Molecular modelling and experimental validation identified a new therapeutic inhibitor of toxoplasmosis.

Toxoplasmosis is a widespread parasitic disease, caused by Toxoplasma gondii, that affects nearly one-third of the human population. The lack of effective treatments drives the demand for novel anti-toxoplasmosis therapeutic options. In the present study, we used computational approaches and experimental validation to identify therapeutic inhibitors of toxoplasmosis. Initially, using the structure of the co-crystallized ligand of T. gondii calcium-dependent protein kinase 1 (TgCDPK1), we retrieved 3000 compounds from the database of COCONUT (COlleCtion of Open Natural ProdUcTs). These compounds were docked against the crystal structure of TgCDPK1 on the Glide Ligand Docking panel of Maestro 12.5 (Schrödinger Suite 2020-3). Based on the docking scores, we assessed promising molecules for toxicity potential on the ProTox-II online server, while the ADME profiling was done on the SwissADME server. Following the computational studies, we selected nine promising compounds for experimental validation against T. gondii in vitro. Of the compounds, C4, C5, C6, and C8 exhibited dose-dependent anti-T. gondii action with EC50 values ranging from 3.3 to 120.2 µg/mL. Host toxicity profiling revealed differential cytotoxic action with a selectivity index (SI) of <1 for the compounds except C5, which had an SI of 1.8. To validate our screening assay, we used sulfadiazine, a standard drug for toxoplasmosis and showed that it inhibited parasite growth. Further experiments showed that C5, an imidazole-based natural compound, has strong but reversible anti-parasitic action that peaks within the first 8 h. In addition, C5 exhibited similar toxic tendencies towards T. gondii within (intracellular) and outside (extracellular) the host, suggesting it likely has a parasite target(s). C5 showed no effect on host invasion but strongly impeded parasite replication and growth, thereby affecting the T. gondii lytic cycle. Furthermore, C5 treatment raised the reactive oxygen species level, but this may be a secondary effect because augmentation with Trolox antioxidant failed to block C5 anti-T. gondii action. In addition, molecular dynamics simulations of C5 and TgCDPK1 complex revealed relative stability within 100 ns run time. Collectively, our findings support the potential of imidazole-based compounds as novel, alternative anti-parasitic agents.

The effectiveness of clinical thinking using a problem-solving model.

[Purpose] No established method for appropriately developing clinical thinking in physical therapy currently exists. This study examined whether clinical thinking can be appropriately developed using a problem-solving model. [Participants and Methods] Physical therapy students were asked to develop clinical thinking in the same two cases: one class using the problem-solving model and the other class using International Classification of Functioning, Disability and Health concepts. Each clinical thought was scored and compared based on consistency. [Results] In both cases, students who used the problem-solving model scored higher. [Conclusion] The hierarchical structure of the problem-solving model clarified the relationship between each element and this was easy to maintain, suggesting that it facilitated appropriate clinical thinking.

ctDNA-based molecular residual disease and survival in resectable colorectal cancer.

The interim analysis of the CIRCULATE-Japan GALAXY observational study demonstrated the association of circulating tumor DNA (ctDNA)-based molecular residual disease (MRD) detection with recurrence risk and benefit from adjuvant chemotherapy (ACT) in resectable colorectal cancer (CRC). This updated analysis with a 23-month median follow-up, including 2,240 patients with stage II-III colon cancer or stage IV CRC, reinforces the prognostic value of ctDNA positivity during the MRD window with significantly inferior disease-free survival (DFS; hazard ratio (HR): 11.99, P < 0.0001) and overall survival (OS; HR: 9.68, P < 0.0001). In patients who experienced recurrence, ctDNA positivity correlated with shorter OS (HR: 2.71, P < 0.0001). The significantly shorter DFS in MRD-positive patients was consistent across actionable biomarker subsets. Sustained ctDNA clearance in response to ACT was an indicator of favorable DFS and OS compared to transient clearance (24-month DFS: 89.0% versus 3.3%; 24-month OS: 100.0% versus 82.3%). True spontaneous clearance rate with no clinical recurrence was 1.9% (2/105). Overall, our findings provide evidence for the utility of ctDNA monitoring for post-resection recurrence and mortality risk stratification that could be used for guiding adjuvant therapy.

Testing the interrater reliability of integrated interpretations using a problem-solving model.

