Immunological Tests Research Articles

OA26 Atypical Multisystem Inflammation related to infection or just plain old polyarteritis nodosa?

Abstract Introduction We present the case of a 5-year-old boy with episodes of pyrexia of unknown origin associated with polyarthritis, central neuroinflammation and mucocutaneous manifestations. His skin biopsy demonstrated a nodular vasculitis which could be consistent with polyarteritis nodosa. Interestingly though, he had additional atypical rashes, ocular inflammation and evidence of acute and past streptococcal infection and his fever and CRP normalised prior to corticosteroid initiation. Case description A five-year-old British African male presented with five-week history of persistent fever, headaches, joint pain and bilateral eye pain. He was transferred to a tertiary paediatric infectious disease’s unit due to unrelenting fever with no fixed pattern, despite nine days of broad-spectrum intravenous antibiotics. He was diagnosed with neuroinflammation of uncertain aetiology, evidenced by leptomeningeal enhancement and bilateral papillitis on brain MRI. CSF microbiology and autoimmune profiles were negative. There was no evidence of primary malignancy. The fever resolved with pulsed IV methylprednisolone and the child was discharged home on a weaning regime of oral prednisolone. After stopping steroid treatment, he represented with high-grade fever, limping, anterior uveitis and new-onset non tender cutaneous lesions, plaques over eyelids and cheek and annular lesions over arms, legs and buttocks. Repeat MRI of the brain and spine showed resolution of previous neuroinflammation. Blood tests revealed raised ANA, IgG and ASOT titre, and his throat swab was positive for Group A Streptococcus. He completed 10 days of intravenous antibiotics and oral high dose ibuprofen (10 mg/kg TDS). On review by tertiary paediatric rheumatology, he was identified to have pleomorphic skin rash and subcutaneous nodules. Skin and deep tissue biopsy confirmed lobular and septal panniculitis with necrotising vasculitis, consistent with a nodular vasculitis. Treatment with high-dose steroid and mycophenolate mofetil (MMF) was initiated, but his CRP and fever had resolved prior to his first dose. He experienced improvement in systemic symptoms and arthritis; however, skin lesions persisted, with appearance of new lesions and hyperpigmentation, on reduction of steroid dose. His course was complicated by acute varicella infection, requiring oral aciclovir. Further treatment trials included topical tacrolimus and topical corticosteroids for his skin lesions. At one biopsy site, he experienced significant delay in wound healing. The genetic panel for primary immunodeficiency panel (R15) demonstrated no pathological variants. Discussion This case report highlights that, whilst the biopsy may be consistent with nodular vasculitis, such as PAN, the clinical manifestations are not perfectly aligned. Discussion within the rheumatology community would support optimal patient care. Childhood-onset Polyarteritis Nodosa (c-PAN) is a rare systemic vasculitis. Although the aetiology is unknown, associations with infections like group A streptococcus and hepatitis B suggest a postinfectious autoimmune response. There are no clear genetic links to PAN, but PAN-like vasculitis is part of reported monogenic autoinflammatory conditions. Key atypical features in this case include uveitis at presentation, nonspecific central neuroinflammation, unusual cutaneous lesion morphology, improvement of acute phase reactants before immunomodulatory treatment, and delayed biopsy wound healing. While ocular findings in PAN may include episcleritis, scleritis, keratitis, retinal vascular occlusions, and ocular ischemic syndrome, uveitis has not been reported in c-PAN. The patient also showed nonspecific inflammatory changes on initial MRI. Neurological involvement in c-PAN typically includes cerebrovascular accidents, cranial nerve palsies, and mononeuritis multiplex, with meningeal involvement being less common (reported 4% in a study of 69 children). Additionally, the patient experienced delayed wound healing following the biopsy, which is undocumented in c-PAN, but may be related to immune modulating treatment. A positive streptococcal throat swab could suggest an immune reaction as part of bacterial tonsilitis, or Acute Rheumatic Fever. However, he did not meet Jones Criteria, there was no response to prolonged antibiotics and the chronic remitting nature made these less likely. An autoinflammatory disorder, Deficiency of Adenosine Deaminase 2 (DADA2), was also considered but he had no evidence of livedo racemosa, strokes and no CECR1 mutation was identified. Our case has been discussed within a multidisciplinary team and we speculate that it could represent a previously undefined immune sensitivity within the autoimmune, autoinflammatory and immune deficiency spectrum giving an exaggerated response to infection. Key learning points • This case presents a significant diagnostic challenge due to its atypical clinical features and inconclusive extensive investigations, including immunologic testing, tissue biopsy, and genetic analysis. • While the patient’s symptoms improved with high-dose steroids and MMF, the recurrence of symptoms upon tapering steroids underscores the persistent inflammatory nature of the disease. • The detection of group A Streptococcus in the patient’s throat swab suggests a potential postinfectious autoimmune trigger, highlighting the role infections can play in the pathogenesis of vasculitis. The negative results from the R15 Primary Immunodeficiency panel suggest that more advanced genetic and immunologic testing may be beneficial. • We invite discussion on whether additional genetic or immunologic testing could optimise this patient’s care and contribute to the broader understanding of atypical vasculitis syndromes.

