Kupffer cell diversity maintains liver function in alcohol-associated liver disease.

BACKGROUND AND AIMS: Liver macrophages are heterogeneous and play an important role in alcohol-associated liver disease (ALD) but there is limited understanding of the functions of specific macrophage subsets in the disease. We used a Western diet alcohol (WDA) mouse model of ALD to examine the hepatic myeloid cell compartment by single cell RNAseq and targeted KC ablation to understand the diversity and function of liver macrophages in ALD. APPROACH AND RESULTS: In the WDA liver, KCs and infiltrating monocytes/macrophages each represented about 50% of the myeloid pool. Five major KC clusters all expressed genes associated with receptor-mediated endocytosis and lipid metabolism, but most were predicted to be noninflammatory and antifibrotic with 1 minor KC cluster having a proinflammatory and extracellular matrix degradation gene signature. Infiltrating monocyte/macrophage clusters, in contrast, were predicted to be proinflammatory and profibrotic. In vivo, diphtheria toxin-based selective KC ablation during alcohol exposure resulted in a liver failure phenotype with increases in PT/INR and bilirubin, loss of differentiated hepatocyte gene expression, and an increase in expression of hepatocyte progenitor markers such as EpCAM, CK7, and Igf2bp3. Gene set enrichment analysis of whole-liver RNAseq from the KC-ablated WDA mice showed a similar pattern as seen in human alcoholic hepatitis. CONCLUSIONS: In this ALD model, KCs are anti-inflammatory and are critical for the maintenance of hepatocyte differentiation. Infiltrating monocytes/macrophages are largely proinflammatory and contribute more to liver fibrosis. Future targeting of specific macrophage subsets may provide new approaches to the treatment of liver failure and fibrosis in ALD.

Banff 2022 Liver Group Meeting report: Monitoring long-term allograft health.

The Banff Working Group on Liver Allograft Pathology met in September 2022. Participants included hepatologists, surgeons, pathologists, immunologists, and histocompatibility specialists. Presentations and discussions focused on the evaluation of long-term allograft health, including noninvasive and tissue monitoring, immunosuppression optimization, and long-term structural changes. Potential revision of the rejection classification scheme to better accommodate and communicate late T cell-mediated rejection patterns and related structural changes, such as nodular regenerative hyperplasia, were discussed. Improved stratification of long-term maintenance immunosuppression to match the heterogeneity of patient settings will be central to improving long-term patient survival. Such personalized therapeutics are in turn contingent on a better understanding and monitoring of allograft status within a rational decision-making approach, likely to be facilitated in implementation with emerging decision-support tools. Proposed revisions to rejection classification emerging from the meeting include the incorporation of interface hepatitis and fibrosis staging. These will be opened to online testing, modified accordingly, and subject to consensus discussion leading up to the next Banff conference.

Morphologic and immunophenotypic evaluation of liver allograft biopsies with contemporaneous serum DSA measurements.

BACKGROUND: Acute antibody mediated rejection is increasingly identified in liver allografts as a unique form of alloimmune injury associated with donor specific antibodies (DSA). This manifests pathologically as microvascular injury and C4d uptake. Despite the liver allograft's relative resistance to alloimmune injury, liver allografts are not impervious to cellular and antibody-mediated rejection. METHODS: In this blinded control study, we evaluated CD163 immunohistochemistry and applied the Banff 2016 criteria for diagnosis of acute AMR on a group of indication allograft liver biopsies from DSA positive patients and compared them to indication biopsies from DSA negative controls. RESULTS: Most DSA positive patients were females (75%, p = .027), and underwent transplantation for HCV infection. Significant histopathological predictors of serum DSA positivity were Banff H-score (p = .01), moderate to severe cholestasis (p = .03), and CD163 score > 2 (p = .029). Other morphologic features that showed a trend with DSA positivity include Banff portal C4d-score (p = .06), bile ductular reaction (p = .07), and central perivenulitis (p = .07). The odds of DSA sMFI ≥5000 was 12.5 times higher in those with a C4d score >1 than those with a C4d score ≤ 1 (p = .04). Incidence of definite for aAMR in the DSA positive cohort was 25% (n = 5), and 0% in the DSA negative cohort. A group of 5 DSA positive cases were not classifiable by the current scheme. CONCLUSION: Sinusoidal CD163, Banff H-score, and diffuse C4d are predictors of serum DSA, and facilitate recognition of histopathological features associated with serum DSA and tissue-antibody interaction.