[Purpose] A method for evaluating integrated interpretation skills, which is an important educational goal in clinical practice, is yet to be established. This study examined the interrater reliability of a problem-solving model that was developed as a novel assessment method. [Participants and Methods] Three students created problem-solving models using fictitious case data. Ten clinical practice supervisors with at least 3 years of experience were asked to score the models, and the intraclass correlation coefficient (2, 1) was calculated. [Results] The intraclass correlation coefficient was 0.88, which indicates a good result. [Conclusion] Good interrater reliability was obtained using the problem-solving model, which suggests that this model may be used to assess integrated interpretation skills in clinical practice.

Efficacy of medicinal plants and their derived biomolecules against Plasmodium falciparum.

Many apicomplexan pathogens pose significant threats to humans and domestic animals, with the lack of effective drugs and drug resistance representing major challenges in disease management. To address this, the search for new and potent antimalarial drugs is crucial. Plant-based formulations offer a promising alternative for such drug development. Here, we evaluated the in vitro antiplasmodial activity of nine plant extracts, traditionally used to treat fever-like symptoms in Bangladesh. We assessed the antimalarial activity of plant extracts by using the Plasmodium falciparum 3D7 growth inhibition assay, an invasion assay, and a cytotoxicity assay. Of the nine plants studied, ethanolic and methanolic leaf extracts of Ficus hispida, Streblus asper, and Boerhavia repens exhibited high antiplasmodial activity, with IC50 values of 9.31, 4.13, 9.63 µg/ml (ethanolic) and 15.12, 6.63, 7.58 µg/ml (methanolic), respectively, and minimal toxicity (cell viability >80%). Clerodendrum viscosum displayed antiplasmodial effects with IC50 values of 28.90 µg/ml (ethanolic) and 30.57 µg/ml (methanolic). Adhatoda vasica, Mussaenda corymbosa, and Amaranthus spinosus ethanolic extracts showed antimalarial effects with IC50 values of 61.78 µg/ml, 66.31 µg/ml, and 64.14 µg/ml, respectively. However, methanolic extracts of A. vasica and A. spinosus had IC50 values >100 µg/ml. The ethanolic and methanolic extracts of A. vasica, A. spinosus, F. hispida, S. asper, and B. repens significantly reduced parasitemia by inhibiting invasion into erythrocytes. This study highlights the robust antimalarial activity and low cytotoxicity of leaf extracts of F. hispida, S. asper, and B. repens, indicating the presence of antimalarial compounds that warrant further investigation.

Toxoplasma gondii chitinase-like protein TgCLP1 regulates the parasite cyst burden.

Toxoplasma, an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Although Toxoplasma secretory proteins during acute infection (tachyzoite, which divides rapidly and causes inflammation) have been extensively characterized, those involved in chronic infection (bradyzoite, which divides slowly and is surrounded by a cyst wall) remain uncertain. Regulation of the cyst wall is essential to the parasite life cycle, and polysaccharides, such as chitin, in the cyst wall are necessary to sustain latent infection. Toxoplasma secretory proteins during the bradyzoite stage may have important roles in regulating the cyst wall via polysaccharides. Here, we focused on characterizing the hypothetical T. gondii chitinase, chitinase-like protein 1 (TgCLP1). We found that the chitinase-like domain containing TgCLP1 is partially present in the bradyzoite microneme and confirmed, albeit partially, its previous identification in the tachyzoite microneme. Furthermore, although parasites lacking TgCLP1 could convert from tachyzoites to bradyzoites and make an intact cyst wall, they failed to convert from bradyzoites to tachyzoites, indicating that TgCLP1 is necessary for bradyzoite reactivation. Taken together, our findings deepen our understanding of the molecular basis of recrudescence and could contribute to the development of novel strategies for the control of toxoplasmosis.

Comprehensive molecular epidemiology of Cryptosporidium species in Japan.

Cryptosporidium species, causing diarrheal illnesses in humans and animals worldwide, are under investigation for their molecular epidemiology in Japan. The study focuses on detecting Cryptosporidium species in humans, animals, water, and the environment, revealing three species in people: C. parvum, C. meleagridis, and C. hominis. Subtype IIa of the C. parvum gp60 gene is prevalent, indicating potential zoonotic transmission. Animal studies identified sixteen species, mainly cattle and pets, with C. parvum (subtype IIa) common in cattle and C. canis and C. felis prevalent in pets. Additionally, C. bovis and C. ryanae were found in cattle and sika deer. Knowledge gaps exist, particularly in water and environmental source typing, with limited research revealing five species and five genotypes, suggesting a significant role of water in transmission. Further research is needed to understand the molecular diversity and transmission dynamics across humans, animals, water, and the environment in Japan.

