Coagulopathy associated with COVID-19 - Perspectives & Preventive strategies using a biological response modifier Glucan.

Direct endothelial injury by viruses and dysregulation of clotting mechanisms due to cytokine storm are the major precipitating factors of mortality in COVID-19; both are attributed to a fundamental dysregulation of the immune system. While immune dysregulation can be attributed to several factors, the risk of associated thrombogenic disruption varies across individuals. This variation depends on several factors, such as comorbidities, including diabetes, hypertension, and cardiovascular diseases. When considering ethnic variations, the vulnerability of Caucasians, African Americans and Hispanics needs to be addressed before arriving at strategies to handle thromboembolic complications, which have been identified in recent reports as the leading causes of mortality in COVID-19. Although evaluation of D-dimer and prothrombin during admission is considered to predict prognosis and mortality, there are no preventive or prophylactic strategies before hospital admission. Herein, we present our perspectives on the effect of regular supplementation with the biological response modifier beta glucan based on its relevance to immune modulation. This effect is of paramount importance in decreasing the development of severe COVID-19 and reducing mortality against the background of coagulopathy, especially in vulnerable populations.

CD93 is a cell surface lectin receptor involved in the control of the inflammatory response stimulated by exogenous DNA.

Bacterial DNA contains CpG oligonucleotide (ODN) motifs to trigger innate immune responses through the endosomal receptor Toll-like receptor 9 (TLR9). One of the cell surface receptors to capture and deliver microbial DNA to intracellular TLR9 is the C-type lectin molecule DEC-205 through its N-terminal C-type lectin-like domain (CTLD). CD93 is a cell surface protein and member of the lectin group XIV with a CTLD. We hypothesized that CD93 could interact with CpG motifs, and possibly serve as a novel receptor to deliver bacterial DNA to endosomal TLR9. Using ELISA and tryptophan fluorescence binding studies we observed that the soluble histidine-tagged CD93-CTLD was specifically binding to CpG ODN and bacterial DNA. Moreover, we found that CpG ODN could bind to CD93-expressing IMR32 neuroblastoma cells and induced more robust interleukin-6 secretion when compared with mock-transfected IMR32 control cells. Our data argue for a possible contribution of CD93 to control cell responsiveness to bacterial DNA in a manner reminiscent of DEC-205. We postulate that CD93 may act as a receptor at plasma membrane for DNA or CpG ODN and to grant delivery to endosomal TLR9.

Unique properties of cluster of differentiation 93 in the umbilical cord blood of neonates.

It has previously been reported by these authors that cluster of differentiation (CD) 93 is co-expressed on naive T-lymphocytes (CD4(+) CD45RA(+) cells) in neonatal umbilical cord blood cells (UCBCs) but not on normal adult peripheral blood cells (PBCs). In this study, expression of CD93 on other lymphocyte subsets and the concentration of soluble formed CD93 (sCD93) in serum or culture supernatants from neonatal umbilical cord blood (UCB) was examined. It was found that CD93 is also co-expressed on CD2(+) , CD16(+) , CD56(+) or CD25(+) cells in the lymphocyte population of neonatal UCBCs, but not on normal adult PBCs. The concentrations of sCD93 in serum and culture supernatants from neonatal UCB were significantly greater than those from normal adult peripheral blood. The concentrations of sCD93 in culture supernatants from neonatal UCBCs and normal adult PBCs treated with phorbol 12-myristate 13-acetate (PMA) were significantly enhanced compared with those without PMA treatment. The degree of enhancement of sCD93 by PMA in culture supernatants from neonatal UCBCs was significantly greater than that of normal adult PBCs and enhancement of sCD93 by PMA in the culture supernatants from neonatal UCBCs and normal adult PBCs was significantly suppressed by PKC inhibitor. Interestingly, the high concentration of serum sCD93 in neonates was significantly decreased in sera from infants at 1 month after birth. Expression of CD93 on the lymphocyte population of PBCs from infants at 1 month after birth was also significantly decreased, compared with that for neonatal UCBCs. These findings indicate that CD93 in neonatal UCB has unique properties as an immunological biomarker.

Antibody dependent disease enhancement (ADE) after COVID-19 vaccination and beta glucans as a safer strategy in management.

