Association of Vitamin D with Severity and Outcome of COVID-19: Clinical and Experimental Evidence.

INTRODUCTION: The role of vitamin in COVID-19 remains controversial. We investigated the association between endogenous vitamin D and the severity of COVID-19 as well as the mechanisms of action of vitamin D supplementation. METHODS: 25(OH)D3 in serum was associated with disease severity and outcome in 190 COVID-19 patients. In a COVID-19 animal model using intravenous injection of plasma from patients with COVID-19 acute respiratory distress syndrome into C57/BL6 mice, mice were treated with 0.25 µg human 1,25(OH)D3 or vehicle. Mice were sacrificed on day 4. Cytokines and myeloperoxidase (MPO) in tissues were measured. Changes in gene expression after vitamin D supplementation were measured. RESULTS: Vitamin D deficiency and insufficiency were associated with increased severity and unfavorable outcome after 28 days. Vitamin D levels were negatively associated with biomarkers of COVID-19 severity. Vitamin D supplementation after challenge of mice with COVID-19 plasma led to reduced levels of TNFα, IL-6, IFNγ, and MPO in the lung, as well as down-regulation of pro-inflammatory pathways. CONCLUSION: Normal levels of endogenous vitamin D are associated with reduced severity and risk of unfavorable outcome in COVID-19, possibly through attenuation of tissue-specific hyperinflammation.

Deficiency of hydrogen sulfide production and pregnancy rate in an experimental model: Association with preterm delivery.

PROBLEM: Pro-inflammatory phenomena drive preterm delivery (PTD). Hydrogen sulfide is a gasotransmitter with anti-inflammatory properties produced through the activity of the enzyme cystathionine-γ-lyase (CSE), and its impact was studied in models of normal delivery and PTD in mice. METHOD OF STUDY: Female CSE+/+ and CSE-/- mice were mated with male CSE+/+ mice; mating was done with drinking water unsupplemented and supplemented with cysteine. The pregnancy rate was monitored. PTD was induced by the intraperitoneal injection of bacterial lipopolysaccharide (LPS) on day 14.5 of pregnancy. Mice were sacrificed for tissue collection and splenocyte isolation after 6 and 12 h. Isolated splenocytes were stimulated for the production of tumor necrosis factor-alpha (TNFα), interleukin (IL)-10 and interferon-gamma (IFNγ); TNFα and vascular endothelial growth factor (VEGF) were measured in the fetuses and the placenta. RESULTS: The successful pregnancy rate was lower in CSE-/- mice and it was restored with cysteine supplementation. CSE deficiency was associated with higher tissue concentrations of TNFα in the fetuses, attenuated IL-10 responses and higher IFNγ production from splenocytes. CSE deficiency was not associated with PTD. Following PTD induction, CSE-/- mice did not show attenuated IL-10 responses but the production of TNFα and IFNγ was lowered over-time; placental VEGF was also increased over-time. CONCLUSIONS: CSE deficiency has an unfavorable impact on pregnancy. H2 S deficiency through CSE does not drive PTD but mediates pro-inflammatory phenomena in fetuses.

An active new formulation of iron carried by aspartyl casein for iron-deficiency anemia: results of the ACCESS trial.

Oral iron supplementation is the cornerstone for the management of iron-deficiency anemia. A new oral formulation of iron conjugated with N-aspartyl-casein (Fe-ASP) (Omalin®, Uni-Pharma) is studied in the ACCESS double-blind, double-dummy randomized clinical trial; 60 patients were randomized to 12-week oral treatment twice every day either with oral ferrous sulfate (FeSO4) delivering 47 mg elementary iron or oral Fe-ASP delivering 40 mg elementary iron. Participants had hemoglobin less than 10 g/dl, decreased red blood cell (RBC) count, and ferritin lower than 30 ng/ml; patients with a medical history of malignancy were excluded. The primary endpoint was the increase of Hb in the first 4 weeks of treatment, and the study was powered for non-inferiority. A new score of global improvement was introduced where all participants were given one point for any at least 10% increase of Hb, RBC, and reticulocytes. At week 4, the mean (SE) change of Hb was 0.76 g/dl in the FeSO4 group and 0.83 g/dl in the Fe-ASP group (p: 0.876). The odds for worse allocation of the global score were 0.35 in the Fe-ASP group compared to the FeSO4 group. Patients in the Fe-ASP group experienced a significant decrease in the number of IDA-related physical signs by week 4. No differences were found between the two groups in any of the patient-reported outcomes of fatigue and of gastrointestinal adverse events either at week 4 or at week 12. ACCESS is the most recent clinical trial showing the non-inferiority of Fe-ASP to FeSO4 for the primary endpoint of the Hb change.

