Baricitinibe para tratamento de pacientes adultos com Covid-19 hospitalizados que necessitam de oxigênio por máscara ou cateter nasal, ou que necessitam de alto fluxo de oxigênio ou ventilação não invasiva / Baricitinib for the treatment of hospitalized adult patients with Covid-19 who require oxygen by mask or nasal catheter, or who require high flow oxygen or non-invasive ventilation

INTRODUÇÃO: O baricitinbe, um imunomodulador que atua sobre a atividade da IL-6 (citocina pró-inflamatória), pode representar uma estratégia para o tratamento de pacientes com COVID-19 que tiveram comprometimento pulmonar devido a resposta hiperinflamátoria desencadeada pela tempestade de citocinas característica na infecção causada pelo vírus SARS-COV2. TECNOLOGIA: Baricitinibe (Olumiant®). EVIDÊNCIAS CLÍNICAS: Para seleção das evidências clínicas foi conduzida uma revisão sistemática da literatura em busca de ensaios clínicos randomizados (ECR), estudos observacionais (mundo real) e revisões sistemáticas que avaliassem os efeitos do baricitinibe como monoterapia ou associado aos cuidados usuais - definidos aqui como 'terapia padrão' (corticoesteróides sistêmicos, anticoagulantes, antimicrobianos/antivirais) no tratamento de pacientes adultos com COVID-19, hospitalizados e que necessitam de suplementação de oxigênio (máscara ou cateter nasal, alto fluxo de oxigênio ou ventilação não invasiva). As buscas eletrônicas foram realizadas nas bases de dados: the Cochrane Library, MEDLINE via Pubmed, Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS), EMBASE e Centre for Reviews and Dissemination (CRD). O risco de viés dos estudos primários incluídos foi avaliado pelas ferramentas Risk of Bias versão 2 da Cochrane (para ECR) ou ROBINS-I (para estudos observacionais), e a qualidade metodológica das revisões sistemáticas foi avaliada pela ferramenta AMSTAR-2. A qualidade da evidência foi avaliada pelo sistema GRADE. Seis artigos foram incluídos na presente revisão, sendo dois deles referentes a um ensaio clínico randomizado (ECR), um estudo observacional e três revisões sistemáticas com meta-análise (RSMA), sendo uma

Surface morphology and protective effect of Hericium erinaceus polysaccharide on cyclophosphamide-induced immunosuppression in mice.

The aim of this study was to investigate the surface morphological features and in vivo immunomodulatory activities of a hetero polysaccharide fraction (HEP-W) from Hericium erinaceus. SEM and AFM images revealed that HEP-W displayed a flexible random coil conformation, and these flexible winding chains further formed continuous fiber network structure. Meanwhile, Congo red assay and XRD further proved that HEP-W mainly exhibited amorphous structure with non-triple-helical conformation in solution. In vivo immunomodulatory experiments demonstrated that HEP-W possessed protective effects against cyclophosphamide-induced immunosuppression in mice by significantly enhancing immune organ index, splenocyte proliferation, NK cell activity, IL-2 production as well as improving the macrophage phagocytosis. These findings suggest that HEP-W could be explored as a natural and effective immunomodulatory agent.

The STAT3 inhibitor galiellalactone inhibits the generation of MDSC-like monocytes by prostate cancer cells and decreases immunosuppressive and tumorigenic factors.

