The Adrenal Cortex, an Underestimated Site of SARS-CoV-2 Infection.

Background: The majority of the critically ill patients may have critical illness-related corticosteroid insufficiency (CIRCI). The therapeutic effect of dexamethasone may be related to its ability to improve cortical function. Recent study showed that dexamethasone can reduce COVID-19 deaths by up to one third in critically ill patients. The aim of this article is to investigate whether SARS-CoV-2 can attack the adrenal cortex to aggravate the relative adrenal insufficiency. Methods: We summarized the clinical features of COVID-19 reported in currently available observational studies. ACE2 and TMPRSS2 expression was examined in human adrenal glands by immunohistochemical staining. We retrospectively analyzed serum cortisol levels in critically ill patients with or without COVID-19. Results: High percentage of critically ill patients with SARS-COV-2 infection in the study were treated with vasopressors. ACE2 receptor and TMPRSS2 serine protease were colocalized in adrenocortical cells in zona fasciculata and zona reticularis. We collected plasma cortisol concentrations in nine critically ill patients with COVID-19. The cortisol levels of critically ill patients with COVID-19 were lower than those in non-COVID-19 critically ill group. Six of the nine COVID-19 critically ill patients had random plasma cortisol concentrations below 10 µg/dl, which met the criteria for the diagnosis of CIRCI. Conclusion: We demonstrate that ACE2 and TMPRSS2 are colocalized in adrenocortical cells, and that the cortisol levels are lower in critically ill patients with COVID-19 as compared to those of non-COVID-19 critically ill patients. Based on our findings, we recommend measuring plasma cortisol level to guide hormonal therapy.

[Structural changes of the adrenal cortex in experimental genital herpes virus infection].

The reactive changes in the adrenal gland cortex were studied in mature female guinea pigs (n=5) in an experimental model of acute genital herpes virus infection. The methods of light and transmission electron microscopy were used. To confirm the presence of viral antigen in the corticosterocytes (CSC), the methods of immunfluorescence and electron microscopic immunocytochemistry were used. It was shown that at day 7 of an acute process, focal CSC reactive changes appeared in the glomerular zone - at the light microscopic level, CSC had intact nuclei and optically empty cytoplasm, while at the electron microscopic level, these CSC demonstrated the damaged membranous organelles, and various membranous structures which were not found in the normal cells. The aggregates of hypertrophied CSC were found in the fasciculate zone. The changes described were reversible, as they practically disappeared by the onset of spontaneous recovery (day 21 after inoculation). The regeneration of CSC of glomerular and fasciculate zones of the adrenal cortex involves both intracellular and cellular mechanisms.

Human cytomegalovirus productively infects adrenocortical cells and induces an early cortisol response.

Following our recent findings on the presence of human cytomegalovirus (HCMV) in the normal human adrenal cortex and in adrenocortical tumors, especially in cortisol-secreting tumors, aim of the present study was to investigate the direct effects of HCMV infection on human adrenocortical cells. To this aim, both clinical isolates and laboratory strains of HCMV were used to assess the early effects of infection on human adrenocortical cell morphology, proliferation, gene expression, and steroidogenic function. Both clinical and laboratory HCMV strains could infect and replicate in primary human adrenocortical cell cultures and in adrenocortical carcinoma cell lines, leading to cytopathic changes. Most importantly, in the first hours post-infection (p.i.), adrenocortical cells showed a significant increase of cortisol and estrogen production, paralleled by up-regulation of steroidogenic acute regulatory protein and expression of steroidogenic enzymes involved in the last steps of adrenal steroidogenesis. This effect was probably due to HCMV immediate-early gene expression, since it was most evident in the early phases p.i. and UV-inactivated viral particles did not affect hormone production. Moreover, the effect on steroidogenesis was HCMV specific, since it was not observed after infection with herpes simplex virus. These data suggest that human adrenocortical cells are permissive to HCMV infection and acutely respond to infection with increased cortisol production. An acute glucocorticoid response is typically triggered by infections and is considered to be critical to host defense against pathogens, although, in the case of HCMV infection, it might also enhance viral replication and reactivation from latency.