Immunomodulation Through Beta-D-glucan in Chemically-induced Necrotizing Pancreatitis.

BACKGROUND: Although the ability of ß-D-glucan and monophosphoryl lipid A (MPLA) to modulate immune responses has been studied in human primary cells, their effect on sterile inflammation models such as necrotizing pancreatitis has never been investigated. MATERIALS AND METHODS: 85 male New Zealand rabbits were assigned into following groups: A: control, B: pretreatment with ß-D-glucan 3 d before pancreatitis, C: pretreatment with MPLA 3 d before pancreatitis, D: pretreatment with ß-D-glucan and laminarin 3 d before pancreatitis, E: treatment with ß-D-glucan 1 d after pancreatitis, and F: MPLA 1 d after pancreatitis. Pancreatitis was induced by sodium taurocholate injection into the pancreatic duct and parenchyma. Survival was recorded for 21 d. On days 1, 3, and 7, blood was collected for amylase measurement. Peripheral blood mononuclear cells were isolated and stimulated for tumor necrosis factor alpha and interleukin 10 production. Pancreatic necrosis and tissue bacterial load were assessed. RESULTS: 21-d survival was prolonged after pretreatment or treatment with ß-D-glucan; this benefit was lost with laminarin administration. At sacrifice, pancreatic inflammatory alterations were more prominent in the control group. Bacterial load was lower after pretreatment or treatment with ß-D-glucan and MPLA. Tumor necrosis factor alpha production from stimulated peripheral blood mononuclear cells was significantly decreased, whereas interleukin 10 production remained unaltered after pretreatment or treatment with ß-D- glucan. CONCLUSIONS: ß-D-glucan reduces mortality of experimental pancreatitis in vivo. This is mediated through attenuation of cytokine production and prevention of bacterial translocation.

Susceptibility profiles and clinical efficacy of antifungals against candida bloodstream isolates from critically ill patients: Focus on intravenous itraconazole.

In vitro and clinical data were analysed to evaluate the susceptibility profile of itraconazole in light of the new cut-off points. The in vitro activity of itraconazole was compared with that of eight comparators against 119 Candida bloodstream isolates from 2015 to 2018. Minimum inhibitory concentrations (MICs) were measured by the colorimetric MICRONAUT-S assay. The content of wells without any color change was sub-cultured to measure killing efficacy. No major differences were found against Candida albicans. Itraconazole, posaconazole and amphotericin B were the most active agents against Candida parapsilosis. Of the 32 isolates of C. parapsilosis that were resistant to fluconazole, 96.9%, 78.1% and 93.8% were susceptible to itraconazole, voriconazole and posaconazole, respectively. The ratio of the minimum fungicidal concentration (MFC) to the MIC of itraconazole was lower than for the other azoles against C. parapsilosis and C. glabrata. Itraconazole achieved greater inhibition over-time of the growth of C. parapsilosis than fluconazole. Seventy-three critically ill patients who were unresponsive to antibiotics received intravenous empirical treatment with itraconazole (n = 28) or comparators (n = 45). Case-control matching was conducted for severity, comorbidities, risk factors for candidemia, administered antibiotics and days of antifungal treatment. Breakthrough candidemia was found in 3.6% of patients treated with itraconazole and in 32.1% of patients treated with comparators (P: 0.020); breakthrough candidemia by C. parapsilosis was found in 3.6% and 28.6% of patients, respectively. Results indicate that itraconazole retains a valuable susceptibility profile against Candida isolates, particularly C. parapsilosis. This superior profile may explain the clinical efficacy in the occurrence of breakthrough candidemia and warrants further clinical investigation.

Daptomycin as adjunctive treatment for experimental infection by Acinetobacter baumannii with resistance to colistin.

The emergence of Acinetobacter baumannii with resistance to colistin (ABRC) led to the investigation of daptomycin as an adjunctive to colistin for these isolates. In this study, one ABRC carbapenemase-producing bloodstream isolate was examined. Minimum inhibitory concentrations (MICs) were >512, >512 and 8 µg/mL for imipenem, daptomycin and colistin, respectively. First, a 'humanised' model of the pharmacokinetics of daptomycin and colistin was developed in 18 male C57BL/6 mice. Then, 112 mice were infected by intraperitoneal injection of the ABRC isolate and were randomly assigned into four groups of once-daily treatment for 7 days: group A, controls treated with saline; group B, treated with 20 mg/kg colistin; group C, treated with 50 mg/kg daptomycin; and group D, treated with both agents. Survival was recorded for 7 days in ten mice per group. The remaining mice were sacrificed at regular time intervals following bacterial challenge and the bacterial outgrowth in the liver, lung and right kidney was determined. Mean serum concentrations of daptomycin at 15, 30 and 60 min post-dose were 121.8, 110.3 and 100.4 µg/mL, respectively. The respective concentrations of colistin were 13.9, 9.1 and 7.5 µg/mL. The 7-day mortality in groups A, B, C and D was 100%, 50%, 100% and 0%, respectively. Tissue outgrowth of the right kidney was significantly decreased in group D compared with group B after 72 h. Daptomycin used in combination with colistin leads to prolonged survival in an experimental infection by ABRC. Failure of colistin alone is probably related to rebound of tissue outgrowth.