Corrigendum: Delphi: A Democratic and Cost-Effective Method of Consensus Generation in Transplantation.

[This corrects the article DOI: 10.3389/ti.2023.11589.].

Corrigendum: Thrombotic Microangiopathy in the Renal Allograft: Results of the TMA Banff Working Group Consensus on Pathologic Diagnostic Criteria.

[This corrects the article DOI: 10.3389/ti.2023.11590.].

Delphi: A Democratic and Cost-Effective Method of Consensus Generation in Transplantation.

The Thrombotic Microangiopathy Banff Working Group (TMA-BWG) was formed in 2015 to survey current practices and develop minimum diagnostic criteria (MDC) for renal transplant TMA (Tx-TMA). To generate consensus among pathologists and nephrologists, the TMA BWG designed a 3-Phase study. Phase I of the study is presented here. Using the Delphi methodology, 23 panelists with >3 years of diagnostic experience with Tx-TMA pathology listed their MDC suggesting light, immunofluorescence, and electron microscopy lesions, clinical and laboratory information, and differential diagnoses. Nine rounds (R) of consensus resulted in MDC validated during two Rs using online evaluation of whole slide digital images of 37 biopsies (28 TMA, 9 non-TMA). Starting with 338 criteria the process resulted in 24 criteria and 8 differential diagnoses including 18 pathologic, 2 clinical, and 4 laboratory criteria. Results show that 3/4 of the panelists agreed on the diagnosis of 3/4 of cases. The process also allowed definition refinement for 4 light and 4 electron microscopy lesions. For the first time in Banff classification, the Delphi methodology was used to generate consensus. The study shows that Delphi is a democratic and cost-effective method allowing rapid consensus generation among numerous physicians dealing with large number of criteria in transplantation.

Thrombotic Microangiopathy in the Renal Allograft: Results of the TMA Banff Working Group Consensus on Pathologic Diagnostic Criteria.

The Banff community summoned the TMA Banff Working Group to develop minimum diagnostic criteria (MDC) and recommendations for renal transplant TMA (Tx-TMA) diagnosis, which currently lacks standardized criteria. Using the Delphi method for consensus generation, 23 nephropathologists (panelists) with >3 years of diagnostic experience with Tx-TMA were asked to list light, immunofluorescence, and electron microscopic, clinical and laboratory criteria and differential diagnoses for Tx-TMA. Delphi was modified to include 2 validations rounds with histological evaluation of whole slide images of 37 transplant biopsies (28 TMA and 9 non-TMA). Starting with 338 criteria in R1, MDC were narrowed down to 24 in R8 generating 18 pathological, 2 clinical, 4 laboratory criteria, and 8 differential diagnoses. The panelists reached a good level of agreement (70%) on 76% of the validated cases. For the first time in Banff classification, Delphi was used to reach consensus on MDC for Tx-TMA. Phase I of the study (pathology phase) will be used as a model for Phase II (nephrology phase) for consensus regarding clinical and laboratory criteria. Eventually in Phase III (consensus of the consensus groups) and the final MDC for Tx-TMA will be reported to the transplantation community.

A novel antibody against cancer stem cell biomarker, DCLK1-S, is potentially useful for assessing colon cancer risk after screening colonoscopy.

