Optimizing mesenchymal stem cell extracellular vesicles for chronic wound healing: Bioengineering, standardization, and safety.

Chronic wounds represent a significant global burden, afflicting millions with debilitating complications. Despite standard care, impaired healing persists due to factors like persistent inflammation and impaired tissue regeneration. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) offer an innovative regenerative medicine approach, delivering stem cell-derived therapeutic cargo in engineered nanoscale delivery systems. This review examines pioneering bioengineering strategies to engineer MSC-EVs into precision nanotherapeutics for chronic wounds. Emerging technologies like CRISPR gene editing, microfluidic manufacturing, and biomimetic delivery systems are highlighted for their potential to enhance MSC-EV targeting, optimize therapeutic cargo enrichment, and ensure consistent clinical-grade production. However, key hurdles remain, including batch variability, rigorous safety assessment for potential tumorigenicity, immunogenicity, and biodistribution profiling. Crucially, collaborative frameworks harmonizing regulatory science with bioengineering and patient advocacy hold the key to expediting global clinical translation. By overcoming these challenges, engineered MSC-EVs could catalyze a new era of off-the-shelf regenerative therapies, restoring hope and healing for millions afflicted by non-healing wounds.

Exploring SSEA3 as an emerging biomarker for assessing the regenerative potential of dental pulp-derived stem cells.

Background: Human dental pulp-derived stem cells (hDPSCs) have emerged as a promising source for adult stem cell-based regenerative medicine. Stage-specific embryonic antigen 3 (SSEA3) is a cell surface marker associated with Multilineage-differentiating stress-enduring (Muse) cells, a subpopulation of human bone marrow-derived stem cells (hBMSCs), known for their potent regenerative potential and safety profile. In this study, we investigated the influence of the prolonged culture period and the number of culture passages on the regenerative capacity of hDPSCs and explored the association between SSEA3 expression and their regenerative abilities. Methods: hDPSCs were isolated and cultured for up to 20 passages. Cell proliferation, migration, and osteogenic, adipogenic and neurogenic differentiation potential were assessed at passages 5, 10, and 20. Flow cytometry and immunofluorescence were employed to analyze SSEA3 expression. RNA sequencing (RNA-seq) was performed on SSEA3-positive and SSEA3-negative hDPSCs to identify differentially expressed genes and associated pathways. Results: Our findings demonstrated a progressive decline in hDPSCs proliferation and migration capacity with increasing passage number. Conversely, cell size exhibited a positive correlation with passage number. Early passage hDPSCs displayed superior osteogenic and adipogenic differentiation potential. Notably, SSEA3 expression exhibited a significant negative correlation with passage numbers, reflecting the observed decline in differentiation capacity. RNA-seq analysis revealed distinct transcriptional profiles between SSEA3-positive and SSEA3-negative hDPSCs. SSEA3-positive cells displayed upregulation of genes associated with ectodermal differentiation and downregulation of genes involved in cell adhesion. Conclusions: This study elucidates the impact of passaging on hDPSC behavior and suggests SSEA3 as a valuable biomarker for evaluating stemness and regenerative potential. SSEA3-positive hDPSCs, functionally analogous to Muse cells, represent a promising cell population for developing targeted regenerative therapies with potentially improved clinical outcomes.

Autologous Rib Cartilage Reconstruction After Silicone Implant Removal in a Patient With Bilateral Cleft Lip and Palate: A Case Report.

Cleft lip rhinoplasty (CLR) corrects nasal deformities in cleft lip and palate patients. However, limitations exist in some countries like Japan regarding the use of silicone implants for CLR. While historical reports mention their use since the 1980s, long-term data is lacking. This case report describes a 53-year-old Japanese woman with bilateral cleft lip and palate who received a CLR with a silicone implant over 30 years ago. The implant calcified, causing nasal dorsum skin hardening and thinning, raising concerns of extrusion. To prevent potential extrusion, the implant was removed and replaced with autologous seventh rib cartilage grafts. Various grafting techniques were used for basal support, dorsal augmentation, and nasal tip refinement. The postoperative evaluation showed excellent results with no complications. This case highlights the importance of long-term follow-up after CLR with silicone implants and advocates for autologous rib cartilage as a reliable alternative. Reporting such cases is crucial for informing patient management and research on the long-term safety of silicone implants in CLR.