Developmental expression of HERV-R (ERV3) and HERV-K in human tissue.

The human endogenous retroviruses (HERVs), ERV3 (HERV-R) and HERV-K, are both known to be transcriptionally active in human placenta. In the case of ERV3 there is also indirect evidence for its participation in cellular differentiation. In this study we examined the expression of ERV3 (HERV-R) and HERV-K in human normal fetal tissues by in situ hybridization. The highest level of ERV3 env expression was detected in primitive adrenal cortex. Elevated levels of expression were also found in the following developing tissues: kidneys (tubules), tongue, heart, liver, and central nervous system. Tissue-specific expression was found for HERV-K rec (former cORF) but not for pol/int transcripts. The highest rec expression was found in placenta and levels slightly higher than sense control were found in the rest of the tissues examined. Pol/Int was not possible to quantitate. It appears that ERV3 is expressed in an organ-specific way during embryogenesis and might suggest a possible role in the development and differentiation of human tissues.

Tissue-specific high-level expression of human endogenous retrovirus-R in the human adrenal cortex.

In an attempt to clarify the biological nature of a human endogenous retrovirus (HERV), HERV-R, which is a single-copy type of HERVs and is conserved as a full-length viral sequence, the expression of HERV-R mRNA in normal autopsied systemic organs was examined by Northern blot analysis. The expression showed different levels among individuals, with the adrenal glands expressing the highest level of HERV-R among all organs tested, except for the placenta. In various adrenal tumors, HERV-R was expressed at high levels in all cortical adenomas but less so in pheochromocytomas. In situ hybridization revealed the expression of HERV-R to be localized in all layers of the adrenal cortex, but not in the medulla. This high-level expression of HERV-R in the adrenal cortex may possibly relate to differentiation and/or steroid production by adrenocortical cells.

Herpesvirus infection in stranded Pacific harbor seals of coastal California.

Histopathological examination revealed multifocal acute to chronic adrenal necrosis in 74 of 162 (45%) Pacific harbor seal pups (Phoca vitulina richardsi) dying during rehabilitation following live stranding along the coast of central and northern California (USA). Necrotic adrenal cells contained amphophilic, smudgy intranuclear inclusion bodies that were stained positive for DNA. Fifty of these seals also had lesions typical of sepsis, bacterial omphalophlebitis, pneumonia or gastroenteritis. Twenty four seals had no lesions other than thymic atrophy and occasional multifocal hepatic necrosis. Prior to death, affected seals had a marked lymphopenia. Electron microscopy revealed unenveloped intranuclear hexagonal to round viral particles approximately 100 nm in diameter, and cytoplasmic enveloped virions approximately 160 nm in diameter. These were morphologically consistent with herpesvirus. Inoculation of phocine adrenal and kidney cell lines with an adrenal tissue homogenate from affected animals produced a cytopathic effect in 5 days. Electron microscopy of cell cultures showing this cytopathic effect revealed similar viral particles to those observed in affected adrenal glands. Cases with characteristic inclusion bodies were observed in 42 of 95 (44%) male and 32 of 67 (47%) female seals. Affected animals had been in rehabilitation 0 to 63 days and were below average birth weight for this species.

[Tissue-specific expression of human endogenous retrovirus mRNA and its regulation by cytokines in vitro].

To investigate biological roles of human endogenous retroviruses (ERVs), the author examined the viral mRNA expression in the normal systemic organs in vivo and its regulation by cytokines in cultured cells. The following evidence suggesting biological activities of a human ERV, ERV3, was obtained. First, the ERV3 mRNA was demonstrated at different levels in organs, and at consistently high levels in adrenal glands from all individuals and in all adrenocortical adenomas examined, by Northern hybridization. In situ hybridization revealed that the ERV3 expression was localized in all three layers of the adrenal cortex, but not in the medulla. These results suggest that the ERV3 expression may relate to the cellular differentiation and/or steroid production of adrenocortical cells. Second, the amount of ERV3 mRNA in cultured endothelial cells from human umbilical vein was significantly increased with any of TNF-alpha, IL-1 beta or IL-1 alpha stimulation but decreased with IFN-gamma treatment, by a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with competitive PCR. The collective evidence suggests that the ERV3 expression may be upregulated at the inflammatory sites of vessels in vivo, and that the ERV3 expression may, therefore, play certain pathogenic roles in diseases, including collagen and vascular diseases in man.