Efficacy and safety of tumor necrosis factor inhibitors for systemic juvenile idiopathic arthritis: a systematic review.

OBJECTIVES: This systematic review assessed the efficacy and safety of tumor necrosis factor (TNF) inhibitors in patients with systemic juvenile idiopathic arthritis (JIA). METHODS: Studies were searched using PubMed, Embase, Cochrane, Ichushi-Web, and clinical trial registries (from 2000 to 2021). The risk of bias was assessed using the Cochrane Risk of Bias version 2 for randomized controlled trials (RCTs) and the manual for development clinical practice guidelines by Minds, a project promoting evidence-based medicine in Japan, for observational studies. RESULTS: One RCT and 22 observational studies were included. In the RCT on infliximab, the American College of Rheumatology pediatric (ACR Pedi) 30/50/70 responses at 14 weeks were 63.8%/50.0%/22.4%, with relative risks of 1.30 (95% confidence interval [CI]: 0.94-1.79)/1.48 (95% CI: 0.95-2.29)/1.89 (95% CI: 0.81-4.40), respectively. In the observational studies, ACR Pedi 30/50/70 responses for etanercept at 12 months were 76.7%/64.7%/46.4%, respectively. Infliximab treatment caused anaphylaxis in 17% and an infusion reaction in 23% of patients. The incidence of macrophage activation syndrome, serious infection and malignancy caused by TNF inhibitors was 0%-4%. CONCLUSIONS: Thus, although TNF inhibitors were relatively safe, they were unlikely to be preferentially administered in patients with systemic JIA because of their inadequate efficacy. Further studies, particularly well-designed RCTs, are necessary to confirm the efficacy and safety of TNF inhibitors for systemic JIA.

Efficacy and safety of abatacept for systemic juvenile idiopathic arthritis: a systematic review.

OBJECTIVES: This systematic review assessed the efficacy and safety of abatacept in patients with systemic juvenile idiopathic arthritis (JIA). METHODS: Studies published between 2000 and 2021 were searched using PubMed, Embase, Cochrane, Ichushi-Web and clinical trial registries. The risk of bias was assessed according to the manual for development clinical practice guidelines by Minds, a project to promote evidence-based medicine in Japan. RESULTS: Seven observational studies were included. American College of Rheumatology pediatric 30/50/70 responses at 3, 6 and 12 months were 64.8%/50.3%/27.9%, 85.7%/71.4%/42.9% and 80.0%/50.0%/40.0%, respectively. Outcomes on systemic symptoms, joint symptoms and activities of daily living were not obtained. No macrophage activation syndrome or infusion reaction occurred. Serious infection occurred in 2.6% of cases. CONCLUSIONS: Abatacept improved the disease activity index. In addition, abatacept was as safe as interleukin-6 (IL -6) and IL-1 inhibitors. However, both the efficacy and safety data in this systematic review should be reviewed with caution because their quality of evidence is low or very low. Further studies are needed to confirm the efficacy and safety of abatacept for systemic JIA, especially its efficacy on joint symptoms.

Examining the molecular epidemiology of Giardia and Eimeria species in Japan: a comprehensive review.

Globally, animals and humans suffer from diarrheal illnesses due to protozoan parasites such as Giardia and Eimeria species. The molecular epidemiology of these parasites in Japan is summarized in this review. In humans, researchers found only one main species of Giardia, which is most referred to as G. lamblia, but it's also known by different names like G. duodenalis or G. intestinalis. However, within this species, six assemblages (A, B, C, D, E, and F) were found in animals, and assemblage B was frequently recorded in human and monkey populations, whereas assemblages A and E were predominant in calves. Assemblage A was found in sika deer and assemblages A, C, D, and F were predominant in dogs, cats, and ferret. Eimeria bovis, E. zuernii, and other species found in animals made up the group of species known as Eimeria spp., with E. bovis and E. zuernii being the most common in cattle. Our review highlighted a notable lack of data investigations regarding these two pathogens in water and environmental sources. Giardia cysts were found in the few studies that have been done on water sources, suggesting that water may play a significant role in the transmission of Giardia species. Our review suggests that further research is necessary to fully comprehend the molecular diversity and dynamics of transmission of Giardia spp. and Eimeria spp. in humans, animals, and environmental sources in Japan.