Efficacy of intramuscular moxifloxacin in the treatment of experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus.

Fluoroquinolones have been well studied in the treatment of chronic osteomyelitis due to their beneficial pharmacokinetic (PK) and pharmacodynamic profiles. The purpose of this study was to determine the efficacy of intramuscular (IM) moxifloxacin administration in the treatment of experimental osteomyelitis by methicillin-resistant Staphylococcus aureus. Following an experimental osteomyelitis animal model described previously, three groups of rabbits (A = control; B = IM moxifloxacin administration; C = PK study of moxifloxacin penetration into bone) were evaluated. Three weeks after bacterial inoculation, surgical debridement was performed in all animals and IM treatment commenced for Groups B and C. Sacrifice was performed in an A:B group animal ratio of 1:2 at weekly intervals from 7th to 42nd day post debridement and from 21st to 56th day post debridement for Groups A and B, respectively (including 2-week interval without antibiotics for Group B). Cancellous bone was harvested for microbiological and histopathological analyses at re-operation and sacrifice for Groups A and B. Cortical bone moxifloxacin levels were measured in Group C following 7, 14, 35 and 42 days of treatment. In Group A, bacterial growth after surgical debridement was significant, whereas high eradication rates were observed in Group B. Radiological abnormalities and histopathological findings were evaluated. Moxifloxacin bone levels, observed in Group C, were approximately 43 times higher than the minimum inhibitory concentration, with no difference found between infected and healthy tibial bone. The therapeutic protocol was very effective in this model of experimental osteomyelitis. However, further evaluation of these results in clinical studies is crucial.

Thalidomide prolongs survival after experimental musculoskeletal injury, through an effect on mononuclear apoptosis.

BACKGROUND: This study was conducted to investigate the effects of intravenous thalidomide administration in an experimental model of musculoskeletal trauma. We hypothesized that because thalidomide inhibits secretion of tumor necrosis factor alpha (TNF-α), survival of animals that received thalidomide would be significantly prolonged. MATERIAL AND METHODS: After an open fracture of the right femur, 24 rabbits were randomly assigned to control and thalidomide groups. Intravenous therapy with thalidomide was started 30 min after fracture. Hemodynamic monitoring of all animals was performed for 4 h. Survival was recorded and bacterial growth in blood and organs was measured after animal death or sacrifice. Blood was sampled for TNF-α measurement and for isolation of peripheral blood mononuclear cells (PBMCs). Apoptosis of PBMCs was measured by flow cytometry. RESULTS: Survival was significantly prolonged in the thalidomide group. Apoptosis of PBMCs was increased in the control group compared with the thalidomide group at 24 h. There were no differences in vital signs, blood and tissue cultures, and serum TNF-α concentration between the two groups. CONCLUSIONS: Intravenous thalidomide prolonged survival in an experimental model of severe musculoskeletal injury in rabbits. Its mechanism of action did not involve TNF-α suppression but prevention of mononuclear apoptosis. In view of these promising results, further research is needed to clarify the immunomodulatory mechanism of action of thalidomide and its potential use for the management of severe trauma.

Controversies in the management of the critically ill: the role of probiotics.

Probiotics are commercially available, viable, non-pathogenic micro-organisms that, when ingested in sufficient quantities, exert a health benefit to the host derived through modification of the gut flora, local release of antimicrobial factors, maintenance of integrity of the gut barrier, competition for epithelial adherence, prevention of bacterial translocation, and modulation of the local immune response. In critically ill patients, probiotics appear to lead to decreased susceptibility to antibiotic-associated diarrhoea, Clostridium difficile infections, ventilator-associated pneumonia, necrotising enterocolitis, acute severe pancreatitis, sepsis and multiple organ dysfunction syndrome as well as a shortened duration of infections. Current scientific evidence supporting the use of probiotics is not conclusive and is mainly derived from single-centre, not very well designed trials that are limited by many factors including small sample sizes, heterogeneity in the probiotic strains used, effectiveness of the combined strains, optimum dose regimens, frequency and duration of administration, and certainly incomplete knowledge of the mechanism of action of each strain. Probiotics appear to be well tolerated, whilst adverse events are very rare. The most commonly reported adverse events include bacteraemia, fungaemia and sepsis. At present, based on the available evidence and although helpful and relatively safe for certain disease conditions, routine use of probiotics in the critically ill is not recommended.