Metastatic gallbladder cancer presenting as numb chin syndrome: A case report and literature review.

Gallbladder cancer (GBC) is an uncommon malignancy that is highly aggressive in the advanced stages. However, it rarely metastasizes to the mandible. Numb chin syndrome (NCS) is a rare neurological manifestation associated with various underlying causes, including occult primary cancers and distant metastases. It is often considered to be a significant indicator of malignancy, and thorough investigation is essential in the presence of unclear etiology. The current study reported on the case of a 69-year-old Japanese woman who presented with numbness and mild pain in the lower lip and chin area for three months. No other systemic symptoms were observed. Immunocytochemical examination revealed the presence of an adenocarcinoma and TNM staging as per the Union for International Cancer Control and the American Joint Committee on Cancer guidelines confirmed stage IVb GBC. Comprehensive full-body positron emission tomography-computed tomography examination using 18F-fluoro-2-deoxy-D-glucose revealed additional bone and soft-tissue metastases. Palliative chemotherapy and radiation treatment were initiated based on the advanced stage of disease at the time of diagnosis. However, the patient succumbed to multiple organ failure six months later. The simultaneous occurrence of GBC, mandibular metastasis and NCS is rare and associated with poor prognosis. Despite the widespread nature of the disease, it can often manifest as non-specific oral symptoms without any systemic indications. The current study emphasizes the critical importance of timely confirmatory testing for accurate diagnosis and initiation of appropriate management for such complex conditions.

Metastatic colon carcinoma in the maxilla: Highlighting the importance of perioperative oral management: A case report.

Metastatic colorectal carcinoma involving the maxilla is a rare phenomenon, and existing literature regarding the significance of perioperative oral function management (POM) in managing such cases is limited. In the present case report the clinical details of a 58-year-old male referred to the oral and maxillofacial department for POM. The patient had previously undergone segmental bowel resection due to stage IIIb colon cancer. A comprehensive approach encompassing a thorough medical history, meticulous physical examination, radiographic imaging and immunohistopathology was employed, and a definitive diagnosis of metastatic adenocarcinoma in the left maxillary gingiva originating from a colorectal carcinoma was reached. Additionally, concomitant metastases were detected in the lungs and liver. Despite the daunting prognosis associated with the metastases in the oral cavity, the patient's quality of life exhibited discernible improvements owing to the implementation of palliative care interventions. Notably, this interdisciplinary approach facilitated the patient's survival for over a year. The present case report strongly advocates for the prompt integration of POM in the surgical management of cancer patients with oral manifestations, which can optimize both the quality of life and overall survival.

Management of Axillary Contracture in Poland Syndrome: Differentiating Fibrous Band and Skin for Optimal Release.

Poland syndrome (PS), an uncommon congenital unilateral aplasia of chest wall muscles, may exhibit rare accompanying signs, such as axillary webbing or contractures. The existing literature on the specific management of axillary contractures is limited. In this report, we present the case of a 10-year-old girl with PS manifesting an axillary web containing a fibrous band, which was successfully surgically corrected by a double-opposing Z-plasty. Our surgical approach entailed a meticulous distinction between the deep fibrous band and the superficial cutaneous layer, guided by histopathological findings that indicated the presence of tendon-like tissue, ultimately yielding excellent outcomes. This report will help expand knowledge by highlighting the unique manifestation of PS and emphasizing the importance of employing appropriate treatment approaches. Moreover, addressing both tendon and skin components is essential for optimal contracture release in PS.

Exploring stage­specific embryonic antigen 3 involvement in oral cancer progression and as a potential target for taxane­based chemotherapy.