Goethite and Hematite Nanoparticles Show Promising Anti-Toxoplasma Properties.

Toxoplasma gondii is an intracellular parasitic protozoan with a high infection rate in mammals, including humans, and birds. There is no effective vaccine, and treatment relies on antiparasitic drugs. However, existing antiprotozoal drugs have strong side effects and other problems; therefore, new treatment approaches are needed. Metal nanoparticles have attracted increased interest in the biomedical community in recent years because of their extremely high surface area to volume ratio and their unique reactivity that could be exploited for medicinal purposes. Previously, we confirmed the anti-Toxoplasma effects of gold, silver, and platinum nanoparticles, in a growth inhibition test. Here, we asked whether the anti-Toxoplasma effect could be confirmed with less expensive metal nanoparticles, specifically iron oxide nanoparticles (goethite and hematite). To improve the selective action of the nanoparticles, we modified the surface with l-tryptophan as our previous findings showed that the bio-modification of nanoparticles enhances their selectivity against T. gondii. Fourier-Transform Infrared Spectroscopy (FTIR) analysis confirmed the successful coating of the iron oxide nanoparticles with l-tryptophan. Subsequently, cytotoxicity and growth inhibition assays were performed. L-tryptophan-modified nanoparticles showed superior anti-Toxoplasma action compared to their naked nanoparticle counterparts. L-tryptophan enhanced the selective toxicity of the iron oxide nanoparticles toward T. gondii. The bio-modified nanoparticles did not exhibit detectable host cell toxicity in the effective anti-Toxoplasma doses. To elucidate whether reactive oxygen species contribute to the anti-Toxoplasma action of the bio-modified nanoparticles, we added Trolox antioxidant to the assay medium and found that Trolox appreciably reduced the nanoparticle-induced growth inhibition.

Detection of Developmental Asexual Stage-Specific RNA Editing Events in Plasmodium falciparum 3D7 Malaria Parasite.

Transcriptional variation has been studied but post-transcriptional modification due to RNA editing has not been investigated in Plasmodium. We investigated developmental stage-specific RNA editing in selected genes in Plasmodium falciparum 3D7. We detected extensive amination- and deamination-type RNA editing at 8, 16, 24, 32, 40, and 46 h in tightly synchronized Plasmodium. Most of the editing events were observed in 8 and 16 h ring-stage parasites. Extensive A-to-G deamination-type editing was detected more during the 16 h ring stage (25%) than the 8 h ring stage (20%). Extensive U-to-C amination-type editing was detected more during the 16 h ring stage (31%) than the 8 h ring stage (22%). In 28S, rRNA editing converted the loop structure to the stem structure. The hemoglobin binding activity of PF3D7_0216900 was also altered due to RNA editing. Among the expressed 28S rRNA genes, PF3D7_0532000 and PF3D7_0726000 expression was higher. Increased amounts of the transcripts of these two genes were found, particularly PF3D7_0726000 in the ring stage and PF3D7_0532000 in the trophozoite and schizont stages. Adenosine deaminase (ADA) expression did not correlate with the editing level. This first experimental report of RNA editing will help to identify the editing machinery that might be useful for antimalarial drug discovery and malaria control.

Effects of monosaccharides including rare sugars on proliferation of Entamoeba histolytica trophozoites in vitro.

Entamoeba histolytica is a parasitic protozoan with roles in pathogenicity of intestinal amoebiasis. E. histolytica trophozoites lack functional mitochondria and their energy production depends mostly on glycolysis. D-Glucose has a pivotal role in this process and trophozoites store this sugar as glycogen in glycogen granules. Rare sugars, which are defined as sugars present in nature in limited amounts, are of interest as natural low-calorie sweeteners for improving physical conditions of humans. One such rare sugar, D-allose, can be absorbed by a sodium-dependent glucose cotransporter as a substitute for D-glucose, and some rare sugars are known to inhibit growth of cancer cells, Caenorhabditis elegans and Tritrichomonas foetus. Based on these observations, we examined the effects of rare sugars on growth of E. histolytica trophozoites, together with those of D-galactose and D-fructose. The results indicate that treatment with D-allose or D-psicose (D-allulose) alone inhibits proliferation of E. histolytica trophozoites, but that these sugars enhance proliferation of trophozoites in the presence of D-glucose or D-galactose. The trophozoites could take up D-glucose and D-galactose, but not D-fructose, D-allose or D-psicose. Cell sizes of the trophozoites also differed depending on the culture medium.