A potential risk associated with vaccines for COVID-19 is antibody-dependent disease enhancement (ADE) in which vaccine induced antibody mediated immune responses may lead to enhanced SARS CoV- 2 acquisition or increased disease severity. Though ADE has not been clinically demonstrated with any of the COVID-19 vaccines so far, when neutralizing antibodies are suboptimal, the severity of COVID-19 has been reported to be greater. ADE is presumed to occur via abnormal macrophages induced by the vaccine based immune response by antibody-mediated virus uptake into Fc gamma receptor IIa (FcγRIIa) or by the formation of Fc-mediated excessive antibody effector functions. Beta-glucans which are naturally occurring polysaccharides known for unique immunomodulation by capability to interact with macrophages, eliciting a specific beneficial immune-response and enhancing all arms of the immune system, importantly without over-activation are suggested as safer nutritional supplement-based vaccine adjuvants for COVID-19.

Benefits of Gut Microbiota Reconstitution by Beta 1,3-1,6 Glucans in Subjects with Autism Spectrum Disorder and Other Neurodegenerative Diseases.

BACKGROUND: Aureobasidium pullulans (black yeast) AFO-202 strain-produced beta glucan, Nichi Glucan, has been shown to improve the behavior and sleep pattern along with an increase in α-synuclein and melatonin in children with autism spectrum disorder (ASD). OBJECTIVE: In this randomized pilot clinical study, we have evaluated the gut microbiota of subjects with ASD after consumption of Nichi Glucan. METHODS: Eighteen subjects with ASD were randomly allocated: six subjects in the control group (Group 1): conventional treatment comprising remedial behavioral therapies and L-carnosine 500 mg per day, and 12 subjects (Group 2) underwent supplementation with Nichi Glucan 0.5 g twice daily along with the conventional treatment for 90 days. RESULTS: Whole genome metagenome (WGM) sequencing of the stool samples at baseline and after intervention showed that among genera of relevance, the abundance of Enterobacteriaceae was decreased almost to zero in Group 2 after intervention, whereas it increased from 0.36% to 0.85% in Group 1. The abundance of Bacteroides increased in Group 1, whereas it decreased in Group 2. The abundance of Prevotella increased while the abundance of Lactobacillus decreased in both Group 1 and Group 2. Among species, a decrease was seen in Escherichia coli, Akkermansia muciniphila CAG:154, Blautia spp., Coprobacillus sp., and Clostridium bolteae CAG:59, with an increase of Faecalibacterium prausnitzii and Prevotella copri, which are both beneficial. CONCLUSION: AFO-202 beta 1,3-1,6 glucan, in addition to balancing the gut microbiome in children with ASD and its role in effective control of curli-producing Enterobacteriaceae that leads to α-synuclein misfolding and accumulation, may have a prophylactic role in Parkinson's and Alzheimer's diseases as well.

Beneficial effects of 1,3-1,6 ß-glucans produced by Aureobasidium pullulans on non-esterified fatty acid levels in diabetic KKAy mice and their potential implications in metabolic dysregulation.

Objectives: In this study, we used an obese and diabetic mouse model to compare two strains of Aureobasidium pullulans (AFO-202 and N-163) produced beta-glucans (ß-glucans), which alleviate lipotoxicity. Methods: Four groups of KK-Ay mice were used, with six subjects in each group. Group 1: sacrificed on day 0 for baseline values; Group 2: control (drinking water); Group 3: AFO-202 beta glucan-200 mg/kg/day; Group 4: N-163 beta glucan-300 mg/kg/day for 28 consecutive days. Results: Group 4 (N-163) had the lowest non-esterified fatty acids (NEFA) levels and marginally decreased triglyceride levels compared to the other groups. There were no significant differences in blood glucose, hemoglobin A1c (HbA1c), triglycerides, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol levels. N-163 ß-glucans decreased NEFA levels after 28 days. Conclusion: These results, although modest, warrant further in-depth research into lipotoxicity and associated inflammatory cascades in both healthy and diseased subjects for the prevention and management of metabolic dysregulation and associated diseases such as non-alcoholic fatty liver disease (NAFLD).

Beneficial immune-modulatory effects of the N-163 strain of Aureobasidium pullulans-produced 1,3-1,6 Beta glucans in Duchenne muscular dystrophy: Results of an open-label, prospective, exploratory case-control clinical study.

