Diagnostics of lesions of parenchymatic organs in COVID-19 with the application of digital software processing of computer tomography images

BACKGROUND. In the third wave of the pandemic, the coronavirus disease 2019 (COVID-19) was more aggressive. The available information on the pathogenesis of respiratory failure was supplemented with new data. Up-date information about the respiratory failure pathogenesis was acquired. It has been shown that the SARS-CoV-2 virus leads to disappearance of white pulp cells in the spleen. In this tissue immune cells mature and differentiate, among others T- and B-lymphocytes, which are responsible for premunition. The study of the structure and function of the spleen has become even more urgent. Some authors note a change in the size of the spleen during ultrasonography and chest computed tomography (CT), which correlate with indicators of the pneumonia“s severity. The study of the structure and function of the spleen has become even more urgent. OBJECTIVE. To study is to evaluate changes in the structure of solid organs (lungs, liver and spleen) in patients with a complicated community-acquired viral pneumonia COVID-19 by means of software digital processing of CT scan data and their comparison with pathomorphological changes. MATERIALS AND METHODS. The analysis of CT data in patients with a complicated community-acquired viral pneumonia COVID-19, who were treated at the SI “National institute of phthisiology and pulmonology named. F.G. Yanovsky of the NAMS of Ukraine”. CT WGC was performed on an Aquilion TSX-101A «Tochiba» scanner (Japan), followed by digital software processing of CT images using the Dragonfly software. Histological preparations were obtained as a result of traditional alcoholic histological tracing of tissue samples, embedded in paraffin blocks. To obtain micrographs, an Olympus BX51 microscope was used with an Olympus DP73 digital camera and a CellSens computer program for image processing. RESULTS AND DISCUSSION. The obtained results of digital software processing of CT images clearly correlate with autopsy histological examination of tissues of the same solid organs. Changes in the structure of the spleen occur earlier than in other solid organs, which gives reason to use these changes for diagnostic purposes. Digital processing of CT images of the spleen allows determining the severity of the disease, predicting its further course and evaluating the effectiveness of treatment. CONCLUSIONS. In patients with a complicated viral (COVID-19) community-acquired pneumonia changes (which can be determined by digital software processing of CT data) in the structure of solid organs, especially in lungs and spleen, were observed and they correlate with pathomorphological changes.

Anti-Leishmania infantum Antibody-Producing Plasma Cells in the Spleen in Canine Visceral Leishmaniasis

The spleen is involved in visceral leishmaniasis immunopathogenesis, and presents alterations in white-pulp microenvironments that are associated with an increased susceptibility to coinfections and patient death. Plasmacytosis in splenic red pulp (RP) is one observed alteration, but the specificity of antibody-secreting cells and the distribution of them has not yet been evaluated. We biotinylated soluble L. infantum membrane antigens (bSLMA) used as probes in modified immunohistochemistry, and detected the presence of anti-L. infantum antibody-secreting cells. Were used spleens from eight dogs from the endemic area for canine visceral leishmaniasis (CanL), and three healthier controls. The spleen sections were cryopreserved, and we performed modified immunohistochemistry. The ratio of plasma cells which were reactive to bSLMA (Anti-Leish-PC) in the spleen RP and periarteriolar lymphatic sheath (PALS) were calculated. Dogs with CanL present hyperglobulinemia and more plasma cells in their RP than the controls. Furthermore, dogs with CanL presented a lower proportion of Anti-Leish-PC in their RP than in PALS. Likewise, dysproteinemia was related to RP and PALS plasmacytosis, and a more severe clinical profile.

Tissue compartmentalization enables Salmonella persistence during chemotherapy

Antimicrobial chemotherapy can fail to eradicate the pathogen, even in the absence of antimicrobial resistance. Persisting pathogens can subsequently cause relapsing diseases. In vitro studies suggest various mechanisms of antibiotic persistence, but their in vivo relevance remains unclear because of the difficulty of studying scarce pathogen survivors in complex host tissues. Here, we localized and characterized rare surviving Salmonella in mouse spleen using high-resolution whole-organ tomography. Chemotherapy cleared >99.5% of the Salmonella but was inefficient against a small Salmonella subset in the white pulp. Previous models could not explain these findings: drug exposure was adequate, Salmonella continued to replicate, and host stresses induced only limited Salmonella drug tolerance. Instead, antimicrobial clearance required support of Salmonella-killing neutrophils and monocytes, and the density of such cells was lower in the white pulp than in other spleen compartments containing higher Salmonella loads. Neutrophil densities declined further during treatment in response to receding Salmonella loads, resulting in insufficient support for Salmonella clearance from the white pulp and eradication failure. However, adjunctive therapies sustaining inflammatory support enabled effective clearance. These results identify uneven Salmonella tissue colonization and spatiotemporal inflammation dynamics as main causes of Salmonella persistence and establish a powerful approach to investigate scarce but impactful pathogen subsets in complex host environments.

