Randomized clinical trial of BCG vaccine in patients with convalescent COVID-19: Clinical evolution, adverse events, and humoral immune response.

BACKGROUND: The Bacillus Calmette-Guérin (BCG) vaccine may confer cross-protection against viral diseases in adults. This study evaluated BCG vaccine cross-protection in adults with convalescent coronavirus disease 2019 (COVID-19). METHOD: This was a multicenter, prospective, randomized, placebo-controlled, double-blind phase III study (ClinicalTrials.gov: NCT04369794). SETTING: University Community Health Center and Municipal Outpatient Center in South America. PATIENTS: a total of 378 adult patients with convalescent COVID-19 were included. INTERVENTION: single intradermal BCG vaccine (n = 183) and placebo (n = 195). MEASUREMENTS: the primary outcome was clinical evolution. Other outcomes included adverse events and humoral immune responses for up to 6 months. RESULTS: A significantly higher proportion of BCG patients with anosmia and ageusia recovered at the 6-week follow-up visit than placebo (anosmia: 83.1% vs. 68.7% healed, p = 0.043, number needed to treat [NNT] = 6.9; ageusia: 81.2% vs. 63.4% healed, p = 0.032, NNT = 5.6). BCG also prevented the appearance of ageusia in the following weeks: seven in 113 (6.2%) BCG recipients versus 19 in 126 (15.1%) placebos, p = 0.036, NNT = 11.2. BCG did not induce any severe or systemic adverse effects. The most common and expected adverse effects were local vaccine lesions, erythema (n = 152; 86.4%), and papules (n = 111; 63.1%). Anti-severe acute respiratory syndrome coronavirus 2 humoral response measured by N protein immunoglobulin G titer and seroneutralization by interacting with the angiotensin-converting enzyme 2 receptor suggest that the serum of BCG-injected patients may neutralize the virus at lower specificity; however, the results were not statistically significant. CONCLUSION: BCG vaccine is safe and offers cross-protection against COVID-19 with potential humoral response modulation. LIMITATIONS: No severely ill patients were included.

Autochthonous male urothelial carcinoma immune competent model: from induction to BCG transurethral treatment.

OBJECTIVE: To describe a new animal model of autochthonous urothelial cancer (UC) accessible by transurethral catheter in males, from induction to treatment. Seven-week-old male Fischer 344 rats were used. The first 10 animals were used to overcome and standardize the technical challenges of safe transurethral catheterization of male rats. The remaining 14 animals underwent intravesical N-Methyl-Nitrosourea (MNU) instillation for UC induction, of which six were randomized to undergo intravesical BCG treatment. The stretched male rat urethra travels 35 mm in a tortuous "S" shaped trajectory with a 180° angle behind the pubic bone, safely traversed by a 20G 36" 0.8 mm epidural catheter in a stretched, straightened urethra inserted after anterior dilation of the penile urethra with a 24G IV catheter. Histopathologic analysis of the urinary bladder demonstrated Stage pT1, pTa, and pTis lesions in the 8 controls, all with increased cell proliferation by Ki-67 expression and no pT1 or pTis in the animals 6 treated with BCG. This pioneering study describes an autochthonous, effective, and accessible transurethral animal model of immune-competent UC in males, and may help with understanding of the biology, immunology, and treatment of UC, which predominates in males.

Boosting bladder cancer treatment by intravesical nitazoxanide and bacillus calmette-guérin association.

PURPOSE: Nitazoxanide (NTZ) has shown a promising antitumoral effect, the current study compared the anti-neoplastic effects of intravesical NTZ and BCG plus NTZ in NMIBC animal model. METHODS: 30 rats, Fisher 344 were instilled with 4 intravesical doses of 1.5 mg/kg of N-methyl-N-nitrosourea (MNU) every 15 days for BC induction. The animals were divided into 3 groups (Group BCG 106 UFC - 1 mg of BCG; Group NTZ - 300 mg/kg of NTZ; Group NTZ + BCG - simultaneous treatment of BCG and NTZ) and received weekly intravesical treatment for 6 consecutive weeks. Animals were submitted to ultrasound imaging and euthanasia, their bladders were collected and histopathological, immunohistochemical tests (ki67 e c-Myc) and Western Blotting (PI3K, mTOR, and p-4E-BP) were performed. RESULTS: Histopathological tests showed 66.67%, 62.5% and 37.5% incidence of BC in animals treated with BCG, NTZ, and NTZ + BCG, respectively. Nuclear positivity for ki-67 in BC animals were 12.4% (IC 10.1-14.6%), 13.2% (IC 10.5-15.9%) and 8.8% (IC 6.0-11.6%) in BCG, NTZ and NTZ + BCG group, respectively (p = 0.063). Between animals with carcinoma, c-Myc strong positive was 40.10% in NTZ, 32.2% in BCG and 19.90% in the NTZ + BCG group (p < 0.001). Blotting has shown mTOR (p = 0.0473) and PI3K inhibition (p = 0.0349) in the presence of BCG, added to 4-EBP inhibition in the presence of NTZ (p = 0.0004). CONCLUSIONS: Results show the possible synergy between the gold standard BC treatment BCG and NTZ, in which multiple targets inhibition such as c-Myc and downstream mTOR, p-4E-BP and PI3K might play a role.