Background: This exploratory case-control study is to evaluate the effects of supplementation of Aureobasidium pullulans-N-163 strain produced 1,3-1,- 6 beta glucan in young patients with Duchenne muscular dystrophy (DMD). Methods: Twenty-seven male subjects aged 5-19 years with DMD were included, nine in the control arm and 18 in the treatment arm to receive N-163 beta glucan along with conventional therapies for 45 days. While performing the analysis, steroid usage was also taken into consideration, those not administered steroids (Steroid -ve) (Control, n = 5; treatment, n = 9), those administered steroids (Steroid +ve) (Control, n = 4; treatment, n = 9). Results: IL-6 showed a significant decrease in the treatment groups, especially the N-163 Steroid -ve group. IL-13 decreased in both treatment groups and TGF-ß levels showed a significant decrease in the treatment groups, especially the N-163 Steroid -ve group, (p < 0.05). Dystrophin levels increased by up to 32% in the treatment groups compared to the control. Medical research council (MRC) grading showed slight improvement in muscle strength improvement in 12 out of 18 patients (67%) in the treatment group and four out of nine (44%) subjects in the control group. Conclusion: Supplementation with the N-163 beta glucan food supplement produced beneficial effects: a significant decrease in inflammation and fibrosis markers, increase in serum dystrophin and slight improvement in muscle strength in DMD subjects over 45 days, thus making this a potential adjunct treatment for DMD after validation. Trial registration: The study was registered in Clinical trials registry of India, CTRI/2021/05/033346. Registered on 5th May, 2021.

Controlled modulation of all the arms of the immunity including innate immunity by biological response modifier glucans, a simple yet potential nutritional supplement strategy to fight COVID-19.

Immune modulation, being one of the potential strategies to combat COVID-19 infection, emphasis has been laid on enhancing the innate immune response in a balanced manner. Beta (ß)-glucans have been suggested as nonspecific immunostimulatory adjuvants to beneficially boost protective antiviral immunity. Through this article, we wish to emphasize that ß-glucans not only enhance the innate immunity but also possess the capability to modulate all the arms of the immunity viz., innate, adaptive, TRIM at different sites including those postulated to be the entry site of the SARS-CoV2. Other than immune modulation capabilities, the beneficial metabolic- and coagulation-related effects of ß-glucans, a simple nutritional supplementation strategy, make them be considered for larger clinical studies to validate their prophylactic vaccine adjuvant and nutritional-based therapeutic supplement activities to effectively fight the COVID-19 pandemic. PRACTICAL APPLICATIONS: A 360° wholesome protection from viral infections is possible only when all the arms of the immune system function in a balanced and effective manner which is especially important in COVID-19. Nutritional supplementation using biological response modifier beta (ß)-glucans (BRMGs) is worth considering for large-scale clinical studies based on their track record of safety and their beneficial regulation of all the arms of the immune system.

Integrating the Synergy of the Gut Microbiome into Regenerative Medicine: Relevance to Neurological Disorders.

A new paradigm of cell therapy-based approaches as a solution to several diseases caused by damage or loss of cells/tissues leading to organ failure heralded the birth of a new branch in medicine called regenerative medicine (RM), which was further fueled by in vitro cell expansion and tissue engineering (TE) technologies, including the ability to grow embryonic stem cells, induce pluripotent stem cells, and so on. RM addresses organ failure by repair, regeneration, or restoration, rejuvenation using cells, stem cells, or progenitor cells as tools having added cell-derived products also as a tool, and extracellular matrix component-based support, either direct or indirect (e.g., matrix induced autologous chondrocyte implantation) using scaffolds. Now, the main objective of RM is to solve the functional loss of cells that have evolved from cells as tools to cell-derived factors and scaffolds per se as tools. In this context, an important yet indispensable group of cells that constitute the major portion of the human body in terms of the number of cells having several essential roles to play, both directly and indirectly, starting from digestion and the immune system to the growing evidence of influencing neuronal function, aging, and carcinogenesis has been ignored. We would like to focus on these in this review as they should essentially be considered as a tool of RM, especially for neurological disorders for their vital role. What we are indicating is the second genome or the gut microbiome.

