Visceral and post-Kala-Azar dermal leishmaniasis isolates show significant difference in their in vitro drug susceptibility pattern

Visceral leishmaniasis (VL) remains a major health problem in old world, and India accounts for half of the world burden. The widespread emergence of resistance to standard drug in India poses a major obstacle in the control of leishmaniasis. Post-Kala-Azar dermal leishmaniasis (PKDL) is considered as main source of drug resistance. Experimental data indicate that resistance against newer drugs is also imminent. Therefore, in vitro studies were carried out to test minimum parasiticidal concentration of five conventional and newly introduced anti-leishmanial drugs against 20 field isolates of Leishmania donovani obtained from visceral and post-Kala-Azar dermal leishmaniasis patients of India. Study revealed wide range of variation in minimum inhibitory concentration of sodium antimony gluconate (SAG). PKDL isolates displayed significantly lower susceptibility to SAG and miltefosine than VL isolates with P value of 0.0006 and 0.0243, respectively. All clinical isolates had higher IC₅₀ value for paromomycin and miltefosine as compared to reference strain indicating their vulnerability to develop unresponsiveness. However, isolates were uniformly susceptible to pentamidine and amphotericin B. The results of gene expression analysis of AQP1 were largely in agreement with phenotypic drug sensitivity results. Interestingly, significant down-regulation of AQP1 was observed in PKDL isolates as compared to VL isolates indicating their increased propensity for drug unresponsiveness. However, no significant difference in mRNA expression of LdMT and LdRos3 gene was found for two groups. The present study unravels valuable baseline scientific data showing variation in the drug susceptibility pattern in the L. donovani isolates. The information might have impact on the management and control of Indian visceral leishmaniasis.     

Experimental selection of paromomycin and miltefosine resistance in intracellular amastigotes of Leishmania donovani and L. infantum

Although widespread resistance of Leishmania donovani and L. infantum against miltefosine (MIL) and paromomycin (PMM) has not yet been demonstrated, both run the risk of resistance selection. Unraveling the dynamics and mechanisms of resistance development is key to preserve drug efficacy in the field. In this study, resistance against PMM and MIL was experimentally selected in vitro in intracellular amastigotes of several strains of both species with different antimony susceptibility background. To monitor amastigote susceptibility, microscopic determination of IC₅₀-values and promastigote back-transformation assays were performed. Both techniques were also used to evaluate the susceptibility of field isolates from MIL-relapse patients. PMM-resistance could readily be selected in all species/strains, although promastigotes remained fully PMM-susceptible. Successful MIL-resistance selection was demonstrated only by promastigote back-transformation at increasing MIL-concentrations upon successive selection cycles. Important to note is that amastigotes with the MIL-resistant phenotype could not be visualized after Giemsa staining; hence, MIL-IC₅₀-values showed no shift. The same phenomenon was observed in a set of recent clinical isolates from MIL-relapse patients. This study clearly endorses the need to use intracellular amastigotes for PMM- and MIL-susceptibility testing. When monitoring MIL-resistance, promastigote back-transformation should be used instead of the standard Giemsa staining. In-depth exploration of the mechanistic background of this finding is warranted.     

Evaluation of Enzyme-Linked Immunosorbent Assay for Diagnosis of Post-Kala-Azar Dermal Leishmaniasis with Crude or Recombinant k39 Antigen

The diagnosis of post-kala-azar dermal leishmaniasis (PKDL), a dermatosis that provides the only known reservoir for the parasite Leishmania donovani in India, remains a problem. Timely recognition and treatment of PKDL would contribute significantly to the control of kala-azar. We evaluated here the potential of the enzyme-linked immunosorbent assay (ELISA) as a diagnostic tool for PKDL. Antigen prepared from promastigotes and axenic amastigotes with parasite isolates that were derived from skin lesions of a PKDL patient gave sensitivities of 86.36 and 92%, respectively, in the 88 PKDL cases examined. The specificity of the ELISA test was examined by testing groups of patients with other skin disorders (leprosy and vitiligo) or coendemic infections (malaria and tuberculosis), as well as healthy controls from areas where this disease is endemic or is not endemic. A false-positive reaction was obtained in 14 of 144 (9.8%) of the controls with the promastigote antigen and in 14 of 145 (9.7%) of the controls with the amastigote antigen. Evaluation of the serodiagnostic potential of recombinant k39 by ELISA revealed a higher sensitivity (94.5%) and specificity (93.7%) compared to the other two antigens used. The data demonstrate that ELISA with crude or recombinant antigen k39 provides a relatively simple and less-invasive test for the reliable diagnosis of PKDL.     