Carcinogenesis and Bacillus Calmette-Guérin (BCG) Intravesical Treatment of Non-Muscle-Invasive Bladder Cancer under Tryptophan and Thymine Supplementation.

PURPOSE: Evaluate tryptophan and thymine (TT) impact on carcinogenesis and intravesical BCG bladder cancer treatment. METHODS: After identification of TT in vitro inhibitory effect in multiple cancer cell cultures, bladder cancer animal model was induced by MNU intravesical instillations and randomized into four groups: Control (n = 9), BCG (n = 9), TT (n = 7), and BCG + TT (n = 8). BCG groups received intravesical 106 CFU BCG in 0.2 ml saline for 6 consecutive weeks and TT groups received 1 g/kg (1:1) of TT via daily gavage. After 15 wk of protocol, animals were euthanized and the urinary bladders submitted to histopathology, immunohistochemistry, and Western blotting. RESULTS: Urothelial cancer was identified in 100%, 85.7%, 44.5%, and 37.5% of Control, TT, BCG, and BCG + TT groups, respectively. Cell proliferation marked by nuclear Ki-67 was higher in the Control compared to animals in the other groups (P = 0.03). BCG, TT, and BCG + TT groups showed proliferative cell decline and TLR4/5 labeling increase in the urothelium. BCG decreased the urothelial VEGF labeling, even in TT association. CONCLUSION: TT inhibit urothelial carcinogenesis and potentiate the intravesical BCG in the treatment of bladder cancer by reducing cell proliferation and activating TLRs.

T cells CD4+/CD8+ local immune modulation by prostate cancer hemi-cryoablation.

PURPOSE: Tumors escape from the immune system by decreasing CD8+ and increasing CD4+ T cells' activity, druggable targets. Thermal ablation might activate tumor-specific T cells by raising the presentation of tumor-specific antigens and hindering tumor negative immune regulation. Our aim was to assess T cell infiltrate pre- and post-cryoablation in a prospective observational study. METHODS: A total of 240 sextant prostate biopsies cores (12 cores/patient) were collected from 10 unilateral prostate cancer patients (T1c, PSA density < 0.15 ng/dL, Gleason grade group 1, ≤ 2 cancer biopsy cores, and < 50% cancer core involvement) at diagnosis and 12 months after hemi-cryoablation. Cancer-positive (Diag+) and cancer-negative (Diag-) lobes at diagnosis and the same areas 12 months after hemi-cryoablation (Cryo+ and Cryo-, respectively) were explored by immunohistochemistry for infiltrating CD4+ and CD8+ T cells (in 45 random fields per prostate lobe, 400× magnification). The quantitative analysis of cells/mm2 and CD4+/CD8+ ratio were performed and compared among Diag+, Diag-, Cryo+, and Cryo- using ImageJ software. RESULTS: There was a significant increase in tumor-infiltrating CD8+ T cells/mm2 in the Cryo+ tissue (mean, SD 0.31, 0.30) compared to Diag+ (0.18, 0.15), p = 0.015; confirmed in prostate acini (hot spots), p = 0.029, in which infiltrating CD4+/CD8+ T cells' ratio decreased after hemi-cryoablation, p = 0.006. Infiltrating CD4+ T cells/mm2 presented a trend to decrease in Cryo+ (0.26, 0.27) compared to Diag+ (0.38, 0.32). CONCLUSIONS: This is the first study to show local immune modulation after prostate cancer cryoablation, characterized by decreasing CD4+/CD8+ T cells' ratio, potential for clinical impact by unleashing the T-cell response to cancer. Future studies are necessary to explore different energies and longer follow-up clinical endpoints.

Intravesical Immunomodulatory Imiquimod Enhances Bacillus Calmette-Guérin Downregulation of Nonmuscle-invasive Bladder Cancer.