In Vivo Efficacy of Curcumin and Curcumin Nanoparticle in Trypanosoma congolense, Broden 1904 (Kinetoplastea: Trypanosomatidae)-Infected Mice.

Curcumin (CUR) is known for its wide folkloric effects on various infections; however, its solubility status has remained a hindrance to its bioavailability in the host. This study evaluated the comparative effects of CUR and CUR-nanoparticle in vitro on T. congolense, T. b. brucei, and T. evansi. Additionally, CUR and CUR-nanoparticle anti-Trypanosoma efficacy were assessed in vivo against T. congolense. All the CUR-nanoparticles were two folds more effective on the T. congolense as compared to CUR in vitro, with recorded efficacy of 3.67 ± 0.31; 7.61 ± 1.22; and 6.40 ± 3.07 µM, while the CUR-nanoparticles efficacy was 1.56 ± 0.50; 28.16 ± 9.43 and 13.12 ± 0.13 µM on T. congolense, T. b. brucei, and T. evansi, respectively. Both CUR and CUR-nanoparticles displayed moderate efficacy orally. The efficacy of CUR and CUR-nanoparticles in vivo was influenced by solubility, presence of food, and treatment period. CUR-treated mice were not cured of the infection; however, the survival rate of the orally treated mice was significantly prolonged as compared with intraperitoneal-treated mice. CUR-nanoparticles resulted in significant suppression of parasitemia even though relapsed was observed. In conclusion, CUR and CUR-nanoparticles possess moderate efficacy orally on the trypanosomes as compared to the intraperitoneal treatment.

Bilateral video-assisted thoracic surgery for esophageal cancer with left inferior pulmonary vein invasion following chemoradiation therapy.

BACKGROUND: The surgical strategy for thoracic esophageal cancer that invades the lungs is controversial. In particular, invasion of the pulmonary vein is often regarded unresectable. We successfully applied bilateral video-assisted thoracic surgery (VATS) in esophagectomy for esophageal cancer with left inferior pulmonary vein invasion following induction chemoradiotherapy (CRT), with a favorable response. CASE PRESENTATION: A 64-year-old woman was diagnosed with squamous cell carcinoma of the lower third of the esophagus. Computed tomography (CT) revealed that the tumor was suspected to be invading the main trunk of the left lower pulmonary vein and left lower lung. We initiated induction CRT comprising 5-fluorouracil, cisplatin, and concurrent radiotherapy at 50.4 Gy/28Fr. CT revealed shrinkage of the tumor, and the main trunk of the left inferior pulmonary vein was released from the tumor invasion. We considered the tumor to be completely resectable. VATS esophagectomy is usually performed using a right-sided approach. However, the right-sided approach is inappropriate for evaluating tumors around the left inferior pulmonary vein. We started with left-sided VATS to determine tumor resectability and dissected between the esophagus and the main trunk of the left inferior pulmonary vein. We only needed to perform partial resection of the left lower lobe. We then performed a right-sided VATS esophagectomy and lymphadenectomy with partial en bloc resection of the left lower lobe. Following this, we performed hand-assisted laparoscopic proximal gastrectomy and reconstruction using the gastric remnant. The postoperative course was uneventful. The patient was discharged on postoperative day 14. Histopathological examination of the surgical specimen revealed a complete pathological response without any remnant tumor or lymph node metastasis. There were no signs of recurrence or metastasis at the 1-year follow-up. CONCLUSIONS: Curative resection for thoracic esophageal cancer that invades the pulmonary vein could be possible via the bilateral VATS approach following induction CRT with a favorable response.

Antiplasmodial and interferon-gamma-modulating activities of the aqueous extract of stone breaker (Phyllanthus niruri Linn.) in malaria infection.

