Correlation between levels of anti-cytokine antibodies, recombination activity of the mutant RAG proteins and clinical phenotype in patients with RAG deficiency

RAG mutations cause various phenotypes: SCID, Omenn syndrome (OS), leaky SCID (LS) and combined immunodeficiency (CID). We had previously reported autoantibodies targeting IFN-alpha, IFN-omega in patients with RAG deficiency. However, how the presence of such antibodies correlated with the severity of the clinical phenotype and with the recombination activity of the mutant proteins was unknown. To address this, we have studied anti-cytokine antibodies in 118 patients with RAG defects (SCID, n = 28;OS, n = 29;LS, n = 29;CID, n = 32), and in 42 controls (protocols NCT03394053 and NCT03610802). RAG mutant proteins associated with CID and LS retained 35.6 +/- 4.3 (mean +/- SE) and 29.8 +/- 5.1% recombination activity respectively, compared to wildtype protein, which was significantly higher than the recombination activity of the mutant RAG proteins associated with OS (4.1 +/- 1.5%) and SCID (5.7 +/- 2.1%) (p < 0.0001). Among 32 CID patients, 24 tested positive for anti-IFN-alpha and 21 for anti-IFN-omega antibodies. Among 29 LS patients, 15 had high levels of anti-IFN-alpha and 13 of anti-IFN-omega antibodies. A minority of the CID and LS patients had also high levels of anti-IFN-beta and anti-IL-22 antibodies. By contrast, none of the OS patients tested positive for anti-cytokine antibodies. High levels of anti-IFN-alpha and anti-IFN-omega antibodies correlated with their neutralizing activity as demonstrated in vitro by analysis of STAT1 phosphorylation upon stimulation of healthy donor monocytes in the presence of the appropriate cytokine and patient's or control plasma. Severe viral infections were recorded in 26/41 patients with CID and LS who tested positive and in 7/20 who tested negative for anti-IFN-alpha and/or anti-IFN-omega antibodies (p <0.05). Among those with anti-IFN antibodies, EBV (n = 8), CMV (n = 6), HSV (n = 5), VZV (n = 4) and adenovirus (n = 4) infections were more common. Two patients had COVID-19, which was fatal in one. Presence of the rubella virus was documented in 5 patients with anti-type I IFN antibodies. These results demonstrate that high levels of neutralizing anti-IFN-alpha and anti-IFN-omega antibodies are common in patients with RAG mutations manifesting as CID and LS, but not in those with OS, and that their presence is associated with a high risk of serious viral infections.Copyright © 2023 Elsevier Inc.

Plasma cytokine profile in patients with severe COVID-19

Background/Objectives: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease (COVID-19) enters the lung tissue through exocytosis, leading to the release of a large amount of pro-inflammatory cytokines called 'cytokine storm'. The aim was to provide more insight into relationship between plasma cytokines profile and fatal outcome of COVID-19. Method(s): Plasma cytokines (IL-17F,GM-CSF,IFNg,IL-10,CCL20/ MIP3a,IL-12P70,IL-13, IL-15,IL-17A,IL-22,IL-9,IL-1b,IL-33,IL-2,IL-21,IL-4,IL-23,IL-5,IL-6,IL-17E/IL-25,IL-27,IL-31,TNFa,TNFb,IL-28A) were detected in 30 patients with severe COVID-19 by a Luminex assay system with Milliplex Human Th17 Magnetic Premix 25 Plex Kit (HT17MG-14K-PX-25, Merk-Millipore, USA) according to the instructions. Patients were followed up for 30 days since admission to intensive care. 18 patients died and 12 patients survived during the period of observation. The control group comprised 10 individuals who had never been diagnosed with COVID-19. Result(s): IL-10 and CCL20/MIP3a plasma levels were elevated in non-survivors patients with COVID-19 compared to controls (p = 0.0027, p = 0.012, respectively). IL-15, IL-6, IL-27 plasma levels were higher in survivors with COVID-19 compared to controls (p = 0.049, p = 0.026, p = 0.00032, respectively). Interestingly, IL-15, IL-27 plasma levels were increased in non-survivors with COVID-19 compared to controls and survivors with severe COVID-19 (IL-15: p = 0.00098, p = 0.00014, respectively;IL-27: p = 0.011, p < 0.0001, respectively). Receiver operating characteristic (ROC) analysis has been conducted for IL-15 and IL-27. Cut-off value was estimated as 25.50 pg/ml for IL-15 and 1.51 pg/ml for IL-27. Conclusion(s): Our study demonstrated a more pronounced immune response in non-surviving patients with severe COVID-19. IL-15, IL-27 could be considered as a sensitive biomarker of the fatal outcome from COVID-19.