DCLK1 expression is critically required for maintaining growth of human colon cancer cells (hCCCs). Human colorectal tumors (CRCs) and hCCCs express a novel short isoform of DCLK1 (DCLK1-S; isoform 2) from ß-promoter of hDCLK1 gene, while normal colons express long isoform (DCLK1-L; isoform 1) from 5'(α)-promoter, suggesting that DCLK1-S, and not DCLK1-L, marks cancer stem cells (CSCs). Even though DCLK1-S differs from DCLK1-L by only six amino acids, we succeeded in generating a monospecific DCLK1-S-Antibody (PS41014), which does not cross-react with DCLK1-L, and specifically detects CSCs. Subcellular localization of S/L-isoforms was examined by immune-electron-microscopy (IEM). Surprisingly, besides plasma membrane and cytosolic fractions, S/L also localized to nuclear/mitochondrial fractions, with pronounced localization of S-isoform in the nuclei and mitochondria. Sporadic CRCs develop from adenomas. Screening colonoscopy is used for detection/resection of growths, and morphological/pathological criteria are used for risk assessment and recommendations for follow-up colonoscopy. But, these features are not precise and majority of the patients will never develop cancer. We hypothesized that antibody-based assay(s), which identify CSCs, will significantly improve prognostic value of morphological/pathological criteria. We conducted a pilot retrospective study with PS41014-Ab, by staining archived adenoma specimens from patients who developed (high-risk), or did not develop (low-risk) adenocarcinomas within 10-15 years. PS41014-Ab stained adenomas from initial and follow-up colonoscopies of high-risk patients, at significantly higher levels (three to fivefold) than adenomas from low-risk patients, suggesting that PS41014-Ab could be used as an additional tool for assessing CRC risk. CRC patients, with high DCLK1-S-expressing tumors (by qRT-PCR), were reported to have worse overall survival than low expressers. We now report that DCLK1-S-specific Ab may help to identify high-risk patients at the time of index/screening colonoscopy.

Chemotherapy-induced Sinusoidal Injury (CSI) score: a novel histologic assessment of chemotherapy-related hepatic sinusoidal injury in patients with colorectal liver metastasis.

BACKGROUND: Preoperative neoadjuvant therapy for colorectal liver metastases (CRLM) is increasing in use and can lead to chemotherapy-induced damage to sinusoidal integrity, namely sinusoidal obstruction syndrome (SOS). SOS has been associated with an increased need for intraoperative blood transfusions, increased length of hospitalization post-surgery, decreased tumor response, and a shorter overall survival after resection due to liver insufficiency. It is critical for clinicians and pathologists to be aware of this type of liver injury, and for pathologists to include the status of the background, non-neoplastic liver parenchyma in their pathology reports. In this study, expression of CD34 by sinusoidal endothelial cells (SECs), increased expression of smooth muscle actin (SMA) by hepatic stellate cells (HSCs), and aberrant expression of glutamine synthetase (GS) by noncentrizonal hepatocytes were semiquantitatively evaluated in liver resection or biopsy specimens from patients with CRLM to determine their diagnostic value for assessing chemotherapy-induced sinusoidal injury (CSI). METHODS: The expression of each marker was compared among 22 patients with CRLM with histologically evident SOS (SOS+) and 8 patients with CRLM who had not undergone chemotherapy. Each case was given a histologic grade using the sinusoidal obstruction syndrome index score (SOS-I) to assess the likelihood of SOS. Cases were also given an immunohistochemical grade using the total CSI score calculated as the sum of CD34, SMA, and GS scores. RESULTS: Abnormal staining patterns for CD34 and SMA were significantly more frequent and extensive in SOS+ cases than in the controls (81.8% vs. 25%, P < 0.01; 72.7% vs. 25%, P = 0.03). Aberrant GS expression in midzonal and periportal hepatocytes was only observed in SOS+ cases (31.8% vs. 0%), but this difference did not reach statistical significance. The CSI score was significantly higher in the SOS+ cases when compared to controls (P < 0.01), and was associated with a higher SOS histologic grade (P = 0.02). CONCLUSIONS: The CSI score, calculated using an immunohistochemical panel consisting of CD34, SMA, and GS, may serve as an objective marker of chemotherapy-induced sinusoidal injury and could help diagnose this peculiar form of liver injury.