A Review on Splenic Diffuse Red Pulp Small B-Cell Lymphoma

Splenic diffuse red pulp small B-cell lymphoma (SDRPL) is a rare disease, representing <1% of all non-Hodgkin lymphomas (NHL). The most common clinical manifestations include splenomegaly, lymphocytosis, and hemocytopenia. A diagnosis of SDRPL can be challenging, as it shares multiple clinical and laboratory features with splenic marginal zone lymphoma (SMZL), hairy cell leukemia (HCL), and HCL variant (HCL-v). Obtaining splenic tissue remains the gold standard for diagnosis. In the cases where splenic tissue is not available, diagnosis can be established by a review of peripheral blood and bone marrow studies. SDRPL is characterized by a diffuse involvement of the splenic red pulp by monomorphous small-to-medium sized mature B lymphocytes effacing the white pulp. The characteristic immunophenotype is positive for CD20, DBA.44 (20 to 90%), and IgG, and typically negative for CD5, CD10, CD23, cyclin D1, CD43, annexin A1, CD11c, CD25, CD123, and CD138. The Ki-67 proliferative index is characteristically low. Cyclin D3 is expressed in the majority of SDRPL in contrast with other types of small B-cell lymphomas, thus facilitating the recognition of this disease. There is no standard treatment regimen for SDRPL. Initial treatment options include splenectomy, rituximab monotherapy, or a combination of both. Chemoimmunotherapy should be considered in patients with advanced disease at baseline or progression.

A New Murine Model of Primary Autoimmune Hemolytic Anemia (AIHA)

Loss of humoral tolerance to red blood cells (RBCs) can lead to autoimmune hemolytic anemia (AIHA), a severe, and sometimes fatal disease. Patients with AIHA present with pallor, fatigue, decreased hematocrit, and splenomegaly. While secondary AIHA is associated with lymphoproliferative disorders, infections, and more recently, as an adverse event secondary to cancer immunotherapy, the etiology of primary AIHA is unknown. Several therapeutic strategies are available; however, there are currently no licensed treatments for AIHA and few therapeutics offer treatment-free durable remission. Moreover, supportive care with RBC transfusions can be challenging as most autoantibodies are directed against ubiquitous RBC antigens; thus, virtually all RBC donor units are incompatible. Given the severity of AIHA and the lack of treatment options, understanding the cellular and molecular mechanisms that facilitate the breakdown in tolerance would provide insight into new therapeutics. Herein, we report a new murine model of primary AIHA that reflects the biology observed in patients with primary AIHA. Production of anti-erythrocyte autoantibodies correlated with sex and age, and led to RBC antigen modulation, complement fixation, and anemia, as determined by decreased hematocrit and hemoglobin values and increased reticulocytes in peripheral blood. Moreover, autoantibody-producing animals developed splenomegaly, with altered splenic architecture characterized by expanded white pulp areas and nearly diminished red pulp areas. Additional analysis suggested that compensatory extramedullary erythropoiesis occurred as there were increased frequencies of RBC progenitors detectable in the spleen. No significant correlations between AIHA onset and inflammatory status or microbiome were observed. To our knowledge, this is the first report of a murine model that replicates observations made in humans with idiopathic AIHA. Thus, this is a tractable murine model of AIHA that can serve as a platform to identify key cellular and molecular pathways that are compromised, thereby leading to autoantibody formation, as well as testing new therapeutics and management strategies.

Splenectomy for Primary Immune Thrombocytopenia Revisited at the Era of Thrombopoietin Receptor Agonists: New Insights for an Old Treatment