Genetic typing reveals monomorphism between antimony sensitive and resistant Leishmania donovani isolates from visceral leishmaniasis or post kala-azar dermal leishmaniasis cases in India

Resistance to pentavalent antimonials has emerged as a major hurdle to the treatment and control of visceral leishmaniasis (VL), also known as kala-azar (KA), caused by Leishmania donovani. In India, over 60?% of KA patients are unresponsive to the first-line drug sodium antimony gluconate (SAG). Resistance determinants in laboratory strains are partly known; however, the mechanism operating in field isolates is not well understood. In this study, we attempted to analyze the genetic polymorphism between SAG sensitive and resistant parasites using a total of 52 L. donovani isolates obtained either from bone marrow of VL patients or from skin lesions of post kala-azar dermal leishmaniasis (PKDL) patients that constitute an important reservoir of parasite. The clinical isolates were analyzed in comparison with L. donovani parasites from reference strains belonging to distinct geographical locations, at internal transcribed spacer 1 region; coding region of gp63 and nine microsatellite repeat regions. Our results demonstrated that both SAG resistant (n?=?26) and sensitive (n?=?19) Indian isolates, whether causing VL or PKDL, were monomorphic at all the genetic loci tested, unlike the L. donovani in East African or Leishmania infantum in Mediterranean countries where intraspecies variations exist at these loci. Further, the Indian isolates were found closest to the Kenyan isolates of L. donovani on the basis of fragment analysis of microsatellite markers.     

Potential of Direct Agglutination Test Based on Promastigote and Amastigote Antigens for Serodiagnosis of Post-Kala-Azar Dermal Leishmaniasis

Post-kala-azar dermal leishmaniasis (PKDL) is a dermal complication, a sequel to kala-azar. Diagnosis of PKDL presents a challenge due to the low parasite burden in the lesions. The direct agglutination test (DAT) based on promastigote and amastigote antigens of Leishmania donovani of indigenous isolates was developed to diagnose PKDL, and the results were compared with those of the rk39 strip test. The sensitivities of DAT for antileishmanial antibody detection, based on promastigote and amastigote antigens at a cutoff titer of 1:800 were 98.5% and 100%, respectively, with corresponding specificities of 96.5% and 100%. DAT could correctly detect 100% polymorphic cases and 95.4% macular PKDL cases. In comparison, the rk39 strip test was able to correctly diagnose 95.6% of polymorphic and 86.0% macular PKDL cases. DAT based on axenic amastigote antigen provided 100% sensitivity and specificity, making it particularly useful for macular PKDL cases, which are often missed by the rk39 strip test. Thus, DAT provides a simple, reliable, and inexpensive test for PKDL diagnosis with potential applicability in field conditions.     

Selenium and lipid subfractions in Egyptian type 2 diabetes patients

The purpose of this study was to examine the relationship between serum selenium (Se) levels and lipid subfraction among Egyptian type 2 diabetes patients and their association with the severity of the disease. The study was conducted on 60 type 2 diabetic adults with BMI <30 divided according to disease duration into two groups: group 1 with disease duration less than 5 years and group 2 with a disease duration more than 5 years. Thirty age- and sex-matched apparently healthy volunteers were considered as the control group. Serum selenium was measured by atomic absorption spectrometry lipid subfractions including small dense low density lipoprotein (sd LDL) which was measured by enzyme-linked immunosorbent assay and glycated hemoglobin (HbA1c) by high-performance liquid chromatography. All participants do not receive Se supplementation. The mean serum Se level in participants with diabetes was as follows: group 2?=?62.70?±?5.73, group 1?=?70.58?±?4.158, and control subjects?=?79.80?±?5.37 μg/l (p?=?0.00). Se was found to be an independent protective factor with an OR of 0.29 and 95 % CI of 0.06–1.3. Mean serum sd LDL in participants with diabetes was as follows: group 2?=?43.81?±?13.70, group 1?=?25.77?±?5.28, and control group?=?15.99?±?5.32 (p?=?0.00). Correlation study, between studied parameters, revealed positive correlation between sd LDL and apolipoprotein B (Apo B) (r?=?0.730, p?=?0.001). On the other hand, negative correlation was encountered between apolipoprotein A (Apo A) and Apo B (r?=??0.514, p?=?0.001) as well as Apo A and sd LDL (r?=??0.697, p?=?0.001). Selenium correlated negatively with both Apo B (r?=??0.669, p?=?0.001) and sd LDL (r?=??0.671, p?=?0.001) and positively with Apo A (r?=?0.513, p?=?0.001). In a sample of the Egyptian population, low serum Se levels were positively associated with the prevalence of diabetes. Until findings from prospective studies and randomized controlled trials are available, Se intake, including Se supplementation, should be recommended for primary or secondary diabetes prevention in populations with inadequate selenium status.     