Hairy Cell Leukemia in a Filipino Male during the COVID-19 Pandemic - Report of a Rare Case

Hairy cell leukemia (HCL) is a rare, chronic, mature B-cell lymphoproliferative disorder accounting for 2% of all leukemias. In this paper, we would like to present our experience in the management of HCL in a financially limited setting where other diagnostic tests and chemotherapy are unavailable. The case report aims to emphasize the recognition of the distinctive morphology of hairy cells in the peripheral blood in the consideration of the initial diagnosis. A 60-year-old Filipino male was incidentally found to have anemia, thrombocytopenia and an absolute neutrophilic count below 1,000 in a pre-operative clearance for elective herniorrhaphy. Blood smear revealed atypical lymphocytes with hair like cytoplasmic projections. CT-scan of the abdomen showed splenomegaly and prominent paraaortic nodes. Flow cytometry of the bone marrow aspirate was consistent with an involvement of a Mature B cell neoplasm markers CD19, CD20, CD22 and surface immunoglobulin lambda and hairy cell leukemia markers CD11c, CD103 and CD25. He responded to six-weekly sessions of Cladribine with remission of the bone marrow and hematologic parameters. HCL is a rare type of a mature B cell neoplasm characterized by pancytopenia, splenomegaly, bone marrow fibrosis and the presence of atypical lymphoid cells with hairy projections in blood, bone marrow and spleen. Immunophenotyping express CD11c, CD103, CD123, and CD25. BRAF V600E mutation is the disease defining genetic event. Cladribine and Pentostatin are the first line of treatment. Cases of leukemia can be easily overlooked because of the mild derangement in the complete blood count. A meticulous differential review of the atypical lymphocyte, is the first step in the diagnosis of this rare disease.Copyright © 2022, Philippine College of Physicians. All rights reserved.

The abundance of Interleukin-22, 37, and 38 post-vaccination and following COVID-19 recuperation

The SARS-CoV-2 virus causes a contagious disease known as Coronavirus Disease 2019 (COVID-19). It began spreading globally in 2019 and is still producing pandemics today. Different COVID-19 vaccinations offer protection against this illness. Pfizer-BioNTech and Sinopharm were the two vaccine manufacturers with the highest usage in Iraq. Both vaccines use a different method to activate the immune system. This study seeks to compare the IL-22, IL-37, and IL-38 levels in those who received either the Sinopharm or the Pfizer-BioNTech COVID-19 vaccination. IL-22, IL-37, IL-38 levels have been shown to be upregulated in COVID-19 patients. In this study, IL-22, IL-37, and IL-38 levels were tested in 80 healthy controls and 100 COVID-19 patients 14-21 days after recovery. Additionally, people who received the Sinopharm or Pfizer-BioNTech vaccine (50 each) were monitored 21 days after the first dosage and 21 days after the second dose. In comparison to controls, serum levels were noticeably higher in recovered patients. Except for the first dosage of Pfizer BioNTech, the first and second doses of Sinopharm and Pfizer BioNTech were linked to considerably higher levels of IL-22, IL-37, and IL-38 compared to controls or recovered patients. where IL-22, IL-37, and IL-38 levels did not show significant differences compared to recovered patients. In conclusion, lower IL-37 and IL-38 molecule levels were linked to recovery from COVID-19, although these levels remained considerably greater in recovered patients compared to uninfected controls. Vaccination with Sinopharm or Pfizer-BioNTech confirmed the up-regulating effects of SARS-CoV-2 on IL-22, IL-37, and IL-38 levels.Copyright © 2023, Codon Publications. All rights reserved.

