HO­1 knockdown upregulates the expression of VCAM­1 to induce neutrophil recruitment during renal ischemia­reperfusion injury.

Heme oxygenase­1 (HO­1) has been reported to be upregulated following renal ischemia­reperfusion injury (IRI) and plays a key cytoprotective role; however, the underlying molecular mechanisms of its protective effects remain poorly understood. In the present study, in order to further elucidate the molecular mechanisms underlying the cytoprotective role of HO­1 in renal IRI, HO­1+/+ and HO­1+/­ mice were subjected to renal ischemia and subsequent reperfusion followed by the analysis of blood urea nitrogen (BUN) and serum creatinine (SCr) levels, the severity of histological changes, HO­1 and vascular cell adhesion molecule­1 (VCAM­1) protein expression, the mRNA expression of inflammatory factors and the effects of VCAM­1 blockade. The results of the present study demonstrated that the upregulated expression levels of VCAM­1 in HO­1+/­ mice during IRI increased the extent of renal tissue damage and activated the inflammatory response. These effects were subsequently reversed following infusion with an anti­VCAM­1 antibody. In addition, the upregulated expression of VCAM­1 in mouse glomerulus vascular endothelial cells isolated from HO­1+/­ mice increased the adhesion and migration of neutrophils, effects which were also reversed upon incubation with an anti­VCAM­1 antibody. These results indicated that HO­1 knockdown may upregulate the expression of VCAM­1 during renal IRI, resulting in increased neutrophil recruitment and the activation of the inflammatory response, thereby exacerbating renal IRI. The present study thus highlights the regulatory mechanisms of HO­1 in renal IRI and provides a potential target for the clinical treatment of IRI following renal transplantation.

Absent immune response to SARS-CoV-2 in a 3-month recurrence of coronavirus disease 2019 (COVID-19) case.

BACKGROUND: The viral persistence in patients with Coronavirus Disease 2019 (COVID-19) remains to be investigated. METHODS: We investigated the viral loads, therapies, clinical features, and immune responses in a 70-year patient tested positive for SARS-CoV-2 for 3 months. FINDINGS: The patient exhibited the highest prevalence of abnormal indices of clinical features and immune responses at the first admission, including fever (38.3 â„ƒ), decreased lymphocytes (0.83 × 109/L) and serum potassium (3.1 mmol/L), as well as elevated serum creatinine (115 µmol/L), urea (8.6 mmol/L), and C-reactive protein (80 mg/L). By contrast, at the second and the third admission, these indices were all normal. Through three admissions, IL-2 increased from 0.14 pg/mL, 0.69 pg/mL, to 0.91 pg/mL, while IL-6 decreased from 11.78 pg/mL, 1.52 pg/mL, to 0.69 pg/mL, so did IL-10 from 5.13 pg/mL, 1.85 pg/mL, to 1.75 pg/mL. The steady declining trend was also found in TNF-α (1.49, 1.15, and 0.85 pg/mL) and IFN-γ (0.64, 0.42, and 0.27 pg/mL). The threshold cycle values of RT-PCR were 26.1, 30.5, and 23.5 for ORFlab gene, and 26.2, 30.6, and 22.7 for N gene, showing the patient had higher viral loads at the first and the third admission than during the middle term of the disease. The patient also showed substantially improved acute exudative lesions on the chest CT scanning images. CONCLUSIONS: The patient displayed declining immune responses in spite of the viral shedding for 3 months. We inferred the declining immune responses might result from the segregation of the virus from the immune system.

Semisynthetic Bioluminescent Sensor Proteins for Direct Detection of Antibodies and Small Molecules in Solution.

Single-step immunoassays that can be performed directly in solution are ideally suited for point-of-care diagnostics. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection in blood plasma. Thus far, LUMABS has been limited to the detection of antibodies recognizing natural peptide epitopes. Here, we report the development of semisynthetic LUMABS sensors that recognize nonpeptide epitopes. The non-natural amino acid para-azidophenylalanine was introduced at the position of the original antibody-recognition sites as a chemical handle to enable site-specific conjugation of synthetic epitope molecules coupled to a dibenzocylcooctyne moiety via strain-promoted click chemistry. The approach was successfully demonstrated by developing semisynthetic LUMABS sensors for antibodies targeting the small molecules dinitrophenol and creatinine (DNP-LUMABS and CR-LUMABS) with affinities of 5.8 pM and 1.3 nM, respectively. An important application of these semisynthetic LUMABS is the detection of small molecules using a competitive assay format, which is demonstrated here for the detection of creatinine. Using a preassembled complex of CR-LUMABS and an anti-creatinine antibody, the detection of high micromolar concentrations of creatinine was possible both in buffer and in 1:1 diluted blood plasma. The use of semisynthetic LUMABS sensors significantly expands the range of antibody targets and enables the application of LUMABS sensors for the ratiometric bioluminescent detection of small molecules using a competitive immunoassay format.