Leishmania amazonensis Amastigotes Trigger Neutrophil Activation but Resist Neutrophil Microbicidal Mechanisms

Neutrophils are the first cells to infiltrate to the site of Leishmania promastigote infection, and these cells help to reduce parasite burden shortly after infection is initiated. Several clinical reports indicate that neutrophil recruitment is sustained over the course of leishmaniasis, and amastigote-laden neutrophils have been isolated from chronically infected patients and experimentally infected animals. The goal of this study was to compare how thioglycolate-elicited murine neutrophils respond to L. amazonensis metacyclic promastigotes and amastigotes derived from axenic cultures or from the lesions of infected mice. Neutrophils efficiently internalized both amastigote and promastigote forms of the parasite, and phagocytosis was enhanced in lipopolysaccharide (LPS)-activated neutrophils or when parasites were opsonized in serum from infected mice. Parasite uptake resulted in neutrophil activation, oxidative burst, and accelerated neutrophil death. While promastigotes triggered the release of tumor necrosis factor alpha (TNF-α), uptake of amastigotes preferentially resulted in the secretion of interleukin-10 (IL-10) from neutrophils. Finally, the majority of promastigotes were killed by neutrophils, while axenic culture- and lesion-derived amastigotes were highly resistant to neutrophil microbicidal mechanisms. This study indicates that neutrophils exhibit distinct responses to promastigote and amastigote infection. Our findings have important implications for determining the impact of sustained neutrophil recruitment and amastigote-neutrophil interactions during the late phase of cutaneous leishmaniasis.     

Genetic susceptibility to visceral leishmaniasis in The Sudan: linkage and association with IL4 and IFNGR1

Longitudinal studies in Sudan show ethnic differences in incidence and clinical phenotypes associated with Leishmania donovani. Immunologically, bias in type 1 vs type 2 cytokine responses is important. To determine whether polymorphisms at IL4/IL9 or IFNGR1 contribute to susceptibility, we examined 59 multicase families of visceral leishmaniasis (VL) with/without post Kala-azar dermal leishmaniasis (PKDL). Multipoint nonparametric analysis (Allegro) linked IL4/IL9 to VL per se (P=0.002). Transmission disequilibrium testing with robust variance estimates confirmed association in the presence of linkage between VL per se and IL4 (P=0.008) but not IL9. Stepwise logistic regression analysis showed both IL4RP2 and IL4RP1 markers contributed significantly to the association, suggesting a common disease-associated haplotype. In contrast, IFNGR1 was linked (P=0.031) and associated (P=0.007) to PKDL but not VL or VL per se. Hence, polymorphism in a type 2 cytokine gene influences underlying susceptibility to VL, whereas IFNGR1 is specifically related to susceptibility to PKDL.     

Human papillomavirus genotype and viral load agreement between paired first-void urine and clinician-collected cervical samples