Introduction Although splenectomy is still considered as the most effective curative treatment for primary immune thrombocytopenia (ITP), its use has significantly declined in the last decade, especially since the emergence of thrombopoietin receptor agonist (TPO-RAs) and anti-CD20 monoclonal antibodies 1-3. The main objective of our study was to evaluate if splenectomy was still as effective in the modern era, particularly for patients who failed to respond to TPO-RAs and rituximab. One of the secondary objectives was to assess, among patients who did not respond to or relapse after splenectomy, the pattern of response to subsequent intervention with treatments used before splenectomy and particularly TPO-RAs. Methods This multicentre retrospective observational study involved adults who underwent surgical splenectomy for primary ITP in France from 2011 (authorization of TPO-RAs in France) to 2020. To be included in the study, patients had to fulfil the following criteria : age ³18 years, primary ITP diagnosis defined according to the usual international criteria 2. Patients with abnormal spleen histology (other than reactional lymphoid hyperplasia, white-pulp hypoplasia or red pulp hyperplasia) or yet definite secondary ITP were excluded. Response was defined according to international criteria 4. Sustained response was defined as the absence of ITP relapse at last visit. We performed univariable and multivariable logistic regression procedures to calculates the odds ratio associated with a sustained response. Results In total,185 patients, 98 (53 %) women, with median age at splenectomy of 43.3 [interquartile range 27.6-64.3] years, were included in 18 French university and general hospitals from the French reference center network. Most of the patients were splenectomised at the chronic phase of ITP (n=150, 81.1%) and only two patients had undergone surgery within 3 months after ITP onset. Of note, 100 (54.1%) and 135 (73.0%) patients received TPO-RAs and/or rituximab prior to the splenectomy, respectively. The median time of follow-up after splenectomy was 39.2 months [16.5-63.0]. Overall, 144 (77.8%) of patients had an initial response and 23 patients (12.4%) relapsed during follow-up leading to an overall rate of sustained response of 65.4%, similar to the one observed in the pre-TPO-RA's era 1. Characteristics of patients according to the period during which occurred the splenectomy is available in Table 1. Among the 14 patients who failed to respond to both eltrombopag and romiplostim prior to splenectomy a sustained response after splenectomy was observed in 7 (50%). Among the 13 patients who had failed after both TPO-RAs and rituximab, we observed a sustained response in 6 (46%). In the multivariate analysis, an older age (60-75 years: OR 0,39 [0,17-0,86], p=0.02; &gt;75 years: OR 0,28 [0,10-0,75], p=0.013) and a history of more than 4 treatment lines for ITP before splenectomy (OR 0.25 [0.08-0.66], p=0.010) were significantly associated with a lack of sustained response after splenectomy. TPO-RAs were used for 57/64 (89.1%) patients who failed to respond to splenectomy. Among them, 21 were treated with one TPO-RA (i.e. eltrombopag or romiplostim) which was previously used before splenectomy without any efficacy and a response was observed in 13 (62%) of them. Conclusions In conclusion, splenectomy seems to be still a relevant option for treating adult primary ITP not responding to TPO-RAs and rituximab. Patients who fail to respond or relapse after splenectomy should be re-challenged with TPO-RAs. 1. Kojouri, K., Vesely, S. K., Terrell, D. R. & George, J. N. Splenectomy for adult patients with idiopathic thrombocytopenic purpura: a systematic review to assess long-term 2. Provan, D. et al. Updated international consensus report on the investigation and management of primary immune thrombocytopenia. Blood Adv. 3.Neunert, C. et al. American Society of Hematology 2019 guidelines for immune thrombocytopenia. Blood Adv. 3, 3829-3866 (2019). 4. Rodeghiero, F. et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood 113, 2386-2393 (2009). Figure 1 Figure 1. Disclosures Terriou: Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ebbo: Grifols: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Octapharma: Other: Attendance Grant; Sobi: Other: Attendance Grant. Viallard: Novartis: Consultancy; Amgen: Consultancy; Grifols: Consultancy; LFB: Consultancy. Jeandel: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Support for congress; Sobi: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Support for congress; GSK: Other: Support for congress; Pharming: Other: support for congress. Michel: Amgen: Consultancy; Novartis: Consultancy; Rigel: Honoraria; UCB: Honoraria; Alexion: Honoraria; Argenx: Honoraria. Godeau: Grifols: Consultancy; Sobi: Consultancy; Amgen: Consultancy; Novartis: Consultancy. Comont: Takeda: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.

TNFα Controls the Delicate Balance between Erythropoiesis and Stem Cell Exhaustion during Inflammatory Stress