Improvement of behavioural pattern and alpha-synuclein levels in autism spectrum disorder after consumption of a beta-glucan food supplement in a randomised, parallel-group pilot clinical study.

BACKGROUND: Autism spectrum disorders (ASDs) are a wide range of behavioural disabilities for which there are no definite interventional modalities available. Remedial therapies remain the only option but with varying outcomes. We have evaluated the Childhood Autism Rating Scale (CARS) and alpha-synuclein levels in this parallel-group, multiple-arm pilot clinical study after supplementation with a biological response modifier beta-glucan food supplement (Nichi Glucan). METHODS: Six subjects with ASD (n=6) Gr. 1 underwent conventional treatment comprising remedial behavioural therapies and L-carnosine 500 mg per day, and 12 subjects (n=12) Gr. 2 underwent supplementation with the Nichi Glucan 0.5 g two times per day along with the conventional treatment. RESULTS: There was a significant decrease in the CARS score in all of the children of the Nichi Glucan Gr.2 compared with the control (p=0.034517). Plasma levels of alpha-synuclein were significantly higher in Gr. 2 (Nichi Glucan) than in the control group Gr. 1 (p=0.091701). CONCLUSION: Improvement of the behavioural pattern CARS score and a correlating alpha-synuclein level, followed by a safe beta-glucan food supplement, warrants further research on other parameters, such as gut-microbiota evaluation, and relevant neuronal biomarkers which is likely to cast light on novel solutions.

Improvement of sleep and melatonin in children with autism spectrum disorder after ß-1,3/1,6-glucan consumption: An open-label prospective pilot clinical study.

INTRODUCTION: Poor sleep quality is a major problem in patients with autism spectrum disorder (ASD), and is attributed to low melatonin levels. Melatonin supplementation is recommended; however, its effectiveness varies. ß-Glucans have previously been shown to improve melatonin levels in animal studies. Herein, we examined the effectiveness of Aureobasidium pullulans (Nichi Glucan), a species of black yeast that contains beta-1,3/1,6-glucan, in a pilot study of children with ASD. METHODS: Thirteen children (age, 2.5-13 years) with ASD were recruited for the study. The control group consisted of four patients (Gr. 1), while nine patients were classified into the treatment group (Gr. 2). Gr. 2 received 1 g of Nichi Glucan along with conventional therapy, whereas the Gr. 1 (control) patients received conventional therapy alone for 90 days. Serum melatonin levels and sleep patterns, assessed using a subjective questionnaire, were evaluated before and after treatment. RESULTS: In Gr. 2, the average serum melatonin level increased from 238.85 ng/L preintervention to 394.72 ng/L postintervention. Eight of nine participants (88%) in Gr. 2 showed improvements in sleep pattern and quality, while no improvement was observed in the participants in Gr. 1. CONCLUSION: The consumption of Nichi Glucan for 90 days resulted in visible improvement in sleep quality, sleep pattern, and serum melatonin levels, which was reported for the first time by our study. A larger multicenter study is required to validate our findings.

Hepatoprotective Effects of Aureobasidium pullulans Derived ß 1,3-1,6 Glucans in a Murine Model of Non-alcoholic Steatohepatitis.

Background: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are highly prevalent conditions characterized by inflammation and fibrosis of the liver, which can progress to cirrhosis and hepatocellular carcinoma if left untreated. Conventional modalities are mainly symptomatic, with no definite solution. Beta-glucan-based biological response modifiers are a potential strategy in lieu of their beneficial metabolic effects. Aureobasidium pullulans strains AFO-202 and N-163 beta-glucans were evaluated for anti-fibrotic and anti-inflammatory hepatoprotective potentials in a NASH animal model in this study. Methods: In the STAM™ murine model of NASH, five groups were studied for 8 weeks: (1) vehicle (RO water), (2) AFO-202 beta-glucan; (3) N-163 beta-glucan, (4) AFO-202+N-163 beta-glucan, and (5) telmisartan (standard pharmacological intervention). Evaluation of biochemical parameters in plasma and hepatic histology including Sirius red staining and F4/80 immunostaining were performed. Results: AFO-202 beta-glucan significantly decreased inflammation-associated hepatic cell ballooning and steatosis. N-163 beta-glucan decreased fibrosis and inflammation significantly (P value < 0.05). The combination of AFO-202 with N-163 significantly decreased the NAFLD Activity Score (NAS) compared with other groups. Conclusion: This preclinical study supports the potential of N-163 and AFO-202 beta-glucans alone or in combination as potential preventive and therapeutic agent(s), for NASH.