BACKGROUND: The transcription factor signal transducer and activator of transcription 3 (STAT3) is implicated in cancer drug resistance, metastasis, and immunosuppression and has been identified as a promising therapeutic target for new anticancer drugs. Myeloid-derived suppressor cells (MDSCs) play a major role in the suppression of antitumor immunity and STAT3 is involved in the accumulation, generation, and function of MDSCs. Thus, targeting STAT3 holds the potential of reversing immunosuppression in cancer. This study aims to investigate the effect of the small molecule STAT3 inhibitor galiellalactone on prostate cancer cell- induced generation of MDSCs from monocytes and the effect on immunosuppressive factors and inflammatory cytokines. METHODS: Primary human monocytes were cocultured with prostate cancer cells (DU145, PC3, and LNCaP-IL6) or with conditioned medium (CM) from prostate cancer cells in the presence or absence of the STAT3 inhibitor galiellalactone. Monocytes were analyzed by flow cytometry for an MDSC-like phenotype (CD14+ HLA-DR-/lo ). The secretion and gene expression of immunosuppressive factors and inflammatory cytokines from prostate cancer cells and monocytes were investigated. RESULTS: Galiellalactone blocked the prostate cancer cell-induced generation of MDSC-like monocytes with an immunosuppressive phenotype ex vivo. Monocytes cultured with CM from prostate cancer cells showed increased expression of phosphorylated STAT3. Prostate cancer cells increased the expression of interleukin1ß (IL1ß), IL10, and IL6 in monocytes which was inhibited by galiellalactone. In addition, galiellalactone decreased indoleamine 2,3-dioxygenase gene expression in monocytes. Galiellalactone reduced the levels of IL8 and granulocyte macrophage-colony stimulating factor in prostate cancer cells per se. CONCLUSION: The STAT3 inhibitor galiellalactone may prevent the prostate cancer cell-induced generation of MDSCs and reverse the immunosuppressive mechanisms caused by the interplay between prostate cancer cells and MDSCs. This is a potential new immunotherapeutic approach for the treatment of prostate cancer.

Fermented Red Ginseng Alleviates Cyclophosphamide-Induced Immunosuppression and 2,4,6-Trinitrobenzenesulfonic Acid-Induced Colitis in Mice by Regulating Macrophage Activation and T Cell Differentiation.

A variety of products have been developed with red ginseng (RG, the steamed roots of Panax ginseng Meyer). To clarify the immunomodulating effects of water-extracted RG (wRG), 50% ethanol-extracted RG (eRG), enzyme-treated eRG (ERG) and probiotic-fermented eRG (FRG), we examined their immunopotentiating and immunosuppressive effects in mice with cyclophosphamide (CP)-induced immunosuppression (CI) or 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis (TC). Oral administration of RG in CI mice significantly increased blood IFN- γ levels. Treatment with RG also increased the tumoricidal effects of CI mouse splenic cytotoxic T (Tc) and NK cells against YAC-1 cells. Treatment with RGs, in particular FRG and wRG, significantly increased Th1 cell differentiation. Treatment with RG except wRG increased Treg cell differentiation. However, wRG alone increased IL-6 and IL-17 expression in the colon of CI mice. Furthermore, RG alleviated colitis in TC mice. FRG most potently suppressed TNBS-induced colon shortening, NF- κ B activation and TNF- α and IL-17 expression and increased IL-10 expression. RGs inhibited TNF- α expression and increased IL-10 expression in lipopolysaccharide-stimulated primary macrophages in vitro while the differentiation of splenic T cells into type 1 T (Th1) and regulatory T (Treg) cells was increased by FRG in vitro. In conclusion, FRG can alleviate immunosuppression and inflammation by inhibiting macrophage activation and regulating Th1 and Treg cell differentiation.

Uptake and metabolism of mizoribine, an immunosuppressant, in L5178Y-R mouse lymphoma cells in vitro and peripheral blood mononuclear cells of rats and kidney transplant recipients in vivo.

The cellular uptake of mizoribine (MZR), an immunosuppressant, and metabolism of MZR to MZR-5'- monophosphate (MZRP), an active metabolite, were evaluated in L5178Y-R mouse lymphoma cells and peripheral blood mononuclear cells (PBMCs) of rats and kidney transplant recipients (KTRs, n = 22). Real-time PCR analysis revealed the expression of ENT1 and ENT2 mRNAs, but not of CNTs, in L5178Y-R cells and rat's PBMCs. In L5178Y-R cells, the uptake of MZR was suppressed by adenosine, a substrate for ENT1 and ENT2, but not by 5-(4-nitrobenzyl)-6-thioinosine (0.1 µM), an ENT1 inhibitor. Saturable metabolism of MZR to MZRP was observed. In rats, peak plasma concentrations of MZR and peak concentrations of MZR and MZRP in PBMCs were observed 3 h after oral administration. MZR disappeared from PBMCs in parallel with plasma MZR, but the disappearance of MZRP from PBMCs appeared to be slow. In KTRs, the mean plasma concentration of MZR 3-4 h after ingestion was 3.14 µg/ml and the mean MZRP concentration in PBMCs was 16.8% of MZR, reflecting the involvement of ENT in the uptake of MZR. A linear relationship was observed between plasma MZR concentrations ranging from 1 to 6 µg/ml and PBMC's MZRP concentrations ranging from 90 to 200 ng/ml.