Anemia of Inflammation (AI) is prevalent in patients with chronic inflammatory states, such as infection, autoimmunity, or cancer. Induced expression of hepcidin by pro-inflammatory cytokines results in iron-restricted anemia. In particular, abnormally elevated levels of the cytokine Tumor Necrosis Factor-α (TNFα) is a hallmark of AI, however its contribution to the pathophysiology of AI is not well understood. In this study, we investigated the role of TNFα in the development of anemia in a TNFα knockout (TNFαKO) mouse model of AI, which is induced with a single intraperitoneal (i.p.) injection of heat-killed Brucella Abortus (BA) (Kim et al. Blood). We hypothesized that TNFαKO mice would show a less severe form of AI compared to WT animals when challenged with BA. Our results showed that WT-BA mice developed severe anemia within 2 weeks, which was resolved by 8 weeks, whereas the TNFαKO mice developed leukocytosis and an irreversible macrocytic, hyperchromic anemia. Serum analysis at 8 weeks showed that erythropoietin (EPO) and iron parameters were elevated in TNFαKO compared to WT mice, which ruled out iron-restriction as the cause for the persistent anemia. However, serum cytokine measurements of TNFαKO mice at 4 weeks showed continual elevation of interleukin (IL)-12p40 and Interferon-γ (IFNγ) compared to WT-BA controls. We hypothesized that TNFα served an anti-inflammatory role that restrained prolonged inflammation after BA, and in its absence, pro-inflammatory macrophages continuously secreted IL-12p40 levels and induced the proliferation of IFNγ secreting cells. To test if concurrent loss of IFNγ would correct the inflammatory phenotype, we crossed TNFαKO with IFNγKO mice (DKO). Indeed, IFNγKO and DKO mice challenged with BA showed complete reversal of the anemic phenotype present in WT-BA and TNFαKO-BA at 2 weeks. Additionally, serum levels of IL-12p40 were normalized by 4 weeks in IFNγKO-BA and DKO-BA compared to TNFαKO-BA mice. Flow cytometry analysis of the bone marrow (BM) and spleen (SPL) at 8 weeks showed T-lymphocytes and macrophages were markedly expanded, whereas erythrocytes and B-lymphocytes were reduced in TNFαKO-BA mice. However, only modest differences in erythrocytes, macrophage, T- and B-lymphocyte in WT-BA, IFNγKO-BA and DKO-BA in the BM and SPL were detected. Additionally, we performed immunohistochemistry using an anti-CD3 antibody in SPL and livers of BA treated mice sacrificed at 8 weeks. We found complete disorganization of the white pulp in the SPL and infiltration of T-Lymphocytes in livers of TNFαKO-BA but not in WT-BA, IFNγKO-BA or DKO-BA animals. These results led us to question if lack of TNFα skewed the BM towards T-Lymphocytes. We investigated the hematopoietic stem cell (HSC) LSK, multipotent myeloid progenitors (MPP) LK, and common lymphoid progenitor (CLP) compartments by flow cytometry. Shockingly, the TNFαKO had over a 30% increase of Lin-cKit+Sca1+ LSKs, compared to TNFαKO controls, while the Lin-cKit+Sca1- LK population was drastically reduced. By contrast, the Lin-CD217-cKit+Sca1+ CLP population was expanded by more than 40% in the TNFαKO-BA compared to TNFαKO controls. Considering this data, it is difficult to disentangle the effects of TNFα in AI in vivo from its role in regulating upstream stem and progenitor cell differentiation using a germline KO strategy. However, our observations reinforce the link between iron homeostasis and HSC self-renewal and provide a new model to study inflammation associated bone marrow failure. The TNFα-BA mice displayed severe anemia, which seems to result from persistent IFNγ elevation. A recent study identified TNFα as a major pro-survival and pro-regeneration factor for HSCs (Yamashita & Passegue, Cell Stem Cell). Other studies have shown that IFNγ restricts HSCs self-renewal (Chen et al. Blood). Our results in the TNFαKO-BA treated mice suggest that TNFα preserves balanced progenitor output by countering the action of IFNγ at the HSC level. Ongoing work aims to understand the relationship between TNFα and IFNγ in regulating HSC quiescence, self-renewal, and overall pool size. Disclosures Paulson: Forma Therapeutics: Consultancy. Rivella: Meira GTx: Consultancy; Ionis Pharmaceuticals: Consultancy.

Erythropoietin improves effects of mesenchymal stem cells in an experimental model of sepsis