Distinct profiles and kinetics of soluble infammatory markers in patients with acute and chronic HCV-infections and SARS-CoV-2-infections: Implications for the long-term consequences of viral infections?

Background and Aims Viral infections occur acutely but can also progress chronically, with the immune system having a central role in immunopathoge-nesis. The question arises whether all alterations in immune responses are reversible after viral elimination (spontaneously or by therapy). Therefore, the aim of this study is to compare soluble infammatory markers (SIM) during and after infection with SARS-CoV-2 and acute and chronic HCV-infections. Patients and Method Patients with acute HCV (n = 29), chronic HCV (n = 54), SARS-CoV-2 (n = 39) and 31 healthy-controls were included. Blood samples were tested at baseline, end of treatment/infection, and follow-up ( >= 9 months after baseline). IL-12p70, IL-1b, IL-4, IL-5, IL-6, IL-8, TNF, IFN-g, IL-10, IL-22, CXCL-10, MCP-1, MIP-1b, ITAC were quantified using the HD-SP-X Imaging and Analysis SystemTM. Results SIM profiles in patients with acute HCV were substantially elevated at baseline and the decrease during follow-up was considerably less compared to the SARS-CoV-2 cohort. In chronic HCV-patients, viral elimination by therapy resulted in a decrease in SIM, although not always to those of controls. Cirrhotic HCV patients had higher SIM levels after HCV elimination than non-cirrhotic chronic HCV-patients. In the SARS-CoV-2 cohort, most SIM returned to levels of controls 3 months after baseline. Conclusions SIM profiles and kinetics after viral elimination difer between blood-borne acute and chronic HCV- and respiratory SARS-CoV-2-infections. The immunologic imprint 9 months after cured HCV-infection (both acute and chronic) appears to be more pronounced than after SARS-CoV-2-infection. Further analysis is needed to correlate the SIM profle with the clinical pheno-type (long-HepC vs. long-COVID-19).

Predictive Value of Specific Cytokines for Lethal Covid-19 Outcome

In our study, we aimed to evaluate the significance of specific cytokines in blood plasma as predictive markers of COVID-associated mortality. Materials and methods. In plasma samples of 29 patients with PCR-confirmed COVID-19 we measured the concentrations of 47 molecules. These molecules included: interleukins and selected pro-inflammatory cytokines (IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A/CTLA8, IL-17-E/IL-25, IL-17F, IL-18, IL-22, IL-27, IFNalpha2, IFNgamma, TNFalpha, TNFbeta/Lymphotoxin-alpha(LTA));chemokines (CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL7/MCP-3, CCL11/Eotaxin, CCL22/MDC, CXCL1/GROalpha, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CX3CL1/Fractalkine);anti-inflammatory cytokines (IL-1Ra, IL-10);growth factors (EGF, FGF-2/FGF-basic, Flt-3 Ligand, G-CSF, M-CSF, GM-CSF, PDGF-AA, PDGFAB/BB, TGFalpha, VEGF-A);and sCD40L. We used multiplex analysis based on xMAP technology (Luminex, USA) using Luminex MagPix. As controls, we used plasma samples of 20 healthy individuals. Based on the results, we applied Receiver Operating Characteristic (ROC) analysis and Area Under Curve (AUC) values to compare two different predictive tests and to choose the optimal division point for disease outcome (survivors/non-survivors). To find optimal biomarker combinations, we as used cytokines concentrations as dependent variables to grow a regression tree using JMP 16 Software.Results. Out of 47 studied cytokines/chemokines/growth factors, we picked four pro-inflammatory cytokines as having high significance in evaluation of COVID-19 outcome: IL-6, IL-8, IL-15, and IL-18. Based on the results received, we assume that the highest significance in terms of predicting the outcome of acute COVID-19 belongs to IL-6 and IL-18. Conclusion. Analyzing concentrations of IL-6 and IL-18 before administering treatment may prove valuable in terms of outcome prognosis. Copyright © Arsentieva N.A. et al., 2022.