Ginseng extract reduces tacrolimus-induced oxidative stress by modulating autophagy in pancreatic beta cells.

We previously reported that long-term treatment with a calcineurin inhibitor impairs autophagy process in pancreatic beta cells. This study investigated the effect of Korean red ginseng extract (KRGE) on autophagy modulated by oxidative stress. In mice with tacrolimus (Tac)-induced diabetes mellitus, KRGE alleviated islet dysfunction and decreased oxidative stress and autophagic vacuoles. In vitro, KRGE decreased autophagosome formation and attenuated lysosomal degradation, accompanied by improved beta cell viability and insulin secretion. Addition of 3-methyladenine (3-MA), an inhibitor of autophagosomes, to KRGE further improved cell viability and insulin secretion, and bafilomycin A (BA), an inhibitor of lysosomal function, reduced the effects of KRGE. At the subcellular level, Tac caused mitochondrial dysfunction (impaired mitochondrial oxygen consumption, ATP production, and increased reactive oxygen species production). But KRGE improved these parameters. The effect of KRGE on mitochondrial function enhanced by 3-MA but decreased by BA, suggesting a causal relationship between KRGE effect and autophagy modulation in Tac-induced mitochondrial dysfunction. These findings indicate that KRGE modulates autophagy favorably by reducing Tac-induced oxidative stress, and this effect is closely associated with improvement of mitochondrial function.

Ameliorative effect of Trametes orientalis polysaccharide against immunosuppression and oxidative stress in cyclophosphamide-treated mice.

The present study evaluated the ameliorative effects of Trametes orientalis polysaccharide (TOP-2) against cyclophosphamide (CP) induced toxicity in mice. Intraperitoneal administration of TOP-2 not only effectively increased the thymus, spleen, heart, liver, and kidney indices, but also significantly enhanced the phagocytic activities of macrophages and splenocyte proliferation, dose-dependently. The lowered nitric oxide level of macrophages after CP treatment was elevated by TOP-2 administration. Impaired splenic natural killer cells and cytotoxic T lymphocytes activities were remarkably enhanced by TOP-2. Furthermore, the levels of interleukin-2, interferon-γ, Immunoglobulin A, immunoglobulin G, and immunoglobulin M were notably reduced by CP, while TOP-2 abolished these effects. TOP-2 could also effectively increase the total antioxidant capacity, superoxidase dismutase, catalase and glutathione peroxidase activities, and inhibit the increase in malondialdehyde level. These results indicate that TOP-2 may be of therapeutic value in ameliorating the immunosuppression and oxidative stress caused by CP treatment, through its immunomodulatory and antioxidant potential.

Costimulatory Blockade and Use of Mammalian Target of Rapamycin Inhibitors: Avoiding Injury Part 1.

Although calcineurin inhibitor drugs have been the mostly used therapy in modern immunosuppression in kidney transplantation, their effect on kidney allograft dysfunction has been suboptimal as far as preservation of kidney function is concerned. Additionally, there are metabolic and other nonmetabolic effects including increased risk of malignancy that has necessitated the use of mammalian target of rapamycin inhibitors to reduce exposure to calcineurin inhibitors. Mammalian target of rapamycin inhibitors, both sirolimus and everolimus, have been studied in several trials to facilitate preservation of kidney function with variable effects on kidney allograft function and immunogenicity. Preservation of kidney function is increasingly becoming the mainstay of immunosuppression not only in kidney transplantation, but also in extrakidney transplantation. The best kidney outcomes have been reported in calcineurin inhibitor withdrawal studies using mammalian target of rapamycin inhibitors, in kidney transplant recipients with stable kidney function. This review article summarizes data from several studies in which mammalian target of rapamycin inhibitors have been used to reduce exposure to or withdraw calcineurin inhibitors in an attempt to preserve kidney function.