Probiotics for infectious diseases: more drugs, less dietary supplementation.

According to current definitions, probiotics are live microorganisms that, when ingested in adequate quantities, exert a health benefit to the host. The action of probiotics in the host is exerted by three mechanisms: modulation of the content of gut microbiota; maintenance of the integrity of the gut barrier and prevention of bacterial translocation; and modulation of the local immune response by the gut-associated immune system. Regarding their role for the prevention and treatment of infectious diseases, adequate evidence coming from randomised clinical trials (RCTs) is available for antibiotic-associated diarrhoea (AAD), Clostridium difficile infection (CDI), acute gastroenteritis and infectious complications following admission to the Intensive Care Unit (ICU). Existing evidence supports their role for decreasing the incidence of AAD and CDI when administered in parallel with antimicrobials. They also shorten the duration of symptoms when administered in paediatric populations with acute gastroenteritis, particularly of rotavirus aetiology. Available evidence is not sufficient to support administration for the management of CDI. Regarding populations of critically ill patients, data from many RCTs suggest a decrease of infectious complications by starting feeding with probiotics following ICU admission, with the exception of patients suffering from severe pancreatitis. However, it should be underscored that all analysed RCTs are characterised by marked heterogeneity regarding the type of administered probiotic species, precluding robust recommendations.

Treatment of experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus with a synthetic carrier of calcium sulphate (Stimulan) releasing moxifloxacin.

The objectives of this study were to assess the efficacy of a synthetic semihydrate form of calcium sulphate (Stimulan) in experimental bone infection caused by methicillin-resistant Staphylococcus aureus (MRSA). Osteomyelitis was induced after inoculation of the test pathogen in the left tibia of 72 New Zealand rabbits assigned to the following groups: 18 control rabbits (Group A); 18 rabbits with Stimulan implanted (Group B); and 36 rabbits with moxifloxacin-impregnated Stimulan implanted (Group C). Rabbits were sacrificed at weekly intervals and cancellous bone was harvested for histopathology and for estimation of bacterial growth and concentrations of moxifloxacin. Bacterial growth from cancellous bone of Group C was significantly lower than the respective growth of Groups A and B on all days of sacrifice. The main histological finding of animals in all three groups was a moderate to intense inflammatory reaction accompanied by fibrosis. The degree of fibrosis was higher in Group C compared with both other groups. Infiltration by giant cells was also observed, which was greater in Group C on Day 42. Antibiotic levels in bone were higher for bone samples closer to the site of implantation. In conclusion, Stimulan admixed with 10% moxifloxacin was very effective in achieving complete eradication of the causative pathogen in experimental osteomyelitis caused by MRSA.

Efficacy and pharmacodynamics of linezolid, alone and in combination with rifampicin, in an experimental model of methicillin-resistant Staphylococcus aureus endocarditis.

OBJECTIVES: To evaluate the efficacy of oral linezolid, with or without rifampicin, on valve vegetations and secondary foci of infection compared with vancomycin, in the absence or presence of rifampicin, in experimental endocarditis caused by methicillin-resistant Staphylococcus aureus. METHODS: Treatment groups were controls (n = 16), linezolid (n = 15), vancomycin (n = 15), linezolid and rifampicin (n = 15), vancomycin and rifampicin (n = 13), linezolid relapse (n = 11) and vancomycin relapse (n = 9). Therapy lasted 5 days in all groups, with survival of animals in the linezolid relapse and vancomycin relapse groups being recorded for an additional 5 days. Blood was drawn to determine the linezolid concentration, and valve vegetations, and kidney, liver, lung and spleen segments were collected for culture. RESULTS: Survival in each individual group was higher than that in the control group; bacterial load in valve vegetations was reduced by all treatment regimens, with linezolid exhibiting bactericidal effects. Bactericidal activity of linezolid was noted in all secondary foci of infection except the lung, where only the combination of rifampicin with linezolid was bactericidal. CONCLUSIONS: Orally administered linezolid is effective in limiting bacterial growth in the secondary foci of endocarditis. Co-administration of rifampicin favoured the suppression of bacterial growth in the lung.

Local treatment of experimental Pseudomonas aeruginosa osteomyelitis with a biodegradable dilactide polymer releasing ciprofloxacin.