Growth factor dysregulation and placental dysfunction

The article is devoted to the study of angiogenic and antiangiogenic vascular growth factors in placental dysfunction, which is of great importance in obstetrics and perinatology. The study of growth factors as predictors of the development of placental dysfunction makes it possible to determine in advance the development of many obstetric pathologies, including preeclampsia and fetal growth retardation, which is of great practical importance. The authors conducted an immunological study of pregnant women with placental dysfunction and pregnant women with a physiological pregnancy. Purpose of the study: to study serum growth factors in women with placental dysfunction. We examined 47 pregnant women with a gestation period of 26-40 weeks, with a diagnosis of placental dysfunction, who were under observation in the obstetric department of the city maternity complex No. 3 of the city of Tashkent. The control group consisted of 35 women with a physiological pregnancy. All women underwent an immunological blood test: cellular and humoral immunity, as well as cytokines (VEGF-A, PlGF, sFlt-1, sFlt-1/PlGF) were studied. Based on the results obtained, the authors suggest that an increased level of soluble sFlt-1 and a simultaneous decrease in the levels of VEGF-A and PlGF in pregnant women at 26-40 weeks of gestation may portend the development of pregnancy complications, including preeclampsia and fetal growth restriction, and increase the risk of premature birth. Ddelivery due to impaired placental blood flow and oxygen deficiency for the fetus, lead to deterioration of microcirculation, and insufficient levels of VEGF-A and PlGF can contribute to vascular endothelial dysfunction, which can also contribute to the development of preeclampsia. The studied angiogenic and antiangiogenic vascular growth factors are important indicators and can serve as markers for predicting pregnancy complications for active medical intervention in maintaining the health of both mother and child.

FEATURES OF ANTIBACTERIAL AND LYMPHOTROPIC THERAPY IN PATIENTS WITH PLEURAL EMPYEMA

Abstract. Acute empyema belongs to the category of severe surgical pathology. The difficulty of its treatment is caused by a number of objective and subjective reasons, in particular such as wide spread of antibiotic-resistant microflora and population allergy. Carefully collected analysis, patient’s examination, study of lungs function indicators, data of laboratory and X-ray studies, performing of serological, immunological and allergic tests do not always allow to identify the cause of acute empyema and are often not sufficient to identify its etiology. In spite of a great number of proposed methods of acute pleural empyema treatment, study of long-term results of all types of treatment shows that they do not guarantee 100% success. The outlined data indicates the necessity of improvement of etiotropic therapy in patients with pleural empyema. Constant search for new effective methods of treatment of the aimed pathology proves the relevance of the theme. The data outlined in this article indicates the necessity of improvement of etiotropic therapy in patients with pleural empyema.

An increase of the oncogenesis frequency is associated with the DAMPs accumulation and the killer cell subpopulations ratio change in patients with long-term post-COVID-19 syndrome

In patients with malignant lesions of the lungs and pancreas, the development of which was facilitated by the long-term post-COVID-19 syndrome (PCS), presence a significant increase (on average by 80%) in the serum content of the cytotoxic oligonucleotide fraction DAMP was found. High antigenic load due to the increase of DAMP contributed to the absorptive function growth and insufficient digestive function of neutrophils; decrease in the NADPH-catalase activity reserve in oxygen-dependent phagocytosis. An increase of CD16+ NK cells of innate immunity and an overexpression of CD8+ killer/suppressor T lymphocyte receptors of adaptive immunity in response to the suppressor coreceptors competitive action were revealed. These changes in cellular immunity were more pronounced in patients with malignant lung lesions and correlated with low serum conductivity. To predict the individual course of the disease, it is advisable to carry out screening measurements of electrical conductivity of blood serum and to determine the control points of innate and adaptive immunity changes in patients with comorbid pathology: PCS and an oncological process. In further studies, attention should be paid to the algorithm development of immunological and screening tests for the oncological diseases course prognosis and the treatment tactics effectiveness.

Sex Differences in Allergic Bronchopulmonary Aspergillosis and its Impact on Exacerbations.

The impact of sex on allergic bronchopulmonary aspergillosis (ABPA) outcomes remains uncertain. We retrospectively included ABPA subjects per the revised International Society for Human and Animal Mycology ABPA working group criteria over 13years. We compared the clinical features, lung function, immunological tests, imaging, and ABPA exacerbation rates between men and women. Our primary objective was to assess whether women experience higher ABPA exacerbations than men. We included 731 ABPA subjects (mean age, 34.5years; 49.5% women). Women with ABPA were older and had underlying asthma more frequently than men. There was no difference in lung function, immunological investigations, and imaging between men and women. ABPA exacerbations occurred in a slightly higher proportion of women than men (44.5% vs. 38.2%) but did not reach statistical significance (p = 0.09). We did not find a significant sex difference in ABPA exacerbation rates. Prospective studies should confirm our findings.