Despite significant advancements in therapeutic approaches, oral neoplasms remain formidable and life­threatening conditions that affect a substantial number of individuals worldwide. Within oral malignancies, a subset of cancer stem cells (CSCs) represent a crucial population responsible for tumor initiation and progression. The identification of reliable markers for the detection and characterization of CSCs in solid tumors, particularly in the context of oral cancers, remains an ongoing challenge. Stage­specific embryonic antigen 3 (SSEA3), previously associated with mesenchymal stem cells and linked to the progression of breast neoplasms and poor prognosis, has yet to be comprehensively elucidated in the context of oral malignancies. The present study aimed to investigate the expression and properties of SSEA3 in 16 distinct subsets of human oral neoplastic cell lines, classified as either CD44 positive (+) or CD44 negative (­). For the first time, SSEA3 was examined as an indicator of tumorigenicity and resistance to taxane­derived chemotherapeutic agents. In the majority of oral neoplastic cell lines analyzed, SSEA3 was expressed in a small population of CD44(+) cells. Significantly, SSEA3(+) cells exhibited heightened proliferative activity and upregulated expression of genes associated with stem cells compared with SSEA3(­) cells. The aforementioned findings suggested that SSEA3 may contribute to the evolution and progression of oral malignancies by fostering tumor growth. Furthermore, SSEA3(+) cells displayed increased sensitivity to taxane­based pharmaceuticals, indicating the potential for SSEA3 to be a viable target in the treatment schema for oral cavity neoplasms. In conclusion, the present study provides novel insight into the role of SSEA3 in the progression and management of oral neoplasms, potentially paving the way for more effective therapeutic approaches.

Management of Rheumatoid Arthritis: Possibilities and Challenges of Mesenchymal Stromal/Stem Cell-Based Therapies.

Rheumatoid arthritis (RA) is a highly prevalent, chronic, and progressive autoimmune disorder primarily affecting joints and muscles. The associated inflammation, pain, and motor restriction negatively impact patient quality of life (QOL) and can even contribute to premature mortality. Further, conventional treatments such as antiinflammatory drugs are only symptomatic. Substantial progress has been made on elucidating the etiopathology of overt RA, in particular the contributions of innate and adaptive immune system dysfunction to chronic inflammation. Although the precise mechanisms underlying onset and progression remain elusive, the discovery of new drug targets, early diagnosis, and new targeted treatments have greatly improved the prognosis and QOL of patients with RA. However, a sizable proportion of patients develop severe adverse effects, exhibit poor responses, or cannot tolerate long-term use of these drugs, necessitating more effective and safer therapeutic alternatives. Mounting preclinical and clinical evidence suggests that the transplantation of multipotent adult stem cells such as mesenchymal stromal/stem cells is a safe and effective treatment strategy for controlling chronic inflammation and promoting tissue regeneration in patients with intractable diseases, including RA. This review describes the current status of MSC-based therapies for RA as well as the opportunities and challenges to broader clinical application.

Exploring olfactory receptor family 7 subfamily C member 1 as a novel oral cancer stem cell target for immunotherapy.

The mortality rate of oral cancer has not improved over the past three decades despite remarkable advances in cancer therapies. Oral cancers contain a subpopulation of cancer stem cells (CSCs) that share characteristics associated with normal stem cells, including self-renewal and multi-differentiation potential. CSCs are tumorigenic, play a critical role in cancer infiltration, recurrence, and distant metastasis, and significantly contribute to drug resistance to current therapeutic strategies, including immunotherapy. Cytotoxic CD8+ T lymphocytes (CTLs) are key immune cells that effectively recognize peptide antigens presented by the major histocompatibility complex class I molecules. Increasing evidence suggests that cancer antigen-specific targeting by CTLs effectively regulates CSCs that drive cancer progression. In this study, we utilized data from public domains and performed various bioassays on human oral squamous cell carcinoma clinical samples and cell lines, including HSC-2 and HSC-3, to investigate the potential role of olfactory receptor family 7 subfamily C member 1 (OR7C1), a seven transmembrane G-protein-coupled olfactory receptor that is also expressed in nonolfactory tissues and was previously reported as a novel marker and target of colon cancer initiating cell-targeted immunotherapy, in CSC-targeted treatment against oral cancer. We found that the OR7C1 gene was expressed only in oral CSCs, and that CTLs reacted with human leukocyte antigen-A24-restricted OR7C1 oral CSC-specific peptides. Taken together, our findings suggest that OR7C1 represents a novel target for potent CSC-targeted immunotherapy in oral cancer.

Management of temporomandibular joint diseases: a rare case report of coexisting calcium pyrophosphate crystal deposition and synovial chondromatosis.