A model of acute renal allograft rejection in outbred Yorkshire piglets.

Pigs represent a desirable animal model for the study of rejection in kidney transplantation with inbred Yucatan miniature swine (YMS) the most commonly studied strain due to well defined swine leukocyte antigen (SLA) genotypes. However, limitations to YMS may include cost and availability. Outbred Yorkshire pigs are widely available and significantly cheaper than YMS. Recent advances in SLA genotyping have allowed its application to outbred strains. On this basis, we theorized that Yorkshire pigs would be a viable alternative to YMS for the study of rejection in kidney transplantation. To address this question, we performed auto (Auto) and allotransplants (Allo) in 24 Yorkshire pigs, and assessed SLA genotypes and acute rejection after 72h. At sacrifice, and when compared to autotransplants, allotransplants had significant elevations in serum creatinine (8.4±1.3 vs 2.8±2.0mg/dL for Allo vs autotransplants, respectively) and BUN (61±9 vs 19.2±15mg/dL for Allo vs autotransplants, respectively). Warm ischemia times between the two groups did not differ (24±2.3 vs 26.4±1.4min for Auto vs Allo, respectively). There were 16 distinct SLA haplotypes identified from pigs undergoing allotransplantion, no matched donor-recipient pairs, and all allografts demonstrated rejection. Type IIA cellular rejection (Banff) was the most common. One allograft demonstrated hyperacute rejection due a blood group incompatibility. Histologically, the expression of regulatory Tcells and dendritic cells was increased in allografts. These data suggest that Yorkshire pigs may be a useful model for the study of acute rejection in experimental kidney transplantation.

Serum TARC levels are strongly correlated with blood eosinophil count in patients with drug eruptions.

BACKGROUND: This study aims to evaluate the relationship between serum thymus and activation-regulated chemokine (TARC) levels with various clinicopathological conditions in patients with drug eruptions. The value of TARC in diagnosing drug-induced hypersensitivity syndrome (DIHS) was also examined. METHODS: Study participants included 84 patients who presented with generalized eruptions suspected to be drug-related, including DIHS, Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN), maculopapular exanthema (MPE), erythema multiforme (EM), erythroderma, and toxicoderma. The correlation coefficients between serum TARC levels and clinical parameters in peripheral blood samples were calculated. RESULTS: Serum TARC levels in patients with DIHS were higher than those found in patients with SJS/TEN, MPE, EM, and toxicoderma. TARC levels had 100% sensitivity and 92.3% specificity in diagnosing DIHS, with a threshold value of 13,900 pg/mL. Serum TARC levels positively correlated with age, white blood cell (WBC) count, neutrophil count, eosinophil count, monocyte count, atypical lymphocyte (Aty-ly) count, serum blood urea nitrogen (BUN) levels, and creatinine (Cr) levels. It negatively correlated with serum total protein (TP), albumin (Alb), and estimated glomerular filtration rate (eGFR). Among these clinical parameters, blood eosinophil counts were most strongly correlated with serum TARC levels, with a correlation coefficient of 0.53. CONCLUSIONS: Serum TARC levels are well correlated with blood eosinophil counts in patients with generalized drug eruptions, indicating that Th2-type immune reactions underlie TARC production. Serum TARC measurements also have potent diagnostic value for DIHS, with high sensitivity and specificity.

Alterations of amino acid metabolism in osteoarthritis: its implications for nutrition and health.

Osteoarthritis (OA) is a common form of arthritis in humans. It has long been regarded as a non-inflammatory disease, but a degree of inflammation is now recognized as being a vital inducer of subpopulation of OA. Besides inflammation, the establishment and development of OA are associated with alterations in metabolism and profiles of amino acids (AA), including glutamate- and arginine-family AA as well as their related metabolites (e.g., creatinine, hydroxyproline, γ-aminobutyrate, dimethylarginines and homoarginine). Functional AA (e.g., glutamine, arginine, glutamate, glycine, proline, and tryptophan) have various benefits (i.e., anti-inflammation and anti-oxidation) in treatment of inflammation-associated diseases, including OA. Thus, these AA have potential as immunomodulatory nutrients for patients with inflammation-induced OA.

Urine oligosaccharide pattern in patients with hyperprolactinaemia.