A biodegradable system of poly-D,L-dilactide releasing ciprofloxacin was assessed in a Pseudomonas aeruginosa osteomyelitis model after inoculation of the test pathogen into the left tibia of 76 New Zealand White rabbits; 31 were controls (group A), and 45 were implanted with the polymer at the infection site (group B). The rabbits were killed on a weekly basis, and cancellous bone was harvested for histopathology and for estimation of bacterial growth and the concentrations of ciprofloxacin. Tibial X ray was performed immediately before the animals were killed. The total number of fistulas with purulent discharge that developed after inoculation of the pathogen was counted, and fistulas with purulent discharge were found in 16 animals in group A (51.6%) and 3 animals in group B (6.7%) (P < 0.0001). The animals in group A had a profound loss of body weight compared to the animals in group B. The main radiological finding was the presence of sequestra in 25 animals (80.6%) in group A and 6 animals in group B (13.3%) (P < 0.0001). The bacterial load in group B was significantly reduced compared to that in group A, possibly due to the prolonged local antibiotic release at concentrations exceeding even 80 times the MIC for the test pathogen. The histology of animals killed after week 49 revealed a mild inflammatory reaction accompanied by diffuse fibrosis and new bone formation in group A animals and the presence of small polymer particles in group B animals. It is concluded that the system described achieved eradication of the pathogen, accompanied by clinical and radiologically confirmed benefits, so this treatment may be a candidate for the management of difficult orthopedic infections.

n-6 polyunsaturated fatty acids enhance the activities of ceftazidime and amikacin in experimental sepsis caused by multidrug-resistant Pseudomonas aeruginosa.

Recent in vitro and ex vivo studies disclosed an enhancement of the activity of antimicrobials on multidrug-resistant Pseudomonas aeruginosa by n-6 polyunsaturated fatty acids (PUFAS); therefore their effect was evaluated in experimental sepsis in 60 rabbits. Solutions of gamma-linolenic acid (GLA) and arachidonic acid (AA) were administered intravenously with ceftazidime and amikacin in rabbits with sepsis caused by one multidrug-resistant isolate. Therapy was started after bacterial challenge in five groups comprising 12 animals in each group: A, normal saline; B, antimicrobials; C, 99% ethanol and antimicrobials; D, GLA and antimicrobials; and E, AA and antimicrobials. Blood was sampled for the estimation of levels of endotoxins in serum (lipopolysaccharide), leukocytes, tumor necrosis factor alpha (TNF-alpha) and antimicrobials. Animals were sacrificed 210 min after bacterial challenge for tissue cultures. All animals had considerable endotoxemia and evolved leukopenia. The number of viable cells in blood, lung, and mesenteric lymph nodes was significantly reduced in groups D and E compared to that in other groups. Levels of antimicrobials in serum were inadequate to achieve bacterial killing due to the level of resistance. n-6 PUFAs did not influence TNF-alpha. It is concluded that intravenous coadministration of n-6 PUFAs and antimicrobials enhanced antimicrobial bacterial killing in experimental sepsis caused by multidrug-resistant P. aeruginosa.

Experimental sepsis using Pseudomonas aeruginosa: the significance of multi-drug resistance.

In order to clarify whether susceptible and multidrug-resistant Pseudomonas aeruginosa differ in the mechanism of induction of sepsis, three different isolates were used; one susceptible (isolate A) and two (isolates B and C) multidrug-resistant. Isolate B had moderately elevated MICs of antipseudomonal antimicrobials and isolate C highly elevated MICs. Each isolate was infused by a catheter inserted into the right jugular vein of six rabbits. Survival was recorded; blood was sampled at regular time intervals for estimation of bacterial blood counts, malondialdehyde (MDA) and tumour necrosis factor-alpha (TNFalpha). Quantitative cultures of various organs were performed after death or sacrifice. Mean survival after challenge by isolates A, B and C was 0.73, 2.58 and 11.00 days, respectively (P of comparisons A versus B, 0.0048; A versus C, 0.0012; B versus C, 0.0005). The number of viable organisms in the blood after challenge using isolates A and B was greater than the viable counts of C. Serum MDA was lower after challenge with B and C compared with A. Serum TNFalpha levels were higher after challenge by isolate A compared with isolate C. The bacterial loads of the liver, lower right lung lobe, spleen and mesenteric lymph nodes were greater after challenge by isolate A than the other isolates. It is concluded that infection by multidrug-resistant P. aeruginosa is accompanied by increased survival compared with infection by susceptible isolates; that finding might be explained by the different mechanisms leading to sepsis. Further studies must be done to clarify the significance of these observations for therapeutics.