IS PSORIATIC ARTHRITIS A RISK FACTOR for SEVERE COVID-19 INFECTION? DATA from the ARGENTINIAN REGISTRY SAR-COVID

Background: Comorbidities, particularly cardio-metabolic disorders, are highly prevalent in patients with psoriatic arthritis (PsA) and they were associated with an increased risk of atherosclerotic cardiovascular disease, which have been associated with higher morbidity and mortality. Whether PsA enhances the risk of SARS-CoV-2 infection or affects the disease outcome remains to be ascertained. Objectives: To describe the sociodemographic, clinical and treatment characteristics of patients with PsA with confrmed SARS-CoV-2 infection from the SAR-COVID registry and to identify the variables associated with poor COVID-19 outcomes, comparing them with those with rheumatoid arthritis (RA). Methods: Cross-sectional observational study including patients ≥18 years old, with diagnosis of PsA (CASPAR criteria) and RA (ACR/EULAR 2010 criteria), who had confrmed SARS-CoV-2 infection (RT-PCR or serology) from the SAR-COVID registry. Recruitment period was between August 13, 2020 and July 31, 2021. Sociodemographic variables, comorbidities, and treatments were analyzed. To assess the severity of the infection, the ordinal scale of the National Institute of Allergy and Infectious Diseases (NIAID)1 was used, and it was considered that a patient met the primary outcome, if they presented criteria of categories 5 or higher on the severity scale. For this analysis, Chi2 test, Fisher's test, Student's test or Wilcoxon test, and binomial logistic regression using NIAID>=5 as dependent variable were performed. Results: A total of 129 PsA patients and 808 with RA were included. Clinical characteristics are shown in Table 1. Regarding PsA treatment, 12.4% of PsA were receiving IL-17 inhibitors, 5.4% IL12-23 inhibitors, one patient apremilast and one abatacept. The frequency of NIAID≥5 was comparable between groups (PsA 19.5% vs RA 20.1%;p=0.976). (Figure 1). PsA patients with NIAID≥5 in comparison with NIAID<5 were older (58.6±11.4 vs 50±12.5;p=0.002), had more frequently hypertension (52.2% vs 23%;p=0.011) and dyslipidemia (39.1% vs 15%;p=0.017). In the multivariate analysis, age (OR 1.06;95% CI 1.02-1.11) was associated with a worse outcome of the COVID-19 (NIAID≥5) in patients with PsA, while those who received methotrexate (OR 0.34;95% CI 0.11-0.92) and biological DMARDs (OR 0.28;95% CI 0.09-0.78) had a better outcome. Conclusion: Although PsA patients have a higher frequency of cardiovascular and metabolic comorbidities than those with RA, the COVID-19 severity was similar. Most of the patients had mild SARS-CoV-2 infection and a low death rate.

Abstracts of the BSG Annual Meeting

The proceedings contain 374 papers. The topics discussed include: optical biopsy with linked color imaging accurately predicts inflammation in ulcerative colitis;predicting use of opiates in patients with inoperable pancreatic cancer: a retrospective cohort study;unbiased clustering of breath signature in NAFLD identifies disease progression high-risk patient phenotype - 5 year study;prevalence of malnutrition screening;the association between IBD and mental ill health: a retrospective primary care cohort study;the interleukin 22//neutrophil axis is associated with treatment resistance in ulcerative colitis;neuromuscular dysfunction in patients with nausea and vomiting syndrome defined by body surface gastric mapping;two-tiered liver fibrosis assessment in primary care annual diabetic screening 3 year follow up;can an algorithm help in the difficult dilemma of upper gastrointestinal bleed and anticoagulant COVID-19 pandemic and alcohol-specific hospital admissions;and upper gastrointestinal hemorrhages and COVID-19: a nationwide cohort study of the pandemic's impact on hospitalizations.

LOSS of TOLERANCE and ANERGY in HUMAN PERIPHERAL B CELLS during SARS-CoV-2 INFECTION