Free milk-type oligosaccharides are produced during pregnancy and lactation and may have an impact on several cells in the immune system. Our aim was to investigate if patients with isolated hyperprolactinaemia, not related to pregnancy, also have increased synthesis and urinary excretion of milk-type oligosaccharides and to compare the excretion pattern with that found during pregnancy. Urine samples were collected as morning sample from 18 patients with hyperprolactinaemia, 13 healthy controls with normal prolactin levels and four pregnant women. After purification, lactose and free oligosaccharides were analysed and quantified by high-performance anion-exchange chromatography with pulsed amperometric detection. The identity of peaks was confirmed by exoglycosidase treatment and comparison with oligosaccharide standards. Prolactin was measured in serum collected between 09 and 11 a.m. by a standardized immunochemical method. Patients with hyperprolactinaemia had higher urinary excretion of lactose than normoprolactinemic controls and urinary lactose correlated positively to prolactin levels (r = 0.51, p < 0.05). Increased levels of the fucosylated oligosaccharides 2-fucosyl lactose and lacto-di-fucotetraose were found in urine from three and two patients, respectively. The acidic oligosaccharide 3-sialyl lactose was found in high amount in urine from two patients with prolactin of >10,000 mU/l. However, pregnant women in their third trimester had the highest concentration of all these oligosaccharides and excretion increased during pregnancy. This study is first to show that both lactose and certain fucosylated and sialylated milk-type oligosaccharides are increased in some patients with hyperprolactinaemia. It remains to elucidate the functional importance of these findings.

Synthesis of 1-(4-aminobutyl)-2-iminoimidazolidin-4-one aimed at preparation of a creatinine-specific antibody.

For the purpose of obtaining a creatinine-specific antibody, a creatinine derivative with 4-aminobutyl, which was served as a linker for preparing the creatinine-bovine serum albumin (BSA) conjugate, was synthesized from 4-benzylaminobutan-1-ol in 8 steps. Production of anti-creatinine antibodies was observed in two rabbits using the creatinine-BSA conjugate, although their titer was rather low.

Association of high post-transplant soluble CD30 serum levels with chronic allograft nephropathy.

The purpose of this study was to evaluate the association of post-transplant soluble CD30 (sCD30) levels, isolated or in combination with of anti-HLA class II antibodies and of serum creatinine levels, with kidney graft loss due to chronic allograft nephropathy (CAN), and type of lesions in graft biopsies for cause. The study comprised 511 first kidney graft recipients, transplanted at a single center, with a graft functioning for at least 2.8 years. A single blood sample was collected from each patient. sCD30 levels were determined by ELISA, and HLA antibodies by Luminex assay. The minimum follow-up after testing was 9.3 years. High sCD30 levels, set at sCD30 ≥ 34.15 ng/mL, the presence of HLA class II antibodies, and serum creatinine ≥ 1.9 mg/dL were independently associated with CAN-graft loss (P values <0.0001, 0.05, <0.0001, respectively), and the combined hazard ratio for CAN-graft loss was 20.2. Analyses of 166 biopsies for cause showed that high sCD30 levels and creatinine were independently associated with interstitial lesions. Post-transplant sCD30 serum levels, especially in conjunction with information regarding HLA class II antibodies and serum creatinine levels, provide valuable information regarding graft outcome and could be useful for the management of kidney transplant recipients.

Regulatory T cells participate in CD39-mediated protection from renal injury.

CD39 is an ecto-enzyme that degrades extracellular nucleotides, such as ATP, and is highly expressed on by the vasculature and circulating cells including Foxp3+ regulatory T (Treg) cells. To study the role of purinergic regulation in renal disease, we used the adriamycin nephropathy (AN) mouse model of chronic renal injury, using human CD39-transgenic (hCD39Tg) and wild-type (WT) BALB/c mice. Effects of CD39 expression by Treg cells were assessed in AN by adoptive transfer of CD4(+) CD25(+) and CD4(+) CD25(-) T cells isolated from hCD39Tg and WT mice. hCD39Tg mice were protected from renal injury in AN with decreased urinary protein and serum creatinine, and significantly less renal injury compared with WT mice. While WT CD25(+) and hCD39Tg CD25(-) T cells conferred some protection against AN, hCD39Tg CD25(+) Treg cells offered greater protection. In vitro studies showed direct pro-apoptotic effects of ATP on renal tubular cells. In conclusion, hCD39 expressed by circulating leukocytes and intrinsic renal cells limits innate AN injury. Specifically, CD39 expression by Treg cells contributes to its protective role in renal injury. These findings suggest that extracellular nucleotides mediate AN kidney injury and that CD39, expressed by Treg cells and other cells, is protective in this model.

Cystatin C is a reliable marker for estimation of glomerular filtration rate in renal transplantation: validation of a new turbidimetric assay using monospecific sheep antibodies.