Investigating Concomitant RAG-2 and LRBA Mutations in SCID and Autoimmunity.

Inborn errors of immunity (IEI) are a large heterogenous group of diseases characterized by immunodeficiency, immune dysregulation, allergy, auto-inflammation and predisposition for malignancies. Most are inherited in an autosomal recessive trait. We studied a patient with severe combined immunodeficiency (SCID) and immune dysregulation who harbored two distinct bi-allelic IEI-associated genetic mutations. Clinical, immunological and genetic data were collected. Genetic investigation included whole exome sequencing on DNA extracted from skin fibroblasts. Family segregation was performed by Sanger sequencing. Immunological evaluation included absolute and functional evaluation of lymphocytes and chimerism analysis post hematopoietic stem cell transplantation (HSCT). Treg subsets, LRBA and CTLA4 expression levels were measured by flow-cytometric analysis. A nineteen-year-old female patient from a consanguine background underwent unconditioned matched sibling related HSCT during infancy due to clinical presentation of SCID with an Omenn phenotype. At that time her underlying genetic defect was not defined. Years after HSCT, severe auto-immune phenomena were noted, including a systemic lupus erythematosus-like syndrome and ophthalmic manifestations. Genetic evaluation revealed bi-allelic homozygous mutations in RAG-2 (c.685C>T, p.Arg229Trp) and a previously undescribed mutation in LRBA (c.3325G>T, p.Asp1109Tyr). LRBA and CTLA4 expression levels were normal, suggesting that the LRBA variant identified in these kindred is unlikely to be pathogenic. Multiple genetic defects causing complex IEIs may be identified in the same individual in highly consanguineous populations. Functional immunological testing is essential for evaluation of novel genetic variants.

B-013 Improving the Workflow of Blood and Coagulation Tests in clinical laboratory through the Utilization of Laboratory Automation Track Systems

Abstract Background The Laboratory Automation Track System (TLA) is an automated system designed for executing highly repetitive tasks within a laboratory, commonly applied in biochemical and immunological testing systems. It aims to reduce human errors in specimen handling, enhance the overall process quality, and provide better Turnaround Time (TAT) from specimen collection to result reporting. For the first time in our laboratory, we have integrated the blood and coagulation systems with TLA. Consequently, we seek to evaluate the differences in the testing workflow and reporting efficiency of the blood and coagulation systems after being connected to TLA. Methods Abbott GLP systems Track was the TLA system for connecting with blood and coagulation systems. Two Sysmex XN-9100 fully automatic hematology systems and two Sysmex CS-5100 fully automatic coagulation analyzers were developed for blood and coagulation system, respectively. TUNYEN Laboratory Information System was established for Laboratory information system (LIS). Results Through the analysis of quality indicators and integration with intelligent information systems, improvements have been made, including the reduction of blood collection tube usage, efficient manpower utilization management, tracking of specimen progress and achievement of timely reporting rates.(1)Approximately 2,915 blood collection test tubes are saved every month, with a converted cost of 0.15 USD per tube, and a total saving of 437 USD. And reduce the carbon emissions of 32 kgCO2e, comply with the spirit of ESG sustainable development.(2)The TLA no longer requires staff to collect and deliver specimens as in the past,reducing staff and enhancing work efficiency.One of manpower was saved and new inspection items were developed.(3)Tracking of specimen progress:The laboratory has set up a specimen tracking system to monitor the specimen delivery location and provide timely response to the clinical status.The one-hour TAT for blood and coagulation increased from 82.0% and 76.9% before improvement to 91.8% and 93.3% after improvement respectively. Conclusions The implementation of Laboratory Automation Track Systems (TLA) leads to a reduction in medical errors and specimen sample volume, while also enhancing accuracy and precision, thereby improving the safety of laboratory staff. Although the introduction of TLA may require an initial investment, it brings the benefits of automation, offering ease and efficiency to laboratory processes.

B-048 Proposal of a new monitoring algorythm for patients with a HIV positive result with the Elecsys HIV DUO test