Type I interferon contributes to noncanonical inflammasome activation, mediates immunopathology, and impairs protective immunity during fatal infection with lipopolysaccharide-negative ehrlichiae.

Ehrlichia species are intracellular bacteria that cause fatal ehrlichiosis, mimicking toxic shock syndrome in humans and mice. Virulent ehrlichiae induce inflammasome activation leading to caspase-1 cleavage and IL-18 secretion, which contribute to development of fatal ehrlichiosis. We show that fatal infection triggers expression of inflammasome components, activates caspase-1 and caspase-11, and induces host-cell death and secretion of IL-1ß, IL-1α, and type I interferon (IFN-I). Wild-type and Casp1(-/-) mice were highly susceptible to fatal ehrlichiosis, had overwhelming infection, and developed extensive tissue injury. Nlrp3(-/-) mice effectively cleared ehrlichiae, but displayed acute mortality and developed liver injury similar to wild-type mice. By contrast, Ifnar1(-/-) mice were highly resistant to fatal disease and had lower bacterial burden, attenuated pathology, and prolonged survival. Ifnar1(-/-) mice also had improved protective immune responses mediated by IFN-γ and CD4(+) Th1 and natural killer T cells, with lower IL-10 secretion by T cells. Importantly, heightened resistance of Ifnar1(-/-) mice correlated with improved autophagosome processing, and attenuated noncanonical inflammasome activation indicated by decreased activation of caspase-11 and decreased IL-1ß, compared with other groups. Our findings demonstrate that IFN-I signaling promotes host susceptibility to fatal ehrlichiosis, because it mediates ehrlichia-induced immunopathology and supports bacterial replication, perhaps via activation of noncanonical inflammasomes, reduced autophagy, and suppression of protective CD4(+) T cells and natural killer T-cell responses against ehrlichiae.

Evaluation of the aspartate aminotransferase/platelet ratio index and enhanced liver fibrosis tests to detect significant fibrosis due to chronic hepatitis C.

BACKGROUND: The assessment of liver fibrosis in chronic hepatitis C patients is important for prognosis and making decisions regarding antiviral treatment. Although liver biopsy is considered the reference standard for assessing hepatic fibrosis in patients with chronic hepatitis C, it is invasive and associated with sampling and interobserver variability. Serum fibrosis markers have been utilized as surrogates for a liver biopsy. METHODS: We completed a prospective study of 191 patients in which blood draws and liver biopsies were performed on the same visit. Using liver biopsies the sensitivity, specificity, and negative and positive predictive values for both aspartate aminotransferase/platelet ratio index (APRI) and enhanced liver fibrosis (ELF) were determined. The patients were divided into training and validation patient sets to develop and validate a clinically useful algorithm for differentiating mild and significant fibrosis. RESULTS: The area under the ROC curve for the APRI and ELF tests for the training set was 0.865 and 0.880, respectively. The clinical sensitivity in separating mild (F0-F1) from significant fibrosis (F2-F4) was 80% and 86.0% with a clinical specificity of 86.7% and 77.8%, respectively. For the validation sets the area under the ROC curve for the APRI and ELF tests was, 0.855 and 0.780, respectively. The clinical sensitivity of the APRI and ELF tests in separating mild (F0-F1) from significant (F2-F4) fibrosis for the validation set was 90.0% and 70.0% with a clinical specificity of 73.3% and 86.7%, respectively. There were no differences between the APRI and ELF tests in distinguishing mild from significant fibrosis for either the training or validation sets (P=0.61 and 0.20, respectively). Using APRI as the primary test followed by ELF for patients in the intermediate zone, would have decreased the number of liver biopsies needed by 40% for the validation set. Overall, use of our algorithm would have decreased the number of patients who needed a liver biopsy from 95 to 24-a 74.7% reduction. CONCLUSIONS: This study has shown that the APRI and ELF tests are equally accurate in distinguishing mild from significant liver fibrosis, and combining them into a validated algorithm improves their performance in distinguishing mild from significant fibrosis.