Beneficial effects of novel aureobasidium pullulans strains produced beta-1,3-1,6 glucans on interleukin-6 and D-dimer levels in COVID-19 patients; results of a randomized multiple-arm pilot clinical study.

OBJECTIVE: In this pilot clinical study, we report the beneficial effects of beta glucans derived from two strains AFO-202 and N-163 of a black yeast Aureobasidium pullulans on the biomarkers for cytokine storm and coagulopathy in COVID-19 patients. METHODS: A total of 24 RT-PCR positive COVID-19 patients were recruited and randomly divided into three groups (Gr): Gr. 1 control (n = 8) - Standard treatment; Gr. 2: Standard treatment + AFO-202 beta glucan (n = 8); and Gr. 3, Standard treatment + combination of AFO-202 and N-163 beta glucans (n = 8) for 30 days. RESULTS: There was no mortality or requirement of ventilation of the subjects in any of the groups. There was a decrease in D-Dimer values (751 ng/ml to 143.89 ng/ml) and IL-6 values (7.395-3.16 pg/ml) in Gr. 1 in 15 days but the levels increased to abnormal levels on day 30 (D-Dimer: 202.5 ng/ml; IL-6 55.37 pg/ml); which steadily decreased up to day 30 in groups 2 (D-dimer: 560.99 ng/dl to 79.615; IL-6: 26.18-3.41 pg/ml) and 3 (D-dimer: 1614 ng/dl to 164.25 ng/dl; IL-6: 6.25-0.5 pg/ml). The same trend was observed with ESR. LCR and LeCR increased while NLR decreased significantly in Gr. 3. CD4 + and CD8 + T cell count showed relatively higher increase in Gr.3. There was no difference in CRP within the groups. CONCLUSION: As these beta glucans are well known food supplements with a track record for safety, larger multi-centric clinical studies are recommended to validate their use as an adjunct in the management of COVID-19 and the ensuing long COVID-19 syndrome.

ß­glucan vaccine adjuvant approach for cancer treatment through immune enhancement (B­VACCIEN) in specific immunocompromised populations (Review).

The incidence of cancer, which is the second leading cause of mortality globally, continues to increase, although continued efforts are being made to identify effective treatments with fewer side­effects. Previous studies have reported that chronic microinflammation, which occurs in diseases, including diabetes, along with weakened immune systems, may ultimately lead to cancer development. Chemotherapy, radiotherapy and surgery are the mainstream approaches to treatment; however, they all lead to immune system weakness, which in turn increases the metastatic spread. The aim of the present review was to provide evidence of a biological response modifier ß­glucan [ß­glucan vaccine adjuvant approach to treating cancer via immune enhancement (B­VACCIEN)] and its beneficial effects, including vaccine­adjuvant potential, balancing metabolic parameters (including blood glucose and lipid levels), increasing peripheral blood cell cytotoxicity against cancer and alleviating chemotherapy side effects in animal models. This suggests its value as a potential strategy to provide long­term prophylaxis in immunocompromised individuals or genetically prone to cancer.

Two unique biological response-modifier glucans beneficially regulating gut microbiota and faecal metabolome in a non-alcoholic steatohepatitis animal model, with potential applications in human health and disease.