Abstract Background Worldwide HIV infections remain a public health problem, contributing significantly to morbidity. In Mexico we have had a progressive adaptation to the HIV epidemic providing free therapy and testing (4th generation tests) for the whole population in anbulatory centers for HIV/AIDS prevention and medical care. During 2022 Mexico reported 12,374 new cases. In the last decade there has been an increasing interest in the identification of patients HIV positives subgroups, based on disease progression and serological features. To date, 6 generations of tests for HIV detection have been developed, strongly reducing the window period for a positive result. Elecsys HIV Duo is a 5th generation immunological test that provides in a separate manner an antibody and antigen results for HIV detection. These results show a better insight of the serologycal status reducing the window period post infection. By using these tests we want to provide an early detection of new cases that gives a better prognosis for the patient and propose a new monitoring for patients with a positive antigen result but that have not yet produced antibodies. Such algorythm is currently lacking in the Mexican public health services. Methods A retrospective statystical analysis of HIV results of samples procesed with the Elecsys HIV DUO test during the january-april 2023 period in the National Reference Center of Salud Digna correlated with western blot results. Results The study population was 6405 patients, 6340 (98.98%) had a negative result in the test and 65 (1.02%) a positive test. Based in antibody/antigen results we categorized the positives results in three grupos: Group 1 Ab positive/Ag negative (16), group 2 Ab positive/Ag positive (32) and group 3 Ab negative/Ag positive (7). When correlated with the WB results all the patients from group 1 and 2 had a positive result while all patients in group 3 had a negative result. Conclusions Actually Mexican public health services do not have access to 5th generation HIV tests like the Elecsys HIV DUO test, therefore, public health profesional are not qualified to interprete these results and do not have a stated algorythm for patientes belonging to group 3. Therefore and based in our results we suggest 5th generation test (or superior if available) and propose a new patient monitoring algorythm with a molecular confirmatory test for viral detection. This proposal is strongly supported by the fact the we have a population that have not generated antibodys during the first weeks of post infection and will test negative in a serologycal confirmatory test like WB. Screening for patients in early infection stages is important because if we provide early therapy which may lead to infection reversal or a less impact in C4+ T cells numbers. Studies like this provide information for the generation of public politics in benefit of the population.

Study the Occurrence of Mycobacterium Bovis in Tuberculosis of Peripheral Lymph Nodes and its Effect on the Course of the Disease.

The incidence of tuberculosis caused by Mycobacterium bovis, not only the pulmonary form, but also the form developing in the extrapulmonary organs, is also increasing from year to year. Despite the large number of EPTB, TBPLN occupies a leading place among diseases of this type and the study of its pathogenic strains is an urgent task in ensuring the effectiveness of treatment. In this regard, the main purpose of the presented manuscript is to determine the frequency of M. bovis in TBPLN, its effect on the development and course of the disease, as well as the effectiveness of treatment. For this purpose, for the first time, the features of education that occur in patients in peripheral lymph nodes using instrumental methods of ultrasound, computed tomography, and magnetic resonance imaging have been identified. In subsequent studies, 110 patients with peripheral lymph node pathology were diagnosed with TBPLN by detecting mycobacteria in pathological material using general hematological, microbiological and gene-molecular (Gene Xpert) methods. In order to ensure the high effectiveness of drugs used for medicinal purposes, strains of the pathogen were detected using histological, cytological studies, BCG test and specific analyzes such as Diaskintest, Quantiferon test, immunological tests. The study showed that about 80% of patients had M bovis in the overall assessment, 76.4% of patients were sensitive to rifampicin, 9.1% of patients had rifampicin-resistant bacteria, and 14.5% of patients did not have mycobacteria. Therapeutic measures were carried out in 2 different modes, such as standard and individual or with replacement, when all patients were divided into 2 groups. During the period from the 56-day intensive phase of standard treatment to the 84-day intensive phase, a total of 40 patients had a sharp decrease in lymph nodes, elimination of purulent inflammation, and after a while 22 patients in this group had a relapse. In the individual treatment regimen, Levofloxacin and linezolid were used instead of pyrazinamide. While the effectiveness of treatment was achieved in 48 patients of group II after 56 and 84 days of the intensive phase, relapses after a certain time were observed in only 6 patients. When choosing an individual treatment regimen in patients diagnosed with M. bovis, a decrease in relapses to 11.5% is achieved. When M. bovis is detected, an individual scheme of antibacterial treatment of tuberculosis is selected, in which, instead of pyrazinamide, it is recommended to choose one of the reserve lines, depending on the sensitivity of the pathogen to drugs.

Immunological Variables In Cattle Infected With Blood Protozoa Transmitted By Ticks And Horseflies In Samarra City

The results of the immunological tests on cattle infested with ticks and horseflies showed significant differences between the groups of infested cattle. There was a highly significant difference in the immunoglobulin IgM, which reached 54, compared to the healthy cattle group. Additionally, there were highly significant differences in the surface markers CD8, CD4, and the MHCII complex in the groups of cattle infested with ticks and horseflies compared to the healthy cattle groups. However, no significant differences were observed between the infested and healthy cattle groups in terms of immunoglobulin IgG and complement G3.