Background: Severe infection with SARS-CoV-2 induces systemic autoreactive antibodies with specificity to Type I IFN, phospholipids, nuclear or tissue specific targets. The wide breadth of targets suggests a system-wide defect in B cell tolerance during viral infection and that the source of autoreactive antibodies is likely a heterogenous subset of B cells. BND cells are mature naïve B cells that do not express IgM but do express IgD and are enriched in autoreactive specificities. BND cells are held in an anergic state in healthy humans as a mechanism of peripheral tolerance, although in vitro evidence suggests anergy can be broken with strong inflammation. We hypothesized that robust inflammation associated with viral infection from SARS-CoV2 may relax peripheral tolerance and promote breakage of BND cell anergy. Methods: Plasma and PBMCs were collected from healthy controls (N=10), subjects immunized with Pfizer BNT162b2-mRNA/Moderna mRNA-1273 (N=10), subjects with mild (N=11) or severe SARS-CoV-2 infection (N=14). BND cells were examined ex vivo for markers of activation by flow cytometry. Phosphorylation of signaling proteins downstream of the BCR were measured in vitro with or without BCR crosslinking. Inflammatory cytokines were measured in plasma by multiplex. For statistical analysis, unpaired t test between populations or paired t test between unstimulated and BCR stimulated conditions were performed. Results: BND cells from severe SARS-CoV-2 infection have lower expression of CD21, associated with loss of anergy, higher expression of activation markers CD68 and CD86 with lower expression of inhibitory receptors CD22 and CD72 when compared to BND cells from other subjects, suggesting a phenotypical breach of anergy. Upon BCR crosslinking, BND cells have higher levels of downstream signaling components of the BCR (pPLCγ2, pBlnk, and pSyk) when compared to healthy controls and immunized subjects, suggesting a functional breach in anergy with infection. Examination of plasma from severe SARS-CoV-2 infection showed higher levels of inflammatory cytokines (IFNγ, TNFα, IL-6 and CRP) where TNFα and CRP correlated with enhanced BCR signaling in BND cells. Conclusion: We demonstrate that SARS-CoV-2 viral infection relaxes peripheral tolerance of BND cells, likely through strong systemic inflammation produced during infection. These autoreactive cells overcome anergy and become activated with increased BCR signaling. Thus BND cells could be a source of autoreactive antibodies during viral infection.

Kombucha - An ancient fermented beverage with desired bioactivities: A narrowed review.

Kombucha, originated in China 2000  years ago, is a sour and sweet-tasted drink, prepared traditionally through fermentation of black tea. During the fermentation of kombucha, consisting of mainly acidic compounds, microorganisms, and a tiny amount of alcohol, a biofilm called SCOBY forms. The bacteria in kombucha has been generally identified as Acetobacteraceae. Kombucha is a noteworthy source of B complex vitamins, polyphenols, and organic acids (mainly acetic acid). Nowadays, kombucha is tended to be prepared with some other plant species, which, therefore, lead to variations in its composition. Pre-clinical studies conducted on kombucha revealed that it has desired bioactivities such as antimicrobial, antioxidant, hepatoprotective, anti-hypercholestorelomic, anticancer, anti-inflammatory, etc. Only a few clinical studies have been also reported. In the current review, we aimed to overhaul pre-clinical bioactivities reported on kombucha as well as its brief compositional chemistry. The literature data indicate that kombucha has valuable biological effects on human health.

The potential of BEN815 as an anti-inflammatory, antiviral and antioxidant agent for the treatment of COVID-19.

Background: The corona virus disease 2019 (COVID-19) pandemic has highlighted the fact that there are few effective antiviral agents for treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Although the very recent development of vaccines is an extremely important breakthrough, it remains unclear how long-lived such vaccines will be. The development of new agents therefore remains an important goal. Purpose: Given the multifaceted pathology of COVID-19, a combinatorial formulation may provide an effective treatment. BEN815, a natural nutraceutical composed of extracts from guava leaves (Psidium guajava), green tea leaves (Camellia sinensis), and rose petals (Rosa hybrida), had previously shown to have a therapeutic effect on allergic rhinitis. We investigated whether BEN815 possesses anti-inflammatory, antiviral and antioxidant activities, since the combination of these effects could be useful for the treatment of COVID-19. Study design: We examined the anti-inflammatory effects of BEN815 and its principal active components quercetin and epigallocatechin gallate (EGCG) in lipopolysaccharide (LPS)-induced RAW264.7 cells and in an LPS-challenged mouse model of endotoxemia. We also assessed the antioxidant activity, and antiviral effect of BEN815, quercetin, and EGCG in SARS-CoV-2-infected Vero cells. Methods: The principal active ingredients in BEN815 were determined and quantified using HPLC. Changes in the levels of LPS-induced pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α were measured by ELISA. Changes in the expression levels of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) were analyzed using western blotting. Antioxidant assay was performed using DPPH and ABTS assay. SARS-CoV-2 replication was measured by immunofluorescence staining. Results: BEN815 significantly suppressed the induction of IL-6 and TNF-α as well as COX-2 and iNOS in LPS-induced RAW264.7 cells. In addition, BEN815 protected against LPS-challenged endotoxic shock in mice. Two major constituents of BEN815, quercetin and EGCG, reduced the induction of IL-6 and TNF-α as well as COX-2 and iNOS synthase in LPS-induced RAW264.7 cells. BEN815, quercetin, and EGCG were also found to have antioxidant effects. Importantly, BEN815 and EGCG could inhibit SARS-CoV-2 replications in Vero cells. Conclusion: BEN815 is an anti-inflammatory, antiviral, and antioxidant natural agent that can be used to prevent and improve inflammation-related diseases, COVID-19.