Plasmodium falciparum parasites are the primary cause of malaria across Africa. The problem of drug resistance to malaria is ever growing and novel therapeutic strategies need to be developed, particularly those targeting the parasite and also the host or host-pathogen interaction. Previous studies have shown that the development of cerebral malaria (CM) is related to dysregulation of the immune system in a murine malaria model of experimental cerebral malaria. It involves a complex interaction of events and interferon-gamma seems to be the unifying factor. Therefore, the antiplasmodial activity targeting the parasite and immunomodulatory strategies that reduce overall host inflammation, with IFN-γ in focus, could delay CM onset and prove beneficial in malaria infection therapy. Phyllanthus niruri is used to treat fever and other symptoms of malaria in Nigeria. Its modes of action as an anti-malarial remedy have not been exhaustively investigated. This study therefore examined the aqueous extract of P. niruri (PE) for its antiplasmodial activity in vitro using the Plasmodium falciparum HB3 strain. Furthermore, in vivo murine malaria model using the Plasmodium berghei ANKA strain was used to investigate its anti-malarial effects. We showed that PE has multiple anti-malarial effects, including anti-parasitic and host immunomodulatory activities. Co-culture of P. falciparum with PE and some of its phytoconstituents drastically reduced parasite number. PE also decreased parasitemia, and increased the survival of infected mice. We also observed that the integrity of the blood-brain barrier was maintained in the PE-treated mice. The results confirmed that PE showed moderate antiplasmodial activity. In vivo murine malaria model using P. berghei ANKA for experimental cerebral malaria revealed that PE suppressed parasite growth, and modulate the production of interferon-gamma. The findings demonstrate that PE affects malaria progression, targeting parasites and host cells.

PYloroplasty versus No Intervention in GAstric REmnant REconstruction after Oesophagectomy: study protocol for the PYNI-GAREREO phase III randomized controlled trial.

BACKGROUND: After esophagectomy for esophageal and esophagogastric cancer, more than half of patients have lost > 10% of their body weight at 12 months. In most cases, the gastric remnant is used for reconstruction after esophagectomy. One of the most serious nutritional complications of this technique is delayed gastric emptying caused by gastric remnant mobilization and denervation of the vagus nerve. The aim of the PYloroplasty versus No Intervention in GAstric REmnant REconstruction after Oesophagectomy (PYNI-GAREREO) trial is to analyze the clinical outcome of modified Horsley pyloroplasty (mH-P) as a method of preventing delayed gastric emptying. METHODS: The PYNI-GAREREO trial is designed as an open randomized, single-center superiority trial. Patients will be randomly allocated to undergo gastric remnant reconstruction with mH-P (intervention group) or no intervention (control group) in parallel groups. All patients with esophageal cancer or esophagogastric cancer planning to undergo curative minimally invasive esophagectomy will be considered for inclusion. A total of 140 patients will be included in the study and randomized between the groups in a 1:1 ratio. The primary outcome is the body weight change at 6 months postoperatively, and the secondary outcomes are the nutritional status, postoperative complications, functional outcome, and quality of life until 1 year postoperatively. DISCUSSION: We hypothesize that mH-P after minimally invasive esophagectomy more effectively maintains patients' nutritional status than no pyloroplasty. TRIAL REGISTRATION: UMIN Clinical Trials Registry UMIN000045104. Registered on 25 August 2021. https://center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000051346 .

A complementary approach for genetic diagnosis of inborn errors of immunity using proteogenomic analysis.

Advances in next-generation sequencing technology have identified many genes responsible for inborn errors of immunity (IEI). However, there is still room for improvement in the efficiency of genetic diagnosis. Recently, RNA sequencing and proteomics using peripheral blood mononuclear cells (PBMCs) have gained attention, but only some studies have integrated these analyses in IEI. Moreover, previous proteomic studies for PBMCs have achieved limited coverage (approximately 3000 proteins). More comprehensive data are needed to gain valuable insights into the molecular mechanisms underlying IEI. Here, we propose a state-of-the-art method for diagnosing IEI using PBMCs proteomics integrated with targeted RNA sequencing (T-RNA-seq), providing unique insights into the pathogenesis of IEI. This study analyzed 70 IEI patients whose genetic etiology had not been identified by genetic analysis. In-depth proteomics identified 6498 proteins, which covered 63% of 527 genes identified in T-RNA-seq, allowing us to examine the molecular cause of IEI and immune cell defects. This integrated analysis identified the disease-causing genes in four cases undiagnosed in previous genetic studies. Three of them could be diagnosed by T-RNA-seq, while the other could only be diagnosed by proteomics. Moreover, this integrated analysis showed high protein-mRNA correlations in B- and T-cell-specific genes, and their expression profiles identified patients with immune cell dysfunction. These results indicate that integrated analysis improves the efficiency of genetic diagnosis and provides a deep understanding of the immune cell dysfunction underlying the etiology of IEI. Our novel approach demonstrates the complementary role of proteogenomic analysis in the genetic diagnosis and characterization of IEI.