The performance and acceptability of first-void urine as specimen for the detection of HPV DNA in a Belgian referral population was evaluated using an optimized sample collection and processing protocol. One hundred ten first-void urine and cervical samples were collected from 25- to 64-year-old women who were referred for colposcopy (January–November 2016). Paired samples were analyzed by the Riatol qPCR HPV genotyping assay. Acceptability data were gathered through questionnaires (NCT02714127). A higher high-risk HPV DNA prevalence was observed in first-void urine (n?=?76/110) compared to cervical samples (n?=?73/110), with HPV31 and HPV16/31 being most prevalent correspondingly. For both any and high-risk HPV DNA, good agreement was observed between paired samples (Cohen’s Kappa of 0.660 (95% CI: 0.486–0.833) and 0.688 (95% CI: 0.542–0.835), respectively). In addition, significant positive correlations in HPV copies (per microliter of DNA extract) between paired samples were observed for HPV16 (r_s?=?0.670; FDR (false discovery rate)-adjusted p?=?0.006), HPV18 (r_s?=?0.893; FDR-adjusted p?=?0.031), HPV31 (r_s?=?0.527; FDR-adjusted p?=?0.031), HPV53 (r_s?=?0.691; FDR-adjusted p?=?0.017), and HPV68 (r_s?=?0.569; FDR-adjusted p?=?0.031). First-void urine sampling using a first-void urine collection device was preferred over a clinician-collected cervical sample. And mostly, first-void urine sampling at home was favored over collection at the clinic or the general practitioner’s office. First-void urine sampling is a highly preferred, non-invasive method that ensures good agreement in HPV DNA (copies) with reference cervical samples. It is particularly interesting as a screening technique to reach non-participants, and its clinical performance should be further evaluated.     

Diagnostic and Prognostic Potential of a Competitive Enzyme-Linked Immunosorbent Assay for Leishmaniasis in India

A Leishmania donovani species-specific monoclonal antibody (monoclonal antibody D2) was evaluated for its diagnostic and prognostic potential by a competitive enzyme-linked immunosorbent assay (C-ELISA) in sera from Indian patients with visceral leishmaniasis (VL) and seven patients with post-kala-azar dermal leishmaniasis (PKDL). These results were compared with those obtained by microscopy with Giemsa-stained tissue smears and a direct enzyme-linked immunosorbent assay (direct ELISA) with crude parasite antigen. Of 121 patients with clinically diagnosed VL examined, 103 (85.1%) were positive and 11 (9.1%) were negative by all three methods. An additional 7 (5.8%) who were negative by microscopy were positive by both C-ELISA and direct ELISA. Seven PKDL patients were also examined and were found to be positive by all three methods. Analysis of the chemotherapeutic response to sodium antimony gluconate of these 110 serologically positive VL patients showed that 57 (51.8%) were drug responsive and 53 (48.2%) were drug resistant. The C-ELISA with sera from 20 longitudinally monitored VL patients before and after chemotherapy showed a significant decrease in percent inhibition of monoclonal antibody D2 in drug-responsive patients. However, in drug-unresponsive patients, the percent inhibition of D2 was unchanged or was slightly increased. Our results therefore indicate (i) the applicability of L. donovani species-specific monoclonal antibody D2 for sensitive and specific serodiagnosis by C-ELISA, (ii) that the C-ELISA is more sensitive than microscopy, especially for early diagnosis, (iii) that L. donovani is still the main causative agent of VL, irrespective of the chemotherapeutic response, and (iv) that the C-ELISA can be used to evaluate the success of drug treatment.     

Evaluation of enzyme-linked immunosorbent assay for diagnosis of post-kala-azar dermal leishmaniasis with crude or recombinant k39 antigen

The diagnosis of post-kala-azar dermal leishmaniasis (PKDL), a dermatosis that provides the only known reservoir for the parasite Leishmania donovani in India, remains a problem. Timely recognition and treatment of PKDL would contribute significantly to the control of kala-azar. We evaluated here the potential of the enzyme-linked immunosorbent assay (ELISA) as a diagnostic tool for PKDL. Antigen prepared from promastigotes and axenic amastigotes with parasite isolates that were derived from skin lesions of a PKDL patient gave sensitivities of 86.36 and 92%, respectively, in the 88 PKDL cases examined. The specificity of the ELISA test was examined by testing groups of patients with other skin disorders (leprosy and vitiligo) or coendemic infections (malaria and tuberculosis), as well as healthy controls from areas where this disease is endemic or is not endemic. A false-positive reaction was obtained in 14 of 144 (9.8%) of the controls with the promastigote antigen and in 14 of 145 (9.7%) of the controls with the amastigote antigen. Evaluation of the serodiagnostic potential of recombinant k39 by ELISA revealed a higher sensitivity (94.5%) and specificity (93.7%) compared to the other two antigens used. The data demonstrate that ELISA with crude or recombinant antigen k39 provides a relatively simple and less-invasive test for the reliable diagnosis of PKDL.     

Targeting the association of calgranulin B (S100A9) with insulin resistance and type 2 diabetes

Calgranulin B (S100A9) was recognized as