Disruption of CCR5 signaling to treat COVID-19-associated cytokine storm: Case series of four critically ill patients treated with leronlimab.

Coronavirus disease 2019 (COVID-19) is associated with considerable morbidity and mortality. The number of confirmed cases of infection with SARS-CoV-2, the virus causing COVID-19 continues to escalate with over 70 million confirmed cases and over 1.6 million confirmed deaths. Severe-to-critical COVID-19 is associated with a dysregulated host immune response to the virus, which is thought to lead to pathogenic immune dysregulation and end-organ damage. Presently few effective treatment options are available to treat COVID-19. Leronlimab is a humanized IgG4, kappa monoclonal antibody that blocks C-C chemokine receptor type 5 (CCR5). It has been shown that in patients with severe COVID-19 treatment with leronlimab reduces elevated plasma IL-6 and chemokine ligand 5 (CCL5), and normalized CD4/CD8 ratios. We administered leronlimab to 4 critically ill COVID-19 patients in intensive care. All 4 of these patients improved clinically as measured by vasopressor support, and discontinuation of hemodialysis and mechanical ventilation. Following administration of leronlimab there was a statistically significant decrease in IL-6 observed in patient A (p=0.034) from day 0-7 and patient D (p=0.027) from day 0-14. This corresponds to restoration of the immune function as measured by CD4+/CD8+ T cell ratio. Although two of the patients went on to survive the other two subsequently died of surgical complications after an initial recovery from SARS-CoV-2 infection.

Computationally predicted SARS-COV-2 encoded microRNAs target NFKB, JAK/STAT and TGFB signaling pathways.

Recently an outbreak that emerged in Wuhan, China in December 2019, spread to the whole world in a short time and killed >1,410,000 people. It was determined that a new type of beta coronavirus called severe acute respiratory disease coronavirus type 2 (SARS-CoV-2) was causative agent of this outbreak and the disease caused by the virus was named as coronavirus disease 19 (COVID19). Despite the information obtained from the viral genome structure, many aspects of the virus-host interactions during infection is still unknown. In this study we aimed to identify SARS-CoV-2 encoded microRNAs and their cellular targets. We applied a computational method to predict miRNAs encoded by SARS-CoV-2 along with their putative targets in humans. Targets of predicted miRNAs were clustered into groups based on their biological processes, molecular function, and cellular compartments using GO and PANTHER. By using KEGG pathway enrichment analysis top pathways were identified. Finally, we have constructed an integrative pathway network analysis with target genes. We identified 40 SARS-CoV-2 miRNAs and their regulated targets. Our analysis showed that targeted genes including NFKB1, NFKBIE, JAK1-2, STAT3-4, STAT5B, STAT6, SOCS1-6, IL2, IL8, IL10, IL17, TGFBR1-2, SMAD2-4, HDAC1-6 and JARID1A-C, JARID2 play important roles in NFKB, JAK/STAT and TGFB signaling pathways as well as cells' epigenetic regulation pathways. Our results may help to understand virus-host interaction and the role of viral miRNAs during SARS-CoV-2 infection. As there is no current drug and effective treatment available for COVID19, it may also help to develop new treatment strategies.