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In experimental house dust mite (HDM)-induced allergic asthma, therapeutic administration of a single dose of adipose tissue-derived mesenchymal stromal cells (MSCs) ameliorates lung inflammation but is unable to reverse remodeling. We hypothesized that multiple doses of MSCs might exert better therapeutic effects by reducing lung inflammation and remodeling but might also result in immunosuppressive effects in experimental asthma. HDM was administered intranasally in C57BL/6 mice. After the last HDM challenge, mice received two or three doses of MSCs (10 5 cells per day) or salineintravenously. An additional cohort of mice received dexamethasone as a positive control for immunosuppression. Two and three doses of MSCs reduced lung inflammation; levels of interleukin (IL)-4, IL-13, and eotaxin; total leukocyte, CD4 + T-cell, and eosinophil counts in bronchoalveolar lavage fluid; and total leukocyte counts in bone marrow, spleen, and mediastinal lymph nodes. Two and three doses of MSCs also reduced collagen fiber content and transforming growth factor-β levels in lung tissue; however, the three-dose regimen was more effective, and it reduced these parameters to control levels, while also decreasing α-actin content in lung tissue. Two and three doses of MSCs improved lung mechanics. Dexamethasone and two and three doses of MSCs similarly increased galectin levels, but only the three-dose regimen increased CD39 levels in the thymus. Dexamethasone and the three-dose, but not the two-dose, regimen also increased the levels of programmed death receptor-1 and IL-10, while reducing CD4 + CD8 low cell percentage in the thymus. In conclusion, multiple doses of MSCs reduced lung inflammation and remodeling while causing immunosuppression in HDM-induced allergic asthma. K E Y W O R D Sadipose stem cells, cellular therapy, immunosuppression, lung, mesenchymal stromal cells

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Leishmania (V.) braziliensis is one of the most important ethiologic agents of the two distinct forms of American tegumentary leishmaniasis (cutaneous and mucosal). The drugs of choice used in leishmaniasis therapy are significantly toxic, expensive and are associated with frequent refractory infections. Among the promising new targets for anti-protozoan chemotherapy are the proteases. In this study, serine proteases were partially purified from aqueous, detergent and extracellular extracts of Leishmania braziliensis promastigotes by aprotinin-agarose affinity chromatography. By zymography, the enzymes purified from the aqueous extract showed apparent activity bands of 60 kDa and 45 kDa; of 130 kDa, 83 kDa, 74 kDa and 30 kDa from the detergent extract; and of 62 kDa, 59 kDa, 57 kDa, 49 kDa and 35 kDa from the extracellular extract. All purified proteases exhibited esterase activity against Nα-benzoyl-l-arginine ethyl ester hydrochloride and Nα-p-tosyl-l-arginine methyl ester hydrochloride (serine protease substrates) and optimal activity at pH 8. 0. Proteases purified from the aqueous and extracellular extracts were effectively inhibited by benzamidine (trypsin inhibitor) and those from the detergent extract were inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (chymotrypsin inhibitor) indicating that all these enzymes are serine proteases. These findings indicate that L. braziliensis serine proteases display some biochemical similarities with L. amazonensis serine proteases, demonstrating a conservation of this enzymatic class in the Leishmania genus. This is the first study to report the purification of a serine protease from Leishmania braziliensis.

We used the recombinant trimeric spike (S) glycoprotein in the prefusion conformation to immunize horses for the production of hyperimmune globulins against SARS-CoV-2. Serum antibody titers measured by ELISA were above 1:10 6 , and the neutralizing antibody titer against authentic virus (WT) was 1:14,604 (average PRNT 90 ). Plasma from immunized animals was pepsin digested to remove the Fc portion and purified, yielding an F(ab’) 2 preparation with PRNT 90 titers 150-fold higher than the neutralizing titers in human convalescent plasma. Challenge studies were carried out in hamsters and showed the in vivo ability of equine F(ab’) 2 to reduce viral load in the pulmonary tissues and significant clinical improvement determined by weight gain. The neutralization curve by F(ab’) 2 was similar against WT and P.2 variant but displaced to higher concentrations by 0.39 log units against P.1 (Gamma) variant. These results support the possibility of using equine F(ab’) 2 preparation for the clinical treatment of COVID patients.

Skin secretions of fishes constitute a rich source of proteins with a broad spectrum of antimicrobial properties. We report here the characterization of proteases from skin mucus of tambacu, an economically important Neotropical hybrid fish. The effects of pH on the proteolytic activities of the mucus acting on various substracts -hemoglobin, casein, bovine serum albumin (BSA) and ovalbumin (OVA) -were tested. Optimal pH values for protease activity on hemoglobin were 4.5 and 8.5, on casein, 8.5, on BSA, 5.0 and 7.5, and on ovalbumin, 4.5 and 6.5. The proteolytic activity was inhibited on all of these substrates in the presence of specific inhibitors: caseinolytic activity was inhibited by inhibitors of serine and metalloproteases; hemoglobinolytic activity was inhibited by serine, aspartic and metalloproteases inhibitors; albuminolytic activity on BSA was inhibited by serine and aspartic proteases inhibitors, and on ovalbumin, by cysteine and aspartic proteases inhibitors. Gelatin zymography revealed that the skin mucus of tambacu consisted primarily of serine and metalloproteases.Hemoglobin zymography showed one proteolytic band inhibited by EDTA, whereas casein zymography showed two proteases inhibited by serine proteases inhibitors. We were able to identify all classes of proteases in the mucus from the skin of tambacu. These, and these results suggest that the proteolytic activities of the skin mucus of fish may play an important role in the defense against microorganisms and ectoparasites.

COVID-19 pandemic caused approximately 750,000 deaths and over 20 million confirmed cases of infection by SARS-CoV-2 within 8 months since the emergence of the virus. While there are no vaccines approved and considering the difficulty in meeting the large vaccination demand worldwide, the potential use of passive immunization should be considered based on existing successful therapies against many diseases. Here we demonstrate that hyperimmune globulin preparations raised in horses against the recombinant trimeric spike (S) glycoprotein of SARS-CoV-2 in the prefusion conformation provide very high ELISA titers as well as highly potent neutralizing activity against SARS-CoV-2. Five horses were subcutaneously inoculated for 6 weeks with the recombinant S protein (ectodomain, residues 1-1208). Four out of the 5 horses presented a strong immune response. Considering the average of all 5 horses, ELISA titers above 1:1,000,000 and neutralizing titers (PRNT90) reaching 1:14,604 were observed. When compared with the plasma of three convalescent COVID-19 patients, sera of immunized horses displayed approximately 140-fold higher neutralizing titers measured as PRNT90. To prevent eventual side effects caused by horse antiserum, IgG was digested with pepsin and purified by fractional salt precipitation to eliminate Fc fragments, a process that is industrially used for the production of passive immunization F(ab')2 concentrates against rabies, tetanus and snake venoms. The high neutralizing titers against SARS-CoV-2 obtained for the unprocessed sera were confirmed for the F(ab')2 fragments and were 150-fold higher than the PRNT90 neutralizing titers of plasma of three COVID-19 convalescent patients. The great advantage of using the recombinant trimeric S glycoprotein is that it is safe and provides quick adaptive immunity in horses. Our data show the perspective of using hyperimmune anti-SARS-CoV-2 F(ab')2 preparations as a passive immunization therapy in humans, similar to therapies that have been safely used for decades against rabies, tetanus and snake venoms.

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