Primary Biliary Cholangitis in Men: A Case Report

Primary biliary cholangitis (PBC) is a rare chronic autoimmune liver disease, especially in men, that causes progressive inflammation and destruction of the intrahepatic bile ducts. This leads to hepatic fibrosis that can progress to cirrhosis. PBC diagnosis is suggested by cholestatic jaundice in the absence of abnormalities in intrahepatic and extrahepatic bile ducts, with alkaline phosphatases (ALP) > 1.5 times the upper limit of normal and gamma-glutamyl transferase (GGT) > 3 times the upper limit of normal, with or without elevated bilirubin levels. Confirmation is made through immunological tests, and liver biopsy (PBH) is necessary if tests are negative. The disease is often diagnosed at an advanced stage. Poor prognostic factors include early diagnosis, male gender, severe symptoms, and biological abnormalities such as elevated bilirubin levels. Specific autoantibodies, such as anti-gp210 and anti-sp100, also predict unfavorable disease progression. The first-line treatment is ursodeoxycholic acid (UDCA), which helps slow disease progression and improve symptoms. If UDCA is ineffective, other options include obeticholic acid (OCA) or fibrates like bezafibrate, often used in combination with UDCA. Liver transplantation is the only curative treatment for severe cases. Research is ongoing for new treatments, including PPAR agonists and bile acid transporter inhibitors.

Impact of Treatment with Direct Antivirals on Coagulation Parameters in Patients with Hepatitis C Virus-Related Liver Cirrhosis and Sustained Virological Response.

Background and Objectives: Sustained virologic responses (SVRs) lead to a decrease in portal hypertension, the regression of fibrosis, and the improvement in the hepatic synthesis of procoagulant and anticoagulant factors. We aimed to assess the influence of SVR on coagulation parameters in cirrhotic patients with HCV treated with DAAs. Methods: We performed a prospective study in the Institute of Gastroenterology and Hepatology Iasi, Romania, between January 2022 and February 2024. We included patients diagnosed with compensated and decompensated HCV-related liver cirrhosis, treated with direct antivirals (PrOD ± RBV or SOF/LED ± RBV) for 12/24 weeks. Blood samples for biochemical, immunological, and coagulation tests were collected at the baseline, end of treatment (EOT), and once sustained virological response had been achieved over a period of 12/24 weeks (SVR12/24). Results: We analyzed a group of 52 patients with HCV-related liver cirrhosis, predominantly female (68.0%), and the degree of severity of cirrhosis placed the patients mainly in Child-Pugh classes B (40%) and C (36%). All patients achieved SVRs. The MELD score decreased at EOT (13.48 ± 4.273; p = 0.001) and SVR (9.88 ± 2.774; p = 0.000), compared to the baseline (14.92 ± 4.707). The FibroScan values decreased at SVR (17.596 ± 3.7276; p = 0.000) compared to the baseline (26.068 ± 7.0954). For all common coagulation parameters (platelets, INR, PT, fibrinogen, aPTT), there was a trend towards improvement during treatment, including changes which were statistically significant for the majority of patients. Factor II was low at the baseline (75.40 ± 7.506) but increased at EOT (87.40 ± 9.587) and, later, at SVR (99.12 ± 11.695; p = 0.000). The FVIII values increased at the baseline (175.52 ± 16.414) and decreased at EOT (151.48 ± 13.703) and SVR (143.40 ± 13.937). The FvW values decreased during treatment (146.84 ± 9.428, at baseline; 141.32 ± 9.690, p = 0.000, at EOT; and 126.68 ± 17.960, at SVR). In regard to the anticoagulant factors (PC, PS, ATIII), a significant improvement was brought on by SVR. Advanced stages of liver disease showed the most diminished FII activity, while at the baseline and in Child-Pugh C patients we recorded the highest values of FVIII and FvW. Conclusions: Our study proved that the "reset" of coagulopathy might be due to the improvement in liver function due to viral eradication secondary to AAD therapy.

Clinical and laboratory pattern of patients with systemic lupus erythematosus seropositive for rheumatoid factor.

The aim of the study was to investigate the associations between the presence and level of rheumatoid factor (RF) in the blood serum and the clinical and laboratory characteristics of patients with systemic lupus erythematosus (SLE). This retrospective tricentric cross-sectional study analyzed a Ukrainian contingent of SLE patients. Medical records of 495 patients were evaluated. Rheumatoid factor serum concentration was tested in 206 of them (41.6%) using turbidimetry technique. Clinical manifestations, routine laboratory parameters, specific immunological tests, disease activity (SLEDAI-2K), and damage indices (SLICC/ACR DI) were evaluated. Our study revealed that RF was elevated in 27.7% of patients. The RF-positive patients experienced a longer delay in SLE diagnosis (2.0 vs. 0.5 years, p = 0.046), less frequent kidney involvement (42.1% vs. 59.4%, p = 0.045) and fever (42.1% vs. 59.2%, p = 0.046), and more frequent lymphadenopathy (59.6% vs. 42.3%, p = 0.039) compared to RF-negative patients. Patients with RF positivity had higher levels of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and antinuclear antibody (ANA) titer, and were more frequently positive for antibodies to Ro/SSA and La/SSB. Rheumatoid factor concentration directly correlated with CRP (r = 0.318; p < 0.01) and ESR (r = 0.228; p = 0.04) levels. However, no associations were found between RF levels and SLEDAI-2K, joint involvement frequency, SLICC/ACR DI or drug therapy content. Univariate logistic regression analysis showed that RF positivity was independently associated with lymphadenopathy, presence of anti-Ro/SSA and anti-La/SSB antibodies, and negatively associated with kidney involvement. In RF-seropositive SLE patients (approximately 28%), the diagnosis is established later compared to RF-seronegative ones; kidney involvement and fever are less common, while lymphadenopathy develops more frequently. Rheumatoid factor seropositivity is associated with higher levels of ESR, CRP, ANA, and the presence of antibodies to Ro/SSA and La/SSB. According to the results of univariate logistic regression analysis, an independent association with RF positivity was confirmed only for kidney involvement, lymphadenopathy, and antibodies to Ro/SSA and La/SSB.