BACKGROUND: The coexistence of calcium pyrophosphate dihydrate crystal deposition (CPP) and synovial chondromatosis (SC) in the temporomandibular joint (TMJ) is rarely reported. CPP disease (CPPD) is complex arthritis synonymous with excessive pyrophosphate production and variable aberrations in mineral and organic phase metabolism of the joint cartilage, leading to local inundated CPP and crystal deposition of partially deciphered predispositions. Meanwhile, SC is a rare benign synovial joint proliferative disease of unclear etiology and has a low risk of malignant transformation. However, SC manifests severe joint disability and dysfunction because of connective tissue metaplasia of the synovial membrane, which forms cartilaginous nodules with or without calcifications or ossifications. These nodules often detach and form intra-articular loose bodies and very rarely within extraarticular spaces. CASE PRESENTATION: We report the case of a 61-year-old man to expand the body of literature on these unusual coexisting arthropathies of the TMJ. The patient presented to our hospital in 2020 with complaints of pain in the right TMJ and trismus for over 6 months. Radiographic assessments of the TMJ provided a preoperative provisional diagnosis of SC. However, the histopathology of the open biopsy revealed tumor-like lesions comprising several deposits of rhomboid and rod-shaped crystals that displayed positive birefringence in polarized light, confirming a coexistence of CPPD. A second-stage operation was performed for the complete removal of the loose bodies and chalk-like lesions including synovectomy. No evidence of recurrence was recorded after a follow-up of nearly 1.5 years. CONCLUSIONS: Isolated CPPD and SC of the TMJ are prevalent in the literature however, monoarticular coexistence of these diseases is rare, due to the lack of consistency in the diagnostic criteria in clinical practice. Moreover, optimal treatment depends on several considerations. This report delineated the molecular etiopathology and underscored the need for continued deciphering of the causal mechanisms of coexisting CPPD and SC of the TMJ. In addition, the importance of confirmatory testing for accurate diagnosis, and appropriate management of these diseases were discussed.

Regenerative medicine strategies for hair growth and regeneration: A narrative review of literature.

Hair loss, or alopecia, is associated with several psychosocial and medical comorbidities, and it remains an economic burden to individuals and the society. Alopecia is attributable to varied mechanisms and features a multifactorial predisposition, and the available conventional medical interventions have several limitations. Thus, several therapeutic strategies for alopecia in regenerative medicine are currently being explored, with increasing evidence suggesting that mesenchymal stem cell (MSC) implantation, MSC-derived secretome treatment, and blood-derived platelet-rich plasma therapies are potential treatment options. In this review, we searched the Cochrane Library, MEDLINE (PubMed), EMBASE, and Scopus using various combinations of terms, such as "stem cell," "alopecia," "hair loss," "Androgenetic alopecia," "male-pattern hair loss," "female-pattern hair loss," "regenerative hair growth," "cell therapy," "mesenchymal stem cells," "MSC-derived extracellular vesicles," "MSC-derived exosomes," and "platelet-rich plasma" and summarized the most promising regenerative treatments for alopecia. Moreover, further opportunities of improving efficacy and innovative strategies for promoting clinical application were discussed.

Current regenerative medicine-based approaches for skin regeneration: A review of literature and a report on clinical applications in Japan.

Current trends indicate a growing interest among healthcare specialists and the public in the use of regenerative medicine-based approaches for skin regeneration. The approaches are categorised in either cell-based or cell-free therapies and are reportedly safe and effective. Cell-based therapies include mesenchymal stem cells (MSCs), tissue induced pluripotent stem cells (iPSCs), fibroblast-based products, and blood-derived therapies, such as those employing platelet-rich plasma (PRP) products. Cell-free therapies primarily involve the use of MSC-derived extracellular vesicles/exosomes. MSCs are isolated from various tissues, such as fat, bone marrow, umbilical cord, menstrual blood, and foetal skin, and expanded ex vivo before transplantation. In cell-free therapies, MSC exosomes, MSC-derived cultured media, and MSC-derived extracellular vesicles are collected from MSC-conditioned media or supernatant. In this review, a literature search of the Cochrane Library, MEDLINE (PubMed), EMBASE, and Scopus was conducted using several combinations of terms, such as 'stem', 'cell', 'aging', 'wrinkles', 'nasolabial folds', 'therapy', 'mesenchymal stem cells', and 'skin', to identify relevant articles providing a comprehensive update on the different regenerative medicine-based therapies and their application to skin regeneration. In addition, the regulatory perspectives on the clinical application of some of these therapies in Japan are highlighted.

