16 and the chemokines CXCL9 and CXCL10 was high, suggestive of granulomas actively involved in T-cell chemotaxis.

Early Pulmonary Lesions in Cattle Infected via Aerosolized Mycobacterium bovis

Vet Pathol

2019 Mar 21

Palmer MV, Wiarda J, Kanipe C and Thacker TC
PMID: 30895908 | DOI: 10.1177/0300985819833454

Mycobacterium bovis is a serious zoonotic pathogen and the cause of tuberculosis in many mammalian species, most notably, cattle. The hallmark lesion of tuberculosis is the granuloma. It is within the developing granuloma where host and pathogen interact; therefore, it is critical to understand host-pathogen interactions at the granuloma level. Cytokines and chemokines drive cell recruitment, activity, and function and ultimately determine the success or failure of the host to control infection. In calves, early lesions (ie, 15 and 30 days) after experimental aerosol infection were examined microscopically using in situ hybridization and immunohistochemistry to demonstrate early infiltrates of CD68+ macrophages within alveoli and alveolar interstitium, as well as the presence of CD4, CD8, and gammadelta T cells. Unlike lesions at 15 days, lesions at 30 days after infection contained small foci of necrosis among infiltrates of macrophages, lymphocytes, neutrophils, and multinucleated giant cells and extracellular acid-fast bacilli within necrotic areas. At both time points, there was abundant expression of the chemokines CXCL9, MCP-1/CCL2, and the cytokine transforming growth factor (TGF)-beta. The proinflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta, as well as the anti-inflammatory cytokine IL-10, were expressed at moderate levels at both time points, while expression of IFN-gamma was limited. These findings document the early pulmonary lesions after M. bovis infection in calves and are in general agreement with the proposed pathogenesis of tuberculosis described in laboratory animal and nonhuman primate models of tuberculosis.

A comparative transcriptomic analysis of replicating and dormant liver stages of the relapsing malaria parasite Plasmodium Cynomolgi

Elife.

2017 Dec 07

Voorberg-van der Wel A, Roma G, Gupta DK, Schuierer S, Nigsch F, Carbone W, Zeeman AM, Lee BH, Hofman SO, Faber BW, Knehr J, Pasini E, Kinzel B, Bifani P, Bonamy GMC, Bouwmeester T, Kocken CHM, Diagana TT.
PMID: 29215331 | DOI: 10.7554/eLife.29605

Plasmodium liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite Plasmodium cynomolgi. Hypnozoites express only 34% of Plasmodium physiological pathways, while 91% are expressed in replicating schizonts. Few known malaria drug targets are expressed in quiescent parasites, but pathways involved in microbial dormancy, maintenance of genome integrity and ATP homeostasis were robustly expressed. Several transcripts encoding heavy metal transporters were expressed in hypnozoites and the copper chelator neocuproine was cidal to all liver stage parasites. This transcriptomic dataset is a valuable resource for the discovery of vaccines and effective treatments to combat vivax malaria.

	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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Advanced Cell Diagnostics

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Advanced Cell Diagnostics China