Soluble Immune Response Suppressor (SIRS): Reassessing the immunosuppressant potential of an elusive peptide.

A previously studied immunosuppressive cytokine, Soluble Immune Response Suppressor (SIRS), may have relevance to current studies of immune suppression in a variety of human disease states. Despite extensive efforts using experimental models, mainly in mice, much remains to be discovered as to how autoimmune cells in mice and humans escape normal regulation and, conversely, how tumor cells evade evoking an immune response. It is the contention of this commentary that the literature pre-2000 contain results that might inform current studies. The broadly immunosuppressive protein, SIRS, was studied extensively from the 1970s to 1990s and culminated in the determination of the n-terminal 21mer sequence of this 15kDa protein which had high homology to the short neurotoxins from sea snakes, that are canonical members of the three finger neurotoxin superfamily (3FTx). It was not until 2007 that the prophylactic administration of the synthetic N-terminal peptide of the SIRS 21mer, identical to the published sequence, was reported to inhibit or delay the development of two autoimmune diseases in mice: experimental allergic encephalomyelitis (EAE) and type I diabetes (T1D). These findings were consistent with other studies of the 3FTx superfamily as important probes in the study of mammalian pharmacology. It is the perspective of this commentary that SIRS, SIRS peptide and the anti-peptide mAb, represent useful, pharmacologically-active probes for the study of the immune response as well as in the potential treatment of autoimmune, inflammatory diseases and cancer.

ß-1,3-Glucan reverses aflatoxin B1-mediated suppression of immune responses in mice.

Aflatoxin B1 (AFB1) is immunotoxic to animals and is a suspected immunosuppressant in humans. ß-1,3-Glucan (BG) consists of glucose polymers and has a variety of stimulatory effects on the immune system. In this study, we investigated the role of BG on the expression of phenotypic markers and cytokine secretion in mice exposed to AFB1. We treated animals with BG (150mg/kg, p.o., once daily) for 7days beginning at the onset of AFB1 exposure. Exposure of animals to AFB1 alone (1250µg/kg, p.o, once daily) for 7days resulted in a decrease in the percentages of lymphocyte subsets (CD4(+), GITR(+), CD8(+), TCR ß(+), CD3(+), Foxp3(+), CD4(+)Foxp3(+), and CD127(+)) as compared to an normal control (NC). However, both BG alone and BG given in conjunction with exposure to AFB1 significantly increased the percentages of these lymphocyte subsets in blood. We also observed that mice exposed to AFB1 showed reduced IL-2, TNF-α, IL-17, and IFN-γ production in the spleen and serum. In contrast, oral administration of BG alone and in conjunction with AFB1 exposure augmented the levels of these cytokines. Moreover, this finding was confirmed through RT-PCR and western blot analysis of mRNA and protein expression in the spleen. Altogether, it can be concluded from these studies that BG enhances the responses of lymphocyte subsets, including cytokine production, even when given following exposure to AFB1 immunotoxin. These data demonstrate that BG carries out its immunomodulating activity by regulating cytokine production. Our findings also provide a direction for development of specific immunomodulating therapy.

A comb-like branched ß-D-glucan produced by a Cordyceps sinensis fungus and its protective effect against cyclophosphamide-induced immunosuppression in mice.

An exopolysaccharide (EPS) was fractionated from fermentation media of a Cordyceps sinensis fungus (Cs-HK1) by ethanol precipitation at 2/5 volume ratio of ethanol/media. Its structural characteristics were elucidated by FT-IR, GC, GC-MS, 1D and 2D NMR combined with periodate oxidation, Smith degradation, partial acid hydrolysis, and methylation analysis. Furthermore, the immunomodulatory activity of EPS was evaluated by the model of cyclophosphamide-induced immunosuppression. The results from monosaccharide composition and partial acid hydrolysis indicated that EPS almost consisted of glucose excluding a trace amount of mannose. GC-MS and NMR analysis further confirmed EPS had a linear backbone of (1→3)-ß-D-glucopyranosyl residues with a single (1→6)-ß-D-glucopyranosyl side-branching unit for every three ß-D-glucopyranosyl residues, showing a comb-like ß-D-glucan with short and intensive branches, which was responsible for high viscosity. Moreover, EPS could significantly enhance immune organs and stimulate the release of major cytokines TNF-α and INF-γ, suggesting that EPS exhibited protective effect in immunocompromised mice.