An unusual case of oral surgical management in a patient with isovaleric acidemia and schizophrenia: A case report.

Oral/dental surgical care in patients with chronic medical comorbidities, such as isovaleric acidemia (IVA), can be challenging. In addition to technical complications, different comorbidities also present a complex range of concerning factors/challenges, which can increase the incidence of morbidity and mortality associated with surgery. IVA, a congenital error of metabolism, is a rare organic acidemia with a predisposition towards acute acidosis and life-threatening metabolic decompensation during stressful conditions, such as prolonged fasting and surgery. In addition, schizophrenia, a major neurological disorder, can result in manifestation of severe dental or periodontal conditions, including pericoronitis. The condition is associated with significant risk factors of postoperative complications, such as dangerous behaviors and adverse interactions between antipsychotic drugs and anesthetic agents. A case of comorbid dental disease with two coexisting chronic and life-threatening medical conditions, one of which is rare, is an unusual encounter in oral/dental surgery that is seldomly published. Moreover, implementing a safe and effective surgical intervention in such patients requires several informed considerations. However, only a few reported experiences or guidelines exist, reporting appropriate perioperative management strategies to minimize risks. Hence, in this case report, our experience of managing one of these rare encounters of a 20-year-old man who suffered from bilaterally partially erupted third molars, associated with chronic pericoronitis and dental caries of both the maxilla wisdom teeth with coexisting IVA and schizophrenia comorbidities is described. Additionally, the presentation and anticipated complications of the comorbid disorders of the patient are briefly reviewed. In this case, the pericoronitis and dental caries were treated by surgically removing the impacted third molars and the antagonist maxilla wisdom teeth under regional anesthesia and application of antibiotics for 3 days. The patient recovered without any postoperative complications after 1 year of follow-up.

Mixed tumour of the skin of the lower lip: A case report and review of the literature.

Mixed tumour of the skin or chondroid syringoma (CS) is a rare and mostly benign neoplasm of the sweat glands. Although CS is frequently located on varied parts of the head and neck region, the lower lip is a rarely reported site. The present report describes a case of CS of the lower lip in a 58-year-old male as an expository case to further emphasise the need for proper diagnosis, appropriate treatment and prognostic evaluation. The patient presented with a round, non-tender, slightly hard and mobile mass beneath the mucocutaneous junction of his left lateral side of the lower lip. Radiology revealed a mass measuring 11x11x7 mm3 in size at a depth of ~2 mm. Furthermore, magnetic resonance T1- and T2-weighted images showed slightly low and high signal intensities, respectively. A provisional diagnosis of benign tumour of the lower lip was made, and surgical excision biopsy taken under local anaesthesia, while considering the patient's cosmetic appearance. Histopathology demonstrated features akin to apocrine gland, chondroid and myxoid stroma consistent with the diagnosis of benign CS. No evidence of recurrence or satellites were recorded after a follow-up of nearly 2 years. Although rare, a high index of suspicion for CS among other cutaneous adnexal tumours of the lower lip is necessary. In addition, interprofessional collaboration in the management of such oral tumours could enhance patient satisfaction amid prevailing intraoral and aesthetic concerns.

Mucoepidermoid carcinoma of the anterior lingual salivary gland: A rare case report.

Mucoepidermoid carcinomas (MECs) are rare head and neck malignant tumours that were originally considered to be benign. It has been estimated that ~20% of MECs in the major salivary glands, such as the parotid gland, and 50% in the several minor salivary glands found in the oral cavity, are malignant. The diagnosis of MECs is mainly based on ancillary and immunohistochemistry testing. However, owing to the difficulty in harvesting adequate material for histological examination, the histopathological diagnosis of intraoral MECs may be particularly challenging. We herein report a rare case of an 82-year-old patient who presented to the Department of Oral and Maxillofacial Surgery of Ryukyu University Hospital with complaints of a progressive swelling and pain in the ventral surface of the apex of the tongue. The patient had previously undergone needle biopsy and the histopathological analysis of the tumour suggested a diagnosis of irritation fibroma. To ensure a more accurate histopathological assessment, an incisional biopsy was performed, in addition to the haematological and radiological assessments. Examination of the obtained surgical specimen confirmed low-grade MEC of the anterior lingual gland. The tumour was surgically excised, the patient healed uneventfully and no recurrence was detected on the regular 3-year follow-up. Although MECs are relatively more common in the minor salivary glands of the oral cavity, they are a rare occurrence in the anterior lingual gland. Therefore, adequate histological material should be surgically harvested to perform a complete evaluation of the morphology and cytology of the tumour and ensure the accuracy of diagnosis.