The learning curve of COVID-19 and organ donation: Potential missed opportunities in the COVID era.

Background: Early in the COVID-19 pandemic, positive COVID-19 status often disqualified potential organ donors due to perceived risks, despite limited evidence. Subsequent studies have clarified that the COVID-19 status of donors, particularly when incidental and not the cause of death, does not adversely affect non-lung transplant outcomes. This study quantifies the potential loss of eligible organ donors and the corresponding impact on organ availability during the initial phase of the pandemic. Methods: In this retrospective analysis, we examined deceased donor referrals to a major organ procurement organization from June 2020 to January 2022. Referrals were categorized as All Referrals, Medically Ruled Out (MRO), or Procured Donors (PD). We used Chi-square tests for categorical comparisons and logistic regression to model additional donors and organs, contrasting COVID-negative and positive cases within age-matched cohorts. Results: Among 9478 referrals, 23.4 % (2221) were COVID-positive. Notably, COVID-positive referrals had a substantially higher MRO rate (80.6 % vs. 29.6 %, p < 0.01) and a markedly lower PD rate (0.2 % vs. 8.2 %, p < 0.01). Potential missed donations of 103 organs from COVID-positive referrals were identified. Conclusion: This OPO-level study demonstrates a substantial impact of COVID-19 status on organ donation rates, revealing significant missed opportunities. Improved management of donor COVID-19 status could potentially increase organ donations nationwide, taking into account evolving evidence and vaccine availability changes.

Beyond prevention: Unveiling the benefits of triple vaccination on COVID-19 severity and resource utilization in solid organ transplant recipients.

OBJECTIVE: Despite the widespread reduction in COVID-19-related morbidity and mortality attributed to vaccination in the general population, vaccine efficacy in solid organ transplant recipients (SOTR) remains under-characterized. This study aimed to investigate clinically relevant outcomes on double and triple-vaccinated versus unvaccinated SOTR with COVID-19. STUDY DESIGN AND SETTING: A retrospective propensity score-matched cohort study was performed utilizing data from the US Collaborative Network Database within TriNetX (n = 117,905,631). We recruited vaccinated and unvaccinated (matched controls) SOTR with COVID-19 over two time periods to control for vaccine availability: December 2020 to October 2022 (bi-dose, double-dose vaccine effectiveness) and December 2020 to April 2023 (tri-dose, triple-dose vaccine effectiveness). A total of 42 factors associated with COVID-19 disease severity were controlled for including age, obesity, diabetes, and hypertension. We monitored 30-day outcomes including acute respiratory failure, intubation, and death following a diagnosis of COVID-19. RESULTS: Subjects were categorized into two cohorts based on the two time periods: bi-dose cohort (vaccinated, n = 462; unvaccinated, n = 20,998); tri-dose cohort (vaccinated, n = 517; unvaccinated, n = 23,061).Compared to unvaccinated SOTR, 30-day mortality was significantly lower for vaccinated subjects in both cohorts: tri-dose (2.0% vs 7.5%, HR = 0.22 [95% CI: 0.11, 0.46]); bi-dose (3.7% vs 8.2%, HR = 0.43 [95% CI: 0.24, 0.76]). Hospital admission rates were similar between bi-dose vaccinated and unvaccinated subjects (33.1% vs 28.6%, HR = 1.2 [95% CI: 0.95, 1.52]). In contrast, tri-dose vaccinated subjects had a significantly lower likelihood of hospital admission (29.4% vs 36.6%, HR = 0.74 [95% CI: 0.6, 0.91]). Intubation rates were significantly lower for triple-vaccinated- (2.3% vs 5.2%, p < 0.05), but not double-vaccinated subjects (3.0% vs 5.2%, p > 0.05). CONCLUSION: In solid organ transplant recipients with COVID-19, triple vaccination, but not double vaccination, against SARS-CoV-2 was associated with significantly less hospital resource utilization, decreased disease severity, and fewer short-term complications. These real-world data from extensively matched controls support the protective effects of COVID-19 vaccination with boosters in this vulnerable population.