Abstract B062: Low dose dual epigenetic therapy utilizing hypomethylating agents and HDAC inhibitors prolong survival in an orthotopic PDAC model and alter the tumor microenvironment

Abstract Background: We have previously studied the effect of hypomethylating agent Decitabine (5- aza-dC; DAC) on pancreatic cancer using the KPC model (KrasG12D/+;Trp53R172H/+;Pdx1- Cre) and observed increased CD4+/CD8+ TILs, greater PD1 expression and slowed tumor growth with increased tumor necrosis. Subsequently we showed a significant survival benefit with DAC+αPD1 compared to either single agent. However, we identified a specific myeloid cell population initially identified by Chi3l3 expression that may be responsible for treatment resistance or escape. Based on prior observations of synergy specifically affecting tumor infiltrating myeloid and lymphoid cells, we focused on identifying a dual epigenetic strategy (a combination of DAC plus histone deacetylase inhibitors (HDACi)) to enhance immunological synergy and test their treatment efficacy with αPD1. Methods: We used a combination primary KPC-derived cell lines, bone marrow (BMDM) and peritoneum derived (PM) myeloid cells and T cells in co-culture systems to assess cytotoxic synergy (Bliss/Lowe consensus) and immune effects (cytokine release, myeloid cell polarization and T cell activation) to evaluate combinations of DAC+HDACi. The KPC orthotopic model relying on US monitoring of tumor size for standardized initiation of therapy was used for survival analysis and profiling in treatment combinations of DAC+HDACi+αPD1. Results: We showed that hypomethylation therapy exerts a primarily paracrine effect on myeloid cells and this polarization results in a decrease in DAC–induced T cell activation. We therefore selected HDAC inhibitors that in combination with DAC alter this paracrine effect. We selected specific HDAC inhibitors based on induction of class of HDAC expression following hypomethylation therapy, highest Bliss synergy scores at low doses and reversal of cytokine release, M2 polarization and T cell inactivation. Three HDAC inhibitors were chosen for in vivo testing, Domatinostat, Pracinostat and Entinostat. Using the treatment paradigm of ultra-low dose DAC+ HDACi (mainly to minimize toxicity) combined with αPD1 in the KPC orthotopic model we show that the triple combination (DAC + Entinostat + αPD1) is well-tolerated and results in a significant survival benefit compared to single and dual agent combinations. In addition, we also demonstrate a reduction in the Chi3l3 expressing myeloid cell population. Conclusions: Our results show that the combination of hypomethylating agent plus HDAC inhibitor has several beneficial effects on the tumor immune response in PDAC. HDAC inhibitors specifically reduce the suppressive myeloid cell population we previously identified after DAC therapy. The combination of dual low dose epigenetic therapy using a hypomethylating agent plus HDAC inhibitor followed by αPD1 results in significant survival benefit in the orthotopic KPC model. Citation Format: Stephen Muzyka, Arturo Orlacchio, Amanda Ackermann, Yasmine Chinda, Yoona Shim, Igor Dolgalev, Tamas Gonda. Low dose dual epigenetic therapy utilizing hypomethylating agents and HDAC inhibitors prolong survival in an orthotopic PDAC model and alter the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B062.

New Insights into Leishmaniasis in Rabbits Raised in the Northeast Region of Brazil.

This study aimed to investigate the occurrence of leishmaniasis in domestic rabbits (Oryctolagus cuniculus) in the state of Sergipe, Brazil, and to evaluate the associated clinical signs. A total of 31 rabbits from urban and rural areas were clinically examined using cytological, immunological, and serological tests. Blood and cytological samples were collected and analysed for the presence of Leishmania parasites and antibodies. Immunochromatographic tests were used to screen for anti-Leishmania antibodies, and cytological analysis of skin lesions was performed to detect the presence of Leishmania amastigotes. Of the rabbits tested, 19.35% were reactive in the anti-Leishmania antibody screening, and 3.33% tested positive for Leishmania amastigotes in skin lesion cytology. Clinical signs included cachexia, lymphadenomegaly, dehydration, apathy, dermatitis, ophthalmopathy, and alopecia. Cytological analysis revealed pyogranulomatous inflammation with Leishmania amastigotes present. The findings suggest that leishmaniasis is present among domestic rabbits in this region CONCLUSION: This study demonstrates the presence of leishmaniasis in domestic rabbits in the Northeast of Brazil. The findings underline the importance of early diagnosis and intervention in preventing the spread of the disease, and highlight the need for further research into the role of rabbits as potential reservoirs of Leishmania.