OBJECTIVE: The gut microbiome and its metabolites are influenced by age and stress and reflect the metabolism and health of the immune system. We assessed the gut microbiota and faecal metabolome in a static animal model of non-alcoholic steatohepatitis (NASH). DESIGN: This model was subjected to the following treatments: reverse osmosis water, AFO-202, N-163, AFO-202+N-163 and telmisartan treatment. Faecal samples were collected at 6 and 9 weeks of age. The gut microbiome was analysed using 16S ribosomal RNA sequences acquired by next-generation sequencing, and the faecal metabolome was analysed using gas chromatography-mass spectrometry. RESULTS: Gut microbial diversity increased greatly in the AFO-202+N-163 group. Postintervention, the abundance of Firmicutes decreased, whereas that of Bacteroides increased and was the highest in the AFO-202+N-163 group. The decrease in the abundance of Enterobacteriaceae and other Firmicutes and the abundance of Turicibacter and Bilophila were the highest in the AFO-202 and N-163 groups, respectively. Lactobacillus abundance was highest in the AFO-202+N-163 group. The faecal metabolite spermidine, which is beneficial against inflammation and NASH, was significantly decreased (p=0.012) in the N-163 group. Succinic acid, which is beneficial in neurodevelopmental and neurodegenerative diseases, was increased in the AFO-202 group (p=0.06). The decrease in fructose was the highest in the N-163 group (p=0.0007). Isoleucine and Leucine decreased with statistical significance (p=0.004 and 0.012, respectively), and tryptophan also decreased (p=0.99), whereas ornithine, which is beneficial against chronic immune-metabolic-inflammatory pathologies, increased in the AFO-202+N-163 group. CONCLUSION: AFO-202 treatment in mice is beneficial against neurodevelopmental and neurodegenerative diseases, and has prophylactic potential against metabolic conditions. N-163 treatment exerts anti-inflammatory effects against organ fibrosis and neuroinflammation. In combination, these compounds exhibit anticancer activity.

Beneficial Immune Regulation by Biological Response Modifier Glucans in COVID-19 and Their Envisaged Potentials in the Management of Sepsis.

Sepsis is a life-threatening condition caused by an abnormal immune response induced by infection with no approved or specific therapeutic options. We present our perspectives for the therapeutic management of sepsis through a four-way approach: (1) infection control through immune enhancement; (2) immune suppression during the initial hyper-inflammatory phase; (3) balanced immune-modulation to counter the later immune-paralysis phase; and (4) advantageous effects on metabolic and coagulation parameters throughout. COVID-19 is a virus-triggered, accelerated sepsis-like reaction that is associated with the rapid progress of an inflammatory cascade involving a cytokine storm and multiorgan failure. Here, we discuss the potential of the biological response modifiers, ß-glucans (BRMGs), in the management of sepsis based on their beneficial effects on inflammatory-immune events in COVID-19 clinical studies. In COVID-19 patients, apart from metabolic regulation, BRMGs, derived from a black yeast, Aureobasidium pullulans strain AFO-202, have been reported to stimulate immune responses. BRMGs, produced by another strain (N-163) of A. pullulans, have been implicated in the beneficial regulation of inflammatory markers and immunity, namely IL-6, C-reactive protein (CRP), D-Dimer, ferritin, neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-C-reactive protein ratio (LCR), leucocyte-to-C-reactive protein ratio (LeCR), and leukocyte-to-IL-6 ratio (LeIR). Agents such as these ß-glucans, which are safe as they have been widely consumed by humans for decades, have potential as adjuncts for the prevention and management of sepsis as they exert their beneficial effects across the spectrum of processes and factors involved in sepsis pathology, including, but not limited to, metabolism, infection, inflammation, immune modulation, immune enhancement, and gut microbiota.

ß-glucans: wide-spectrum immune-balancing food-supplement-based enteric (ß-WIFE) vaccine adjuvant approach to COVID-19.

Conventional vaccines to combat COVID-19 through different approaches are at various stages of development. The complexity of COVID-19 such as the potential mutations of the virus leading to antigenic drift and the uncertainty on the duration of the immunity induced by the vaccine have hampered the efforts to control the COVID-19 pandemic. Thus, we suggest an alternative interim treatment strategy based on biological response modifier glucans such as the Aureobasidium pullulans AFO-202-derived ß-glucan, which has been reported to induce trained immunity, akin to that induced by the Bacille Calmette-Guérin vaccine, by epigenetic modifications at the central level in the bone marrow. These ß-glucans act as pathogen-associated molecular patterns, activating mucosal immunity by binding with specific pathogen recognition receptors such as dectin-1 and inducing both the adaptive and innate immunity by reaching distant lymphoid organs. ß-Glucans have also been used as immune adjuvants for vaccines such as the influenza vaccine. Therefore, until a conventional vaccine is widely available, an orally consumable vaccine adjuvant that acts like biosimilars, termed as the wide-spectrum immune-balancing food-supplement-based enteric (ß-WIFE) vaccine adjuvant approach, with well-reported safety is worth in-depth investigation and can be considered for a clinical trial.