In the last years several studies have shown that mesenchymal stem cells (MSCs) are able to reduce the systemic inflammatory response and mortality in experimental models of sepsis. As recently found, the surface of MSCs have receptors for erythropoietin (EPO). So we hypothesized that the introduction of EPO together with MSCs may enhance their effect and improve the results of sepsis treatment. Aim: То evaluate morphologic and immunologic effects of combined treatment with EPO and MSC in an experimental LPS sepsis model in rats. Methods: 50 Wistar rats were randomized into 5 groups: Group 1 - the healthy controls, Groups 2-5 were intraperitoneally introduced bacterial LPS 20 mg/kg. Two hours after LPS injection animals received the following intravenous treatments: Group 3 - 4xl05 allogeneic MSCs, Group 4 - 8.5 pg of recombinant EPO-beta, Group 5 - MSCs and EPO in the same doses. Surviving animals were euthanased on the 4th day. The morphological study of the liver, spleen, thymus, lungs, kidney tissues was performed. We analyzed the tissue changes, white blood cells count and serum level of IL-l, IL-2, IL-6, TNF-. Results: Mortality in LPS groups did not differ. The highest white blood cells count was found in the group of combined treatment EPO+MSCs (8.15x106 cells/ml) compared with controls (2,15x10s cells/ml) and LPS controls (6,52x10s cells/ml). There were no differences in levels of TNF-, IL-2 and IL-6 between the groups, but serum IL-1 level in groups 2 and 4 was significantly higher than in treated with MSCs and MSCc + EPO animals. Histologically in the group 5 we observed significantly less leukocyte lung interalveolar septal infiltration and kidney tubular dystrophy. The most significant differences in group LPS + EPO were found in the lymphoid tissue - considerable hyperplasia of spleen white pulp and thymus cortex, whereas in the other groups different degrees of atrophy of the corresponding zones were noted. Conclusions: Combined treatment with EPO and MSCs can reduce acute lung injury and kidney damage, cause hyperplasia of lymphoid tissue and enhance the immune response more than separate treatment in an experimental model of sepsis in rats.

Macrophage Depletion via Clodronate Pretreatment Reduces Transgene Expression from AAV Vectors In Vivo

Adeno-associated virus is a popular gene delivery vehicle for gene therapy studies. A potential roadblock to widespread clinical adoption is the high vector doses required for efficient transduction in vivo, and the potential for subsequent immune responses that may limit prolonged transgene expression. We hypothesized that the depletion of macrophages via systemic delivery of liposome-encapsulated clodronate would improve transgene expression if given prior to systemic AAV vector administration, as has been shown to be the case with adenoviral vectors. Contrary to our expectations, clodronate liposome pretreatment resulted in significantly reduced transgene expression in the liver and heart, but permitted moderate transduction of the white pulp of the spleen. There was a remarkable localization of transgene expression from the red pulp to the center of the white pulp in clodronate-treated mice compared to untreated mice. Similarly, a greater proportion of transgene expression could be observed in the medulla located in the center of the lymph node in mice treated with clodronate-containing liposomes as compared to untreated mice where transgene expression was localized primarily to the cortex. These results underscore the highly significant role that the immune system plays in influencing the distribution and relative numbers of transduced cells in the context of AAV-mediated gene delivery.

Postmortem Findings in Patients with COVID19 Using Multiple Organ Core Needle Biopsies

Introduction/Objective The coronavirus disease 2019 (COVID19) pandemic had caused more than 500,000 deaths in the United States. Although it mainly manifests with respiratory symptoms, postmortem examination reveals that it is more of a systemic disease affecting mutliple body organs. Methods/Case Report Postmortem needle core biopsies from multiple organs were obtained from 9 patients who died at our institution in the months of April and May of 2020 due to a confirmed SARS-CoV-2 infection by RT-PCR testing of nasopharyngeal swabs. The core biopsies from body organs included lungs (8), liver (7), kidneys (5), heart (2), spleen (2), and brain (2). Histopathological examination was performed in conjunction with a set of special and immunohistochemical stains. Electron microscopy examination was also done in 4 cases. Results (if a Case Study enter NA) The cohort consisted of 6 males and 3 females with a mean age of 70.4 years (range: 68–79). The majority had comorbidities (8/9) and presented with respiratory symptoms (9/9). The most significant postmortem findings were mainly in the lungs, including alveolar hemorrhage, hyaline membranes, fibrin thrombi, intraalveolar macrophages, type-2 pneumocyte hyperplasia, and interstitial myofibroblast reaction and collagen deposition. Immunohistochemical stains showed predominance of T-lymphocytes with a mixture of CD4 and CD8 positive cells. Examination of liver showed minimal to marked microvesicular and macrovesicular steatosis and centrilobular congestion and necrosis. Tissue from kidneys revealed mild to severe acute tubular injury. Microglial activation and Alzheimer type-II astrocytosis were noted in brain, and mild white pulp depletion was seen in the spleen. Electron microscopy showed the presence of foreign bodies suspicious for viral particles ranging in size from 52.6 to 97.9 nm in 2/4 cases. Conclusion Our findings based on postmortem core needle biopsies confirm the observation that most severely affected patients have significant pulmonary pathology. However, other organs show findings that may lead to a better understanding of this disease. Postmortem examination will continue to be an invaluable tool for studying the pathologic manifestations of COVID-19.