Carbapenemase-Producing Escherichia coli: Comparison of a Novel Rapid Lateral Flow Assay With the Polymerase Chain Reaction (PCR) and Antimicrobial Resistance Pattern.

Background In critically ill patients, carbapenems are often used as the last line of treatment. Carbapenem-resistant Enterobacterales (CRE) present an extreme challenge to treatment due to their resistance to various antibiotics. Optimal therapy for patients and infection control relies on the early and accurate diagnosis of these infections. The K.N.I.V.O. Detection K-Set is a newly developed immunological rapid test developed to identify the presence of carbapenemase in Gram-negative bacteria resistant tomultiple drugs. Objectives This study evaluates a new K.N.I.V.O. Detection K-Set and its application for the rapid detection of isolates of multidrug-resistant Escherichia coli (MDR E. coli) that produce carbapenemase. This test aims to compare the test's performance to the polymerase chain reaction (PCR) method. Methods The study included 150 MDR E. coli isolates that were confirmed to be resistant to at least three groups of antibiotics, including carbapenems. The test followed the manufacturer's instructions using the K.N.I.V.O. Detection K-Set. The outcomes were compared with carbapenemase gene detection (bla-KPC, bla-NDM, bla-OXA-48, bla -VIM, and bla -IMP) using the PCR. The K-Set's sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated and studied. Results The K.N.I.V.O. Detection K-Set showed highly effective diagnostic performance with a 97.1% sensitivity, 97.5% specificity, 97.1% positive predictive value, and 98.7% negative predictive value. Seventy-eight of the 150 isolates were proven to be producers of carbapenemase, with 68 of those cases having an accurate identification. The remaining isolates were found to be non-producers. Within 15 minutes, the rapid testprovided results. Conclusion The K.N.I.V.O. Detection K-Set is an effective and rapid method for identifying carbapenemase producers among MDR E. coli isolates. Its rapid processing time, associated with its high sensitivity and specificity, indicatesthat it can increase the effectiveness of diagnostic laboratories and better patient care in clinical settings. Implementing such rapid screenings could be vital for controlling the spread of drug-resistant infections and enhancing antimicrobial stewardship. This also ensures that patients receive timely treatment and effective care.

A New Immunogenic Structure of Polyepitopic Fusion against Leishmania major: In Silico Study.

The lack of complete protection against leishmaniasis and the challenges of anti-leishmaniasis drug treatment have made the treatment process more difficult. This study aimed to develop a new strategy for preparing a vaccine against cutaneous leishmaniasis using some of the antigenic proteins of the Leishmania parasite. This study was carried out in 2022 at Shahid Chamran University of Ahvaz, Ahvaz, Iran. After preparing suitable epitopes of the Leishmania parasite and examining their antiparasitic properties, the process of making a fusion vaccine was performed and with the help of various bioinformatics tools, physicochemical and structural properties as well as immunological and simulation properties were studied and finally optimized. Construction and cloning were performed in the E.coli K12 system and finally, the docking process was performed with Toll-like receptors (TLRs), major histocompatibility complex I (MHC-I), and MHC-II receptors. With the help of selected epitopes of the Leishmania parasite, which had a high percentage of population coverage, a stable, antigenic, and non-allergenic chimeric vaccine was predicted. The results of the structural analysis of the TLR5\vaccine complex and simulation of its molecular dynamics showed a sufficiently stable binding. It also showed good potential for stimulation and production of active B cells and memory, as well as the potential for CD8+ T, CD4+ T cell production and development of Th2 and Th1-induced immune responses. Computational results showed that the designed immunogenic structure has the potential to adequately stimulate cellular and humoral immune responses against Leishmania parasitic disease. As a result of evaluating the effectiveness of the candidate vaccine through in vivo and in vitro immunological tests, it can be suggested as a vaccine against Leishmania major.

Isolation of donor gamma globulin obtained from multiparous women and its effects upon expression of HLA-G and HLA-DR molecules on lymphocytes from mothers of children with septal congenital heart defects