Flow cytometric identification of CD93 expression on naive T lymphocytes (CD4(+)CD45RA (+) cells) in human neonatal umbilical cord blood.

Human CD93 has a molecular weight of about 100 kDa and is selectively expressed by myeloid cell lineages in peripheral blood (PB) mononuclear cells. Although CD93 was initially identified as a receptor for complement component 1, subcomponent q phagocytosis (C1qRp) involved in the C1q-mediated enhancement of the phagocytosis of various antigens, several recent studies have reported that CD93 is not a receptor for the C1q-mediated enhancement of phagocytosis. The expression patterns of CD93 have been previously investigated in PB mononuclear cells (lymphocytes, monocytes, and granulocytes) from adult PB and neonatal umbilical cord blood (UCB), and the expression of CD93 was not found on lymphocytes from either normal adult PB or neonatal UCB. However, the detection of CD93 expression in neonatal UCB using CD93 monoclonal antibodies (mAbs) that recognize different antigenic epitopes remains poorly understood. In this study, we examined the expression of CD93 on lymphocytes, monocytes, and granulocytes from neonatal UCB using four different types of CD93 mAb detection probes, mNI-11, R139, R3, and X-2, using flow cytometric and western blot analyses. We found that CD93, as defined using all four mAbs, was expressed on monocytes and granulocytes in PB mononuclear cells from adult PB and neonatal UCB. On the other hand, we observed for the first time that the expression of CD93 on lymphocytes in neonatal UCB can only be detected using the mNI-11 mAb, established in our laboratory, and not with commercially available CD93 mAbs (R139, R3, and X-2). However, CD93 expression on lymphocytes from normal adults was not detected using any of the four CD93 mAbs. Two-color flow cytometric analyses showed that the CD93 recognized by mNI-11 mAb was expressed on CD3(+) T lymphocytes (mainly CD4(+) helper T lymphocytes), but not on CD19(+) B lymphocytes or on CD8(+) suppressor/cytotoxic T lymphocytes from neonatal UCB. In addition, CD93 was expressed on CD45RA(+) (naive antigen) lymphocytes from neonatal UCB, but not on CD45RO(+) (memory antigen) lymphocytes from neonatal UCB or on CD45RA(+) and CD45RO(+) lymphocytes from normal adult PB. Three-color flow cytometric analysis showed that CD93 was co-expressed on naive T lymphocytes (CD4(+)CD45RA(+)) from neonatal UCB. In a western blot analysis, the CD93 mAb (mNI-11) immunoprecipitated at a molecular weight of 98 kDa, identified as a CD93 molecule, in the CD4(+)CD45RA(+) cells from neonatal UCB but not from adult PB, similar to the results in the human monocyte-like cell line U937 (human CD93-positive cells). Taken together, these results provide the first direct evidence of a novel/naive cell population (CD4(+)CD45RA(+)CD93(+)) in neonatal UCB that may have an important role in cell biology, transplantation, and immature/mature immune responses.

Anti-human very late antigen-alpha4 (CD49d) monoclonal antibody (BU49) cross-reacts with the canine B-cell leukemia cell line GL-1, resulting in the induction of homotypic cell aggregation.

We have found that the anti-human very late antigen-alpha4 (VLA-alpha4) (CD49d) monoclonal antibody (mAb) BU49 cross-reacts with the canine B-cell leukemia cell line GL-1. Interestingly, the BU49 mAb specifically induced the homotypic cell aggregation of GL-1 cells accompanied by morphological changes. Homotypic cell aggregates induced by BU49 mAb were blocked by the presence of a protein kinase C inhibitor, a protein kinase A inhibitor, an actin filament formation inhibitor, and an EDTA. On the other hand, a protein tyrosine kinase inhibitor, a DNA-synthesis inhibitor, and an anti-canine CD45 mAb did not affect the GL-1 homotypic cell aggregation induced by BU49 mAb. The BU49 mAb immunoprecipitated at a molecular weight of about 150kDa in the GL-1 cells, similar to the results in the human monocyte-like cell line U937. Taken together, our results provide the first evidence that human CD49d recognized by BU49 mAb has unique immunological functions against canine cells.