BACKGROUND: The Toll-like receptor (TLR)2/4 agonist bacillus Calmette-Guérin (BCG), although not failure proof, has been the most efficient immunomodulatory treatment of immunogenic nonmuscle-invasive bladder cancer (NMIBC) for > 40 years. We investigated the role of the immunomodulatory molecule TLR7 agonist imiquimod through the BCG key receptors TLR2/4 and the main downstream molecules of the mammalian target of rapamycin pathway in NMIBC treatment. MATERIALS AND METHODS: A total of 40 Fischer-344 rats, 7 weeks old, received 4 doses of 1.5 mg/kg N-methyl-N-nitrosourea intravesically on weeks 0, 2, 4, and 6 for cancer induction. At week 8, the rats were randomized into 4 groups (10 per group) and treated intravesically once a week for 6 weeks: control (0.2 mL of vehicle); BCG (2 × 106 colony-forming units Connaught strain in 0.2 mL); imiquimod (20 mg/kg in 0.2 mL), and associated treatment BCG plus imiquimod in 0.2 mL. The bladders were extracted and analyzed for histopathology, immunohistochemistry, cell proliferation (Ki-67), apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling [TUNEL]), and immunoblotting for TLR2, TLR4, p-P70S6K, and p-4E-BP1 proteins. RESULTS: The histopathology results showed that BCG and imiquimod decreased bladder tumorigenesis compared with the control group, with a proliferation decrease (Ki-67) and an apoptosis increase (TUNEL). BCG upregulated TLR2/4, imiquimod upregulated TLR4, and both downregulated P70S6K1. CONCLUSION: Imiquimod is able to efficiently decrease bladder carcinogenesis through upregulation of TLR7/4 and downregulation of P70S6K1 protein, generating new perspectives to boost BCG effects in the future.

Intravesical Thalidomide boosts bacillus Calmette-Guérin (BCG) in non-muscle invasive bladder cancer treatment.

The aim of this study was to explore the efficacy of intravesical Thalidomide (immunomodulatory, anti-inflammatory and anti-angiogenic) added to BCG using an immune competent autochthonous orthotopic NMIBC animal model. Female Fischer 344 rats, 7 weeks of age, received every 2 weeks for four times, a dose of 1.5 mg/kg of N-methyl-N-nitrosourea (MNU) intravesically. The rats were randomized into four groups (n = 10 per group) to receive intravesical treatment once a week for 6 weeks as follows: control (0.2 ml vehicle), BCG (2 × 106 CFU of Connaught strain in 0.2 ml), Thalidomide (20 mg/kg in 0.2 ml) and BCG-Thalidomide in 0.2 ml. At week 15, bladders were collected for histopathology, cell turnover index by immunohistochemistry and immunoblotting quantification of 4E-BP1 and p70S6K1 for downstream mTOR proliferation signaling and HIF and VEGF for angiogenesis pathway. Thalidomide-BCG association showed a trend for normal histopathology and down-regulation of cell turnover, p70S6K1, HIF-1 and VEGF. 4E-BP1 was up-regulated by treatment, especially in the Thalidomide groups, supporting that its regulation occurs independently of p70S6K1 on mTOR pathway in NMIBC. Intravesical BCG-Thalidomide might represent a significant increment in NMIBC treatment, suggesting p70S6K1, HIF-1 and VEGF as potential molecular target candidates in a clinically relevant immune competent NMIBC model.

Detection of porcine circovirus genotypes 2a and 2b in aborted foetuses from infected swine herds in the State of São Paulo, Brazil.

BACKGROUND: Porcine circovirus type 2 (PCV2) has been associated with several disease complexes, including reproductive failure. The aim of this study was to identify the subtypes of PCV2 that are associated with reproductive failure in pigs from the State of São Paulo, Brazil and to investigate co-infections with other infectious organisms. FINDINGS: Samples of 168 aborted foetuses or mummified foetuses from five farrow-to-finish swine farms known to be infected with PCV2 and located in the State of São Paulo were tested for PCV2 by polymerase chain reaction (PCR). Positive samples were additionally tested for porcine parvovirus (PPV), Leptospira spp. and Brucella spp. by PCR. PCV2 was detected in 18 of the samples (10.7%). PPV, Brucella spp. and Leptospira spp were found in 2, 10 and 0 cases, respectively. Eleven PCV2 strains were sequenced and determined to be either genotype 2a (n = 1) or 2b (n = 10). CONCLUSIONS: The findings indicate that the frequency of PCV2 infections in aborted porcine foetuses from the State of São Paulo is rather low (10.7%) and that co-infection with other pathogens is common and may be involved in PCV2 associated reproductive failure. No repeatable, characteristic amino acid motifs for regions of the PCV2 capsid protein seemed to be associated with abortion in sows.