A common pathogenetic mechanism of reproductive losses and congenital heart disease (CHD) is associated with immune inflammation in the “mother-embryo” system which affects differentiation and proliferation of cardiovascular progenitor cells. It is hypothesized that this link may be blocked by regulatory auto- and alloimmune antibodies to HLA-G and HLA-DR molecules. Moreover, these antibodies may be present at sufficient amounts in donor immunoglobulins, especially those obtained from the blood of multiparous women. Based on this suggestion, the aim of our study was to obtain enriched gamma globulin fraction from the blood of multiparous women and evaluate its functional effects towards HLA-DR and HLA-G molecules. Isolation of the gammaglobulin fraction (GGF) from the blood plasma of multiparous women was performed using affinity chromatography in several sessions. Purity grade of the resulting protein was analyzed by immunoelectrophoresis, electrophoretic separation of the protein fraction of blood serum and electrophoresis in 4.12% polyacrylamide gel with the addition of SDS (PAGE electrophoresis). PAAG electrophoresis showed that this GGF did not differ from commercial therapeutic intravenous immunoglobulin (IVIG). Assessment of the functional activity of GGF upon HLA-DR and HLA-G molecules was performed in the main group of women and their children with congenital heart disease (n = 38), and control group of women who gave birth to conditionally healthy children (n = 21). To determine the specificity of GGF with respect to HLA-G, HLA-DR molecules, as well as to compare its effect with autologous and allogeneic sera and IVIG, we developed an immunological testing protocol using flow cytometry. The protocol was arranged on the basis of the methodology of “cross-match” approach and Russian patent “Method for determining antibodies to HLA-G”. It was found that the blocking activity of female serum towards autologous (intrinsic) and allogeneic (embryo/fetus/child) HLA-G and HLA-DR molecules may determine the protective effect on development of congenital heart defects in the next generation. Donor human immunoglobulin showed a similar blocking effects to these molecules, possibly due to the presence of alloimmune antibodies to HLA classes I and II. The gammaglobulin fraction obtained from the donor blood of multiparous women has a more pronounced blocking effect on the HLA-G and HLA-DR expression. Hence, this immunobiological preparation can be considered a prototype of therapeutic and prophylactic agent blocking the genesis of congenital heart defects.

Respiratory Worsening at the End of a Hospital Stay for Severe Covid19 Pneumonia Revealing Pulmonary Tuberculosis

The coexistence of a viral and tuberculous respiratory infection increases mortality and the transition to a severe form in both directions. We report an observation for a 55-year-old woman with untreated diffuse interstitial pnuemopathy, investigated by immunological and infectious tests, and found negative for pulmonary tuberculosis. Bronchial biopsies revealed non-specific inflammation, responsible for chronic dyspnea. Having presented ten days previously with myalgias, fatigue, dry cough and fever, with worsening of basic dyspnoea, without digestive or neurological manifestations, she was referred to the emergency department, where physical examination showed SpO2 at 80% on room air, reduced to 92% under 15 L/mn delivered via a high-concentration mask, polypnoea at 40 cycles per minute with bilateral diffuse crackling rales on auscultation, scattering just the anterior quadrants. The initial evolution was favorable, with clinical improvement in oxygen saturation to 94% on two liters per minute of oxygen delivered by nasal cannula, with respiratory rate at 23 cycles per minute with modest effort tolerance. On day 18 of hospitalization, she presented with a febrile peak of 38.3°C, polypnoea at 33 cycles per minute, increased oxygen requirements with a flow rate of 6l/m by face mask to reach an oxygen saturation of 92%, tachycardia at 130 beats per minute, and was therefore readmitted to intensive care with high-flow nasal oxygen therapy and antibiotic therapy with imipenem and amikacin. Multiplex PCR showing no germ, and a PCR test for pulmonary tuberculosis on a sputum sample which was positive, prompting the introduction of antibacillary treatment based on a fixed quadric-association of rifampicin, isoniaside, pyrazinaamide and ethombutol. Non-specific antibiotic therapy was discontinued after improvement of the inflammatory syndrome, and antibacillary treatment is still ongoing. Conclusion: Through this observation we discuss the difficulties in terms of diagnosis, management and outcome of a coinfection by SARS-COV 19 and mycobacterium tuberculosis.

COVID-19 seropositive donors yield comparable post-lung transplant outcomes.

Recent reports have suggested that coronavirus disease 2019 (COVID-19) infection can cause pneumonitis even in the absence of clinical symptoms and COVID-19 associated pulmonary inflammation can persist resulting in long-term fibrosis. This single-center study utilized standardized immunological testing to determine whether lungs from COVID-19 seropositive donors, indicative of past COVID-19 infection, can be safely used for clinical transplantation. The study included 90 consecutive lung transplant procedures incorporating donor serological testing for past COVID-19 infection. Donors were negative for active COVID-19 infection and met institutional criteria to be used for lung transplantation. The outcomes of lung transplant recipients were compared between donors with and without serological evidence of past COVID-19 infection. No significant difference was found in post-transplant survival rates between recipients of lungs obtained from donors with serological evidence compared to those without. Additionally, there were no significant differences in primary graft dysfunction grade 3 rates or other post-transplant clinical parameters, such as operative time, ischemic time, extracorporeal membrane oxygenation use, intensive care unit stay, and hospital stay. Our findings suggest that lungs from COVID-19 seropositive donors, but not active COVID-19 infection are safe and feasible for transplantation, yielding comparable post-transplant outcomes to donors who are negative COVID-19 antibodies. This study supports the utilization of lungs from donors with historic COVID-19 infection as long as they meet current transplant criteria, potentially addressing the concerns related to the use of such organs.