Protective effect of mycophenolate mofetil against nephrotoxicity and hepatotoxicity induced by tacrolimus in Wistar rats.

Tacrolimus (TAC), a calcineurin inhibitor (CNI), is clinically used as an immunosuppressive agent in the transplant recipient; however, the use of TAC is greatly limited by its nephrotoxicity and hepatotoxicity. Mycophenolate mofetil (MMF), an inhibitor of the purine synthesis, has been used in combination with many immunosuppressive drugs such as TAC. The association TAC/MMF was used in organ transplantation to increase the efficiency and reduce acute rejection rates, but the effects of MMF on TAC-induced kidney and liver injuries are still not well investigated. The aims of this study are to explore whether MMF co-administration with TAC has a renoprotective and hepatoprotective effect against TAC-induced renal and hepatic injuries and to check the implication of oxidative stress in the MMF's possible protective effect. Our results showed that MMF (at 50 mg kg(-1) body weight (b.w.)) restored creatinine, in addition to increased AST and ALT levels by TAC (at 60 mg kg(-1) b.w.). Furthermore, MMF decreased DNA damage induced by TAC in the kidney and liver of rats as assessed by comet assay. This renoprotective and hepatoprotective effect of MMF was associated with an antioxidant effect. In fact, MMF co-treatment with TAC decreased oxidative damage induced by TAC. It reduced malondialdehyde (MDA) and protein carbonyl (PC) levels as well as catalase and superoxide dismutase (SOD) activities. We conclude that the co-administration MMF with TAC protect liver and kidney against TAC toxicity via an antioxidant process.

Inhibition of dipeptidyl peptidase IV protects tacrolimus-induced kidney injury.

Accumulating evidence shows that a gut-released hormone, the glucagon-like peptide-1 (GLP-1), has not only a glucose-lowering effect but also a renoprotective effect against kidney injury. In this study, we investigated whether a dipeptidyl peptidase (DPP) IV inhibitor has a protective effect against tacrolimus-induced renal injury. Rats were treated with tacrolimus (1.5 mg/kg, subcutaneously) and the DPP IV inhibitor MK0626 (10 or 20 mg/kg, oral gavage) for 4 weeks. MK0626 treatment attenuated tacrolimus-induced renal dysfunction, tubulointerstitial fibrosis, and arteriolopathy. Moreover, these improvements were accompanied by a reduction in oxidative stress and apoptosis. MK0626 treatment increased the blood level of GLP-1 and the level of its receptor in tissue sections but did not alter the levels of other DPP IV substrates, such as neuropeptide Y and the stromal cell-derived factor-1. These data suggest that DPP IV inhibition has an important role in the renoprotection against tacrolimus-induced nephrotoxicity via antioxidative and antiapoptotic effects and preservation of the GLP-1 system.

Phytotrapy of cyclophosphamide-induced immunosuppression.

Cyclophosphamide (CP) is a cytotoxic drug that can suppress both humoral and cellular immunity. Combining traditional medicinal herbs and chemotherapy drugs are used to improve immunity and quality of life performance status. In this paper, the effects of plant extracts, active components and their derivatives on immunosuppression of CP are discussed. Appropriate keywords were used to search through PubMed, Google Scholar, and Sciverse. All relevant results published from 1990 to date were chosen for final review. Over 50 references were found in which plant extracts, active components and their derivatives have been tested for their immune protective effects against CP-induced immune toxicity. Although there are several plants shown to be effective in animal models, no study was carried out on human subjects. According to the results; we can claim that plants and their active ingredients are good candidates for alternative adjuvant chemotherapy in reducing the immunotoxicity of CP.