African-specific polymorphisms in Plasmodium falciparum serine repeat antigen 5 in Uganda and Burkina Faso clinical samples do not interfere with antibody response to BK-SE36 vaccination.

BK-SE36, based on Plasmodium falciparum serine repeat antigen 5 (SERA5), is a blood-stage malaria vaccine candidate currently being evaluated in clinical trials. Phase 1 trials in Uganda and Burkina Faso have demonstrated promising safety and immunogenicity profiles. However, the genetic diversity of sera5 in Africa and the role of allele/variant-specific immunity remain a major concern. Here, sequence analyses were done on 226 strains collected from the two clinical trial/follow-up studies and 88 strains from two cross-sectional studies in Africa. Compared to other highly polymorphic vaccine candidate antigens, polymorphisms in sera5 were largely confined to the repeat regions of the gene. Results also confirmed a SERA5 consensus sequence with African-specific polymorphisms. Mismatches with the vaccine-type SE36 (BK-SE36) in the octamer repeat, serine repeat, and flanking regions, and single-nucleotide polymorphisms in non-repeat regions could compromise vaccine response and efficacy. However, the haplotype diversity of SERA5 was similar between vaccinated and control participants. There was no marked bias or difference in the patterns of distribution of the SE36 haplotype and no statistically significant genetic differentiation among parasites infecting BK-SE36 vaccinees and controls. Results indicate that BK-SE36 does not elicit an allele-specific immune response.

Effects of hydroxyapatite-coated nonwoven polyethylene/polypropylene fabric on non-mesodermal lineage-specific differentiation of human adipose-derived stem cells.

OBJECTIVE: Compared to other stem cells, the multipotency of human adipose-derived mesenchymal stem cells (ASCs) is limited. Effective approaches that trigger or enhance lineage-specific transdifferentiation are highly envisaged in the improvement of ASCs-based cell therapies. Using Immunofluorescence assays and the secretion of cardiac troponin T (cTnT) protein, we studied the impact of two substrates: Hydroxyapatite (HAp)-coated nonwoven polyethylene (PET)/polypropylene (PP) fabric and glass surfaces, representing 3 dimensional (D) and 2 D environments respectively, on the induction of cardiomyocytes - a non-mesodermal cell type from ASCs for 1-5 weeks. RESULTS: ASCs were successfully isolated from human adipose tissue under cGMP conditions. Within 1-3 weeks, expression of cTnT in the induced 3D cultures was overall significantly higher (P < 0.021) than that in the induced 2D cultures or controls (P < 0.0009). Remarkably, after 3 weeks of culture, cTnT secretion in the induced 3D cultures gradually declined, nearly reaching levels observed in the 2D cultures. The results show that HAp-coated nonwoven PE/PP fabric could enhance lineage-specific differentiation of ASCs toward cardiac-like cells. However, the fabric might suppress growth of the transformed cells. These preliminary findings encourage further interest in validating the fabric's potential in improving ASCs transdifferentiation.

Global Repertoire of Human Antibodies Against Plasmodium falciparum RIFINs, SURFINs, and STEVORs in a Malaria Exposed Population.