BACKGROUND: The potential use of cystatin C was recently assessed in kidney transplantation. A new particle-enhanced turbidimetric immunoassay (PETIA) that uses sheep antibodies (Binding Site human cystatin C immunoassay) has been developed. Analytical performance of this new assay was evaluated. Clinical relevance was determined by comparison with a reference method in a cohort of kidney transplant patients. METHODS: First, the analytical performance of the Binding Site cystatin C kit was tested on SPA(PLUS)® and Hitachi® analyzers. Second, a comparison study was performed using SPA(PLUS)® analyzer against two other cystatin C methods (the Siemens-PENIA method on BNII® and the Dako-PETIA application on Olympus AU640®). Third, the glomerular filtration rate (GFR) was estimated using several predictive cystatin C- and creatinine-based equations and compared to GFR measured by an isotopic method (99(m)Tc-DTPA). These predictive algorithms were analyzed with respect to bias, precision and accuracy. RESULTS: Total intra-assay and inter-assay coefficients of variation were below 5%. Values obtained with the SPA(PLUS)® correlated with the Siemens-PENIA and the Dako-PETIA methods. The creatinine and cystatin C-based equation allowed reliable assessment of GFR in our population of renal transplantation. CONCLUSIONS: The use of algorithms based on cystatin C and creatinine could provide a reliable estimate of GFR in kidney transplantation.

Avaliação do ritmo de filtração glomerular / Assessment of glomerular filtration rate

A medida do ritmo de filtração glomerular (RFG) é a prova laboratorial mais utilizada na avaliação da função renal. Para tanto, usam-se marcadores indiretos, como as determinações de creatinina e cistatina C no sangue, ou procede-se à determinação do RFG propriamente dito, com indicadores como inulina; contrastes iodados, marcados ou não; e outras substâncias. O exame mais solicitado para avaliação do RFG no laboratório de patologia clínica é a dosagem da creatinina sérica. Em algumas condições, entretanto, o resultado encontrado da creatinina sérica deve ser corrigido (através da utilização de fórmulas que levam em consideração características próprias do indivíduo) para ser devidamente interpretado. De fato, a inulina ainda é vista como marcador ideal de filtração glomerular, mas seu uso não se destina à prática clínica, de modo que ainda hoje persiste a busca por testes adequados para uso rotineiro.

Glomerular filtration rate (GFR) determination is the most frequently used laboratorial test to evaluate renal function. Indirect markers as blood determination of creatinine and cystatin C are used with this purpose, as well as the direct determination of GFR, with indicators like inulin; iodated contrasts, radioactive or not; and others. Serum creatinine is the test that is most commonly performed in order to evaluate GFR in the clinical pathology laboratory. However, in some conditions, aiming at the adequate interpretation of the test, the result of serum creatinine must be corrected (by using formulas that include individual characteristics of the subjects). In fact, inulin is still seen as the ideal marker of glomerular filtration, but its use is not directed to clinical practice; then the search for appropriate tests for routine use continues.

Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody.

A procedure is described which allows the characterization of enzyme by a hybrid approach using an enzyme and an antibody. The presented method is related to the affinity determination of antibodies by the 'affinity in solution' procedure for BlAcore. The antibody is used as an indicator for the concentration of substrate, which is also the antigen. A mixture of enzyme, substrate and antibody is incubated, and an aliquot of this solution is injected periodically into a flowcell containing immobilized substrate, which is bound by the antibody, but not cleaved by the enzyme. The chosen initial concentration of substrate inhibits the binding of antibody to the immobilized substrate by 90%. During the enzymatic reaction, increased amounts of antibody bind to the surface, as the substrate concentration is decreased. With this method, the cleavage of creatinine with creatinine iminohydrolase (6 mU/ml) was monitored for up to 11 h. A recently developed monoclonal antibody against creatinine was used as the indicating protein. For the calculation of enzyme activity, the signals were compared with a calibration curve for inhibition of antibody binding to the chip by creatinine in solution.

Dermatomiositis juvenil: un reto diagnóstico / Juvenile dermatomyositis: a challenge diagnoses

La Dermatomiositis Juvenil es una enfermedad inflamatoria y degenerativa del músculo estriado capaz de ocasionar errores diagnósticos y tratamientos inadecuados, debido a que su baja incidencia conduce a una falta de conocimiento entre los médicos. Presentamos un caso, ocurrido en el Hospital Privado Centro Médico de Caracas en Febrero de 1966, en un adolescente de 13 años con un cuadro clínico característico, pero con cifras alarmantes de creatininquinasa. La respuesta terapéutica fue excelente

Neopterin measurement provides evidence of altered cell-mediated immunity in patients with depression, but not with schizophrenia.

Neopterin is a validated marker of the activation of cell-mediated immunity in a variety of disease states. We measured neopterin and creatinine concentrations in the plasma and urine of 22 schizophrenic and 26 depressed patients admitted acutely to hospital, and compared results with those in a large group of normal controls. Neopterin/creatinine ratios were normal in the schizophrenic patients, but significantly elevated in the plasma of depressed patients. In each diagnostic group, the use of psychotropic drugs before admission had no effect on the neopterin ratios observed. Our findings indicate altered cell-mediated immunity in depression.