Cuscuta chinensis Ameliorates Immunosuppression and Urotoxic Effect of Cyclophosphamide by Regulating Cytokines - GM-CSF and TNF-Alpha.

Cancer is the leading cause of death worldwide. Cyclophosphamide (CTX) is commonly used as anticancer drug which causes toxicity by its reactive metabolites such as acroline and phosphoramide mustard. In this study, Cuscuta chinensis (C. chinensis) (family: Convolvulaceae) was assessed for ability to restore mice against CTX-induced toxicity. Coadministration of C. chinensis extract (10 mg/kg BW, IP, daily) for ten consecutive days reduced CTX-induced (25 mg/kg BW, IP, daily) toxicity. Treatment with C. chinensis extract significantly (p < 0.01) increased the relative organ weight and body weight. Moreover, administration of C. chinensis extract significantly increased bone marrow cellulatity and α-esterase activity in CTX-treated mice which suggested its protective role on the hematopoietic system. The GSH content was drastically reduced by CTX administration in urinary bladder which was enhanced by treatment with C. chinensis extract, indicating that preventing acroline-mediated tissue damage or cell toxicity and also the extract decreased the urinary bladder nitric oxide (NO) level which proves recovery over urinary tract injury associated with CTX treatment. The administration of C. chinensis extract decreased serum urea, creatinine, and bilirubin levels when compared to CTX-alone-treated group. Histopathological analysis of the urinary bladder of CTX-alone-treated group showed necrotic damage whereas the C. chinensis-treated group showed normal bladder architecture. The above data clearly demonstrates chemoprotective role of C. chinensis against CTX-induced toxicities by regulating antioxidant and inflammatory mediators.

[PHENOTROPIL AS MODULATOR OF CYTOKINE LEVEL UNDER CONDITIONS OF EXPERIMENTAL IMMUNOPATHOLOGY].

We have studied the effect of phenotropil on the level of cytokines IL-1, IL-4, and IL-6 under conditions of immunosuppression induced by cyclophosphamide (150 mg/kg, single i.p. injection) and under immune system hyperreactivity caused by lipopolysaccharide (LPS of Pseudomonas aeruginosa, 100 gg/kg, single i.p. injection). The experiments were performed on 48 Wistar male rats aged 6-8 months. Each series was carried out according to similar schedule onree groups of animals: group 1, intact animals; group 2 received cyclophosphamide (series 1) or LPS (series 2); group 3 received phenotropil on the background of immunomodulators. Phenotropil (synthesized at the Department of Organic Chemistry of the A. I. Herzen St. Petersburg Pedagogical University) was administered in a dose of 25 mg/kg (i.p.) for 5 days, starting 2 days before the introduction of immune system activity modulatos. The levels of serum cytokines IL-1ß, IL-4, and IL-6 were determined by ELISA "sandwich" with the use of monoclonal antibodies (ELISA kits of BenderMedsystems company; instrument Uniplan AIRF-01 of Picon company, Moscow) at a wavelength of 450 nm. In case of immunodeficiency caused by cyclophosphamide, there was a significant decrease in IL-1ß, IL-4 and IL-6, but the introduction of phenotropil largely restored their levels. Upon stimulation with LPS, the immune response showed inc- rease in pro-inflammatory cytokines IL-ß, IL-6 and decrease in IL-4. Under these conditions, phenotropil restored the normal levels of IL-1ß, IL-6 and caused an increase in the level of IL-4.

Lipid nanoparticles protect from edelfosine toxicity in vivo.

Edelfosine, an alkyl-lysophospholipid antitumor drug with severe side-effects, has previously been encapsulated into lipid nanoparticles (LN) with the purpose of improving their toxicity profile. LN are made of lipids recognized as safe by the Food and Drug Administration (FDA) and, therefore, these systems are generally considered as nontoxic vehicles. However, toxicity studies regarding the use of LN as vehicles for drug administration are limited. In the present study, we investigated the in vivo toxicity of free edelfosine, and the protection conferred by LN. The free drug, non-loaded LN and edelfosine-loaded LN were orally administered to mice. Our results show that the oral administration of the free drug at 4 times higher than the therapeutic dose caused the death of the animals within 72h. Moreover, histopathology revealed gastrointestinal toxicity and an immunosuppressive effect. In contrast, LN showed a protective effect against edelfosine toxicity even at the higher dose and were completely safe. LN are, therefore, a safe vehicle for the administration of edelfosine by the oral route. The nanosystems developed could be further used for the administration of other drugs.

Water channel in the binding site of a high affinity anti-methotrexate antibody.

In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 107 M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻5 s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests.

Tacrolimus-induced hypertension and nephrotoxicity in Fawn-Hooded rats are attenuated by dual inhibition of renin-angiotensin system.

Chronic immunosuppressive therapy is often complicated by the development of both arterial hypertension and renal dysfunction. The principal aim of this study was to assess the effects of dual inhibition of renin-angiotensin system (RAS) and other antihypertensive treatment on blood pressure and renal function in normotensive and hypertensive Fawn-Hooded (FH) strains during chronic calcineurin inhibitor (CNI) administration. Combinations of perindopril (5 mg kg(-1) per day) and losartan (50 mg kg(-1) per day) or amlodipine (6 mg kg(-1) per day) and metoprolol (80 mg kg(-1) per day) were administered to normotensive (FHL) and hypertensive (FHH) rats, fed with diet containing tacrolimus (Tac; 12 mg kg(-1) per day). Tac-induced arterial hypertension in both animal strains (FHL: 151±4; FHH: 198±6 mm Hg) was prevented by dual RAS inhibition (FHL: 132±3 mm Hg, P<0.05; FHH: 153±3 mm Hg, P<0.05) as well as by a combination of amlodipine and metoprolol (FHL: 136±3 mm Hg, P<0.05; FHH: 166±4 mm Hg, P<0.05). However, significant nephroprotection was observed only in animals on dual RAS inhibition where albuminuria was reduced in both FHL (51.1±3.9 vs. 68.3±4.5 µg per day; P<0.05) and FHH rats (13.1±0.3 vs. 18.8±0.7 mg per day; P<0.05). We also found Tac-induced enhancement in renal angiotensin II activity that was significantly reduced by dual RAS inhibition in both FHL (63.5±3.2 vs. 23.1±3.0 fmol g(-1)) and FHH (79.8±8.5 vs. 32.2±5.8 fmol g(-1)). In addition, histological analysis revealed that RAS inhibition noticeably diminished glomerulosclerosis and tubulo-interstitial injury. This study indicates that dual blockade of RAS significantly attenuates Tac-induced arterial hypertension and nephrotoxicity in FH rats and further supports the notion that RAS inhibitors display efficient renoprotective properties during CNI treatment.

Deliberate fingolimod overdose presenting with delayed hypotension and bradycardia responsive to atropine.

INTRODUCTION: Fingolimod is an immunomodulating agent used in multiple sclerosis (MS). It is a sphingosine-1-phosphate (S1P) receptor agonist prescribed for relapsing forms of MS to delay onset of physical disability. As fingolimod is known to cause first-dose bradycardia, telemetry is recommended for the first 6 h post-dose. We present the first reported case of deliberate fingolimod overdose requiring atropine administration for bradycardia and hemodynamic instability. CASE REPORT: A 33-year-old woman ingested 14 mg of fingolimod and 2 g of phenoxymethylpenicillin. After presenting to the emergency department 19 h later, she was initially hemodynamically stable (heart rate (HR) 60, blood pressure (BP) 113/89 mmHg). Two hours later, she then developed bradycardia (HR 48) and hypotension (87/57 mmHg). Despite intravenous fluids, stabilisation was only achieved after administration of atropine (300 µg). She was then admitted to the intensive care unit (ICU) for further monitoring where another episode of bradycardia and hypotension required atropine. She was monitored in the ICU for 48 h and then discharged on day 5 with no further episodes. DISCUSSION: Fingolimod is known to cause bradycardia in the first 6 h post first therapeutic dose. Following intentional overdose, onset of bradycardia occurred at 21 h post-ingestion and was associated with hypotension. Atropine was successful in treating bradycardia and associated hypotension.