Clinical immunity to malaria develops after repeated exposure to Plasmodium falciparum parasites. Broadly reactive antibodies against parasite antigens expressed on the surface of infected erythrocytes (variable surface antigens; VSAs) are candidates for anti-malaria therapeutics and vaccines. Among the VSAs, several RIFIN, STEVOR, and SURFIN family members have been demonstrated to be targets of naturally acquired immunity against malaria. For example, RIFIN family members are important ligands for opsonization of P. falciparum infected erythrocytes with specific immunoglobulins (IgG) acquiring broad protective reactivity. However, the global repertoire of human anti-VSAs IgG, its variation in children, and the key protective targets remain poorly understood. Here, we report wheat germ cell-free system-based production and serological profiling of a comprehensive library of A-RIFINs, B-RIFINs, STEVORs, and SURFINs derived from the P. falciparum 3D7 parasite strain. We observed that >98% of assayed proteins (n = 265) were immunogenic in malaria-exposed individuals in Uganda. The overall breadth of immune responses was significantly correlated with age but not with clinical malaria outcome among the study volunteers. However, children with high levels of antibodies to four RIFINs (PF3D7_0201000, PF3D7_1254500, PF3D7_1040600, PF3D7_1041100), STEVOR (PF3D7_0732000), and SURFIN 1.2 (PF3D7_0113600) had prospectively reduced the risk of developing febrile malaria, suggesting that the 5 antigens are important targets of protective immunity. Further studies on the significance of repeated exposure to malaria infection and maintenance of such high-level antibodies would contribute to a better understanding of susceptibility and naturally acquired immunity to malaria.

Antibodies against a short region of PfRipr inhibit Plasmodium falciparum merozoite invasion and PfRipr interaction with Rh5 and SEMA7A.

Plasmodium falciparum merozoite invasion into erythrocytes is an essential step of the blood-stage cycle, survival of parasites, and malaria pathogenesis. P. falciparum merozoite Rh5 interacting protein (PfRipr) forms a complex with Rh5 and CyRPA in sequential molecular events leading to erythrocyte invasion. Recently we described PfRipr as a conserved protein that induces strain-transcending growth inhibitory antibodies in in vitro assays. However, being a large and complex protein of 1086 amino acids (aa) with 87 cysteine residues, PfRipr is difficult to express in conventional expression systems towards vaccine development. In this study we sought to identify the most potent region of PfRipr that could be developed to overcome difficulties related to protein expression, as well as to elucidate the invasion inhibitory mechanism of anti-PfRipr antibodies. Using the wheat germ cell-free system, Ecto- PfRipr and truncates of approximately 200 aa were expressed as soluble proteins. We demonstrate that antibodies against PfRipr truncate 5 (PfRipr_5: C720-D934), a region within the PfRipr C-terminal EGF-like domains, potently inhibit merozoite invasion. Furthermore, the antibodies strongly block PfRipr/Rh5 interaction, as well as that between PfRipr and its erythrocyte-surface receptor, SEMA7A. Taken together, PfRipr_5 is a potential candidate for further development as a blood-stage malaria vaccine.

Recovery and stable persistence of chloroquine sensitivity in Plasmodium falciparum parasites after its discontinued use in Northern Uganda.

BACKGROUND: Usage of chloroquine was discontinued from the treatment of Plasmodium falciparum infection in almost all endemic regions because of global spread of resistant parasites. Since the first report in Malawi, numerous epidemiological studies have demonstrated that the discontinuance led to re-emergence of chloroquine-susceptible P. falciparum, suggesting a possible role in future malaria control. However, most studies were cross-sectional, with few studies looking at the persistence of chloroquine recovery in long term. This study fills the gap by providing, for a period of at least 6 years, proof of persistent re-emergence/stable recovery of susceptible parasite populations using both molecular and phenotypic methods. METHODS: Ex vivo drug-susceptibility assays to chloroquine (n = 319) and lumefantrine (n = 335) were performed from 2013 to 2018 in Gulu, Northern Uganda, where chloroquine had been removed from the official malaria treatment regimen since 2006. Genotyping of pfcrt and pfmdr1 was also performed. RESULTS: Chloroquine resistance (≥ 100 nM) was observed in only 3 (1.3%) samples. Average IC50 values for chloroquine were persistently low throughout the study period (17.4-24.9 nM). Parasites harbouring pfcrt K76 alleles showed significantly lower IC50s to chloroquine than the parasites harbouring K76T alleles (21.4 nM vs. 43.1 nM, p-value = 3.9 × 10-8). Prevalence of K76 alleles gradually increased from 71% in 2013 to 100% in 2018. CONCLUSION: This study found evidence of stable persistence of chloroquine susceptibility with the fixation of pfcrt K76 in Northern Uganda after discontinuation of chloroquine in the region. Accumulation of similar evidence in other endemic areas in Uganda could open channels for possible future re-use of chloroquine as an option for malaria treatment or prevention.