Adrenocortical activation by herpes virus: involvement of IL-1 beta and central noradrenergic system.

We investigated the involvement of brain cytokines and central monoamines in mediating the effect of the neurotropic herpes simplex virus-1 (HSV-1) on adrenocortical activity in rats. Corneal inoculation with a neurovirulent HSV-1 strain, but not with an avirulent strain, induced interleukin-1 beta (IL-1 beta) gene expression mainly in the pons and hypothalamus, and caused an elevation in serum corticosterone levels. Infectious virus was isolated in low titres only from the trigeminal ganglia and pons. Viral DNA was detected by PCR in these tissues and in other brain regions. Virus-induced adrenocortical activation was abolished in rats in which hypothalamic norepinephrine (NE) was depleted by 6-hydroxydopamine. Depletion of hypothalamic serotonin by 5,7-dihydroxytryptamine did not prevent adrenocortical activation. These results suggest that central IL-1 and NE are involved in HSV-1 induced adrenocortical activation.

Characterization of a new Marburg virus isolated from a 1987 fatal case in Kenya.

In 1987, an isolated case of fatal Marburg disease was recognized during routine clinical haemorrhagic fever virus surveillance conducted in Kenya. This report describes the isolation and partial characterization of the new Marburg virus (strain Ravn) isolated from this case. The Ravn isolate was indistinguishable from reference Marburg virus strains by cross-neutralization testing. Virus particles and aggregates of Marburg nucleocapsid matrix in Ravn-infected vero cells, were visualized by immunoelectron microscopic techniques, and also in tissues obtained from the patient and from inoculated monkeys. The cell culture isolate produced a haemorrhagic disease typical of Marburg virus infection when inoculated into rhesus monkeys. Disease was characterized by the sudden appearance of fever and anorexia within 4 to 7 days, and death by day 11. Comparison of nucleotide sequences for portions of the glycoprotein genes of Marburg-Ravn were compared with Marburg reference strains Musoki (MUS) and Popp (POP). Nucleotide identity in this alignment between RAV and MUS is 72.3%, RAV and POP is 71%, and MUS and POP is 91.7%. Amino acid identity between RAV and MUS is 72%, RAV and POP is 67%, and MUS and POP is 93%. These data suggest that Ravn is another subtype of Marburg virus, analogous to the emerging picture of a spectrum of Ebola geographic isolates and subtypes.

Rapid ras-oncogene-mediated transformation maintains steroidogenic differentiation in adrenocortical parenchymal cells.

While its action as a transforming agent is well known, expression of the ras oncogene may also alter tissue-specific differentiation. We have been examining the relationship of transformation and differentiation in steroidogenic cells of the rat. Infection of adrenocortical zona glomerulosa (GLOM) cells with the v-Ki ras containing Kirsten murine sarcoma virus did not induce focus formation. Instead, diffuse cellular multilayers formed from which loosely adherent, refractile cells emerged. After selective passaging these refractile cells, designated KiGLOM, were morphologically transformed, had reduced serum requirements for growth, greatly increased saturation densities, and they rapidly formed tumours in immunosuppressed hosts. In addition, under conditions where normal cells were no longer steroidogenic (ie. after passaging), KiGLOM cells expressed the steroid-specific cholesterol side chain cleavage cytochrome P-450scc and they produced significant, albeit reduced, amounts of corticosterone in comparison with primary GLOM cultures. Additionally, trophic hormone treatment increased steroid production in Ki-GLOM cells and this increase was partially reversed by lovastatin, a pharmacological inhibitor of ras p21 function. Thus, after a morphological selection that removed normal neighbours, v-Ki ras infected cells transformed rapidly while remaining steroidogenic. These results, combined with previous reports of steroidogenic v-Ki ras transformed adrenocortical fibroblasts and ovarian granulosa cells suggest that the ability of the ras oncogene to co-opt signal transduction pathways associated with both growth and differentiation is a common feature of the steroidogenic phenotype.