Disparate outcomes in Hispanic patients with metabolic dysfunction-associated steatotic liver disease/steatohepatitis and type 2 diabetes: Large cohort study.

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are a growing health burden across a significant portion of the global patient population. However, these conditions seem to have disparate rates and outcomes between different ethnic populations. The combination of MASLD/MASH and type 2 diabetes increases the risk of hepatocellular carcinoma (HCC), and Hispanic patients experience the greatest burden, particularly those in South Texas. AIM: To compare outcomes between Hispanic and non-Hispanic patients in the United States, while further focusing on the Hispanic population within Southeast Texas to determine whether the documented disparity in outcomes is a function of geographical circumstance or if there is a more widespread reason that all clinicians must account for in prognostic consideration. METHODS: This cohort analysis was conducted with data obtained from TriNetX, LLC ("TriNetX"), a global federated health research network that provides access to deidentified medical records from healthcare organizations worldwide. Two cohort networks were used: University of Texas Medical Branch (UTMB) hospital and the United States national database collective to determine whether disparities were related to geographic regions, like Southeast Texas. RESULTS: This study findings revealed Hispanics/Latinos have a statistically significant higher occurrence of HCC, type 2 diabetes mellitus, and liver fibrosis/cirrhosis in both the United States and the UTMB Hispanic/Latino groups. All-cause mortality in Hispanics/Latinos was lower within the United States group and not statistically elevated in the UTMB cohort. CONCLUSION: This would appear to support that Hispanic patients in Southeast Texas are not uniquely affected compared to the national Hispanic population.

A challenging case of eosinophilic myocarditis leading to heart failure and transplantation.

The large spectrum of etiologies, severities, and histologic appearances of eosinophilic myocarditis (EoM) poses challenges to its diagnosis and management. Endomyocardial biopsy is the current gold standard for diagnosis. However, cardiovascular magnetic resonance imaging is becoming more frequently used to diagnose acute myocarditis because of enhanced sensitivity when compared to histopathologic examination, and its less invasive nature. We report a complicated case of EoM in a male in his mid-thirties that led to fulminant cardiogenic shock that required immunosuppressive therapy on day 5 of admission and implantation of a left ventricular assist device (LVAD) on day 30. EoM was diagnosed on histopathologic examination of the resected fragment of the left ventricular myocardium. Nine months after the initial presentation, the patient ultimately required heart transplantation. The explanted heart showed minimal residual interstitial inflammation with evidence of mildly active intimal arteritis and patchy areas of interstitial fibrosis. In this report, we describe our patient's clinical features and correlate them with imaging and histopathologic findings to illustrate the difficulty in diagnosing EoM, particularly in this complicated patient that ultimately required heart transplantation. The diagnosis can be challenging due to the variable histopathologic features, clinical presentation, and utilization of therapeutic medications and devices.

Heterogeneity in intrahepatic macrophage populations and druggable target expression in patients with steatotic liver disease-related fibrosis.

Background & Aims: Clinical trials for reducing fibrosis in steatotic liver disease (SLD) have targeted macrophages with variable results. We evaluated intrahepatic macrophages in patients with SLD to determine if activity scores or fibrosis stages influenced phenotypes and expression of druggable targets, such as CCR2 and galectin-3. Methods: Liver biopsies from controls or patients with minimal or advanced fibrosis were subject to gene expression analysis using nCounter to determine differences in macrophage-related genes (n = 30). To investigate variability among individual patients, we compared additional biopsies by staining them with multiplex antibody panels (CD68/CD14/CD16/CD163/Mac387 or CD163/CCR2/galectin-3/Mac387) followed by spectral imaging and spatial analysis. Algorithms that utilize deep learning/artificial intelligence were applied to create cell cluster plots, phenotype profile maps, and to determine levels of protein expression (n = 34). Results: Several genes known to be pro-fibrotic (e.g. CD206, TREM2, CD163, and ARG1) showed either no significant differences or significantly decreased with advanced fibrosis. Although marked variability in gene expression was observed in individual patients with cirrhosis, several druggable targets and their ligands (e.g. CCR2, CCR5, CCL2, CCL5, and LGALS3) were significantly increased when compared to patients with minimal fibrosis. Antibody panels identified populations that were significantly increased (e.g. Mac387+), decreased (e.g. CD14+), or enriched (e.g. interactions of Mac387) in patients that had progression of disease or advanced fibrosis. Despite heterogeneity in patients with SLD, several macrophage phenotypes and druggable targets showed a positive correlation with increasing NAFLD activity scores and fibrosis stages. Conclusions: Patients with SLD have markedly varied macrophage- and druggable target-related gene and protein expression in their livers. Several patients had relatively high expression, while others were like controls. Overall, patients with more advanced disease had significantly higher expression of CCR2 and galectin-3 at both the gene and protein levels. Impact and implications: Appreciating individual differences within the hepatic microenvironment of patients with SLD may be paramount to developing effective treatments. These results may explain why such a small percentage of patients have responded to macrophage-targeting therapies and provide additional support for precision medicine-guided treatment of chronic liver diseases.

The Lone Star tick, Amblyomma americanum, salivary factors exacerbate the clinical outcome of Heartland virus disease in a small animal model.

Heartland virus was first isolated in 2009 from two patients in Missouri and is transmitted by the Lone Star tick, Amblyomma americanum. To understand disease transmission and pathogenesis, it is necessary to develop an animal model which utilizes the natural route of transmission and manifests in a manner similar to documented human cases. Herein we describe our investigations on identifying A129 mice as the most appropriate small animal model for HRTV pathogenesis that mimics human clinical outcomes. We further investigated the impact of tick saliva in enhancing pathogen transmission and clinical outcomes. Our investigations revealed an increase in viral load in the groups of mice that received both virus and tick salivary gland extract (SGE). Spleens of all infected mice showed extramedullary hematopoiesis (EH), depleted white pulp, and absence of germinal centers. This observation mimics the splenomegaly observed in natural human cases. In the group that received both HRTV and tick SGE, the clinical outcome of HRTV infection was exacerbated compared to HRTV only infection. EH scores and the presence of viral antigens in spleen were higher in mice that received both HRTV and tick SGE. In conclusion, we have developed a small animal model that mimics natural human infection and also demonstrated the impact of tick salivary factors in exacerbating the HRTV infection.

Burkholderia pseudomallei BicA protein promotes pathogenicity in macrophages by regulating invasion, intracellular survival, and virulence.

Burkholderia pseudomallei (Bpm) is the causative agent of melioidosis disease. Bpm is a facultative intracellular pathogen with a complex life cycle inside host cells. Pathogenic success depends on a variety of virulence factors with one of the most critical being the type 6 secretion system (T6SS). Bpm uses the T6SS to move into neighboring cells, resulting in multinucleated giant cell (MNGC) formation, a strategy used to disseminate from cell to cell. Our prior study using a dual RNA-seq analysis to dissect T6SS-mediated virulence on intestinal epithelial cells identified BicA as a factor upregulated in a T6SS mutant. BicA regulates both type 3 secretion system (T3SS) and T6SSs; however, the extent of its involvement during disease progression is unclear. To fully dissect the role of BicA during systemic infection, we used two macrophage cell lines paired with a pulmonary in vivo challenge murine model. We found that ΔbicA has a distinct intracellular replication defect in both immortalized and primary macrophages, which begins as early as 1 h post-infection. This intracellular defect is linked with the lack of cell-to-cell dissemination and MNGC formation as well as a defect in T3SS expression. The in vitro phenotype translated in vivo as ΔbicA was attenuated in a pulmonary model of infection, demonstrating a distinct macrophage activation profile and a lack of pathological features present in the wild type. Overall, these results highlight the role of BicA in regulating intracellular virulence and demonstrate that specific regulation of secretion systems has a significant effect on host response and Bpm pathogenesis. IMPORTANCE Melioidosis is an understudied tropical disease that still results in ~50% fatalities in infected patients. It is caused by the Gram-negative bacillus Burkholderia pseudomallei (Bpm). Bpm is an intracellular pathogen that disseminates from the infected cell to target organs, causing disseminated disease. The regulation of secretion systems involved in entry and cell-to-cell spread is poorly understood. In this work, we characterize the role of BicA as a regulator of secretion systems during infection of macrophages in vitro and in vivo. Understanding how these virulence factors are controlled will help us determine their influence on the host cells and define the macrophage responses associated with bacterial clearance.

Under Disguise: A Concealed Case of Acute Hepatitis A Infection.

Acute hepatitis A virus infection is routinely identified through a thorough patient history in conjunction with liver chemistries and viral serologies. The diagnosis has the potential to be delayed when the clinical picture is obscured with another, seemingly more urgent presenting pathology with overlapping features. Here, we describe the case of a young female who presented with acute calculous cholecystitis with concurrent acute hepatitis A virus infection.

Seronegative Autoimmune Hepatitis: A Rare Manifestation of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus pandemic in 2019, commonly causes hepatic dysfunction. Liver injury ranges from mildly elevated liver enzymes to fulminant liver failure. Interestingly, there are cases that suggest a relationship between autoimmune hepatitis (AIH) in patients who either contracted coronavirus disease in 2019 (COVID-19) or were vaccinated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We present a case of a 39-year-old female without a significant past medical history who presented with two weeks of jaundice, abdominal pain, nausea, and diarrhea. She had significantly elevated liver enzymes and conjugated hyperbilirubinemia. She also tested positive for SARS-CoV-2 but denied any respiratory symptoms; her vaccination status was up to date. She denied taking hepatotoxic agents, and the workup was negative for acute viral hepatitis. The F-actin antibody level was 22 units, but serum immunoglobulin (IgG), anti-nuclear (ANA), anti-smooth muscle, anti-mitochondrial, anti-liver/kidney microsomal-1, anti-soluble liver antigen, and anti-neutrophil cytoplasmic antibodies levels were not elevated. Computerized tomography of the abdomen and pelvis revealed hepatic hemangiomas. Eventually, a liver biopsy was performed, and histology showed active lymphoplasmacytic hepatitis with prominent regenerative changes and areas of confluent necrosis. The histologic findings, along with the patient's clinical course, were suggestive of autoimmune hepatitis. The patient was started on systemic steroids with an improvement of abdominal pain and jaundice, as well as an improvement of her liver chemical profile. She was discharged with plans for hepatology clinic follow-up. Here, we present a rare case of seronegative AIH in a patient with a recent COVID-19 infection and discuss the potential underlying mechanism. We call for further investigation into the relationship between autoimmune dysfunction and COVID-19, as well as the pathophysiology behind it. Analyzing how the virus causes autoimmune dysfunction may allow clinicians to more effectively treat patients suffering from sequelae of COVID-19 infection, and it is important not to exclude autoimmune hepatitis from the differential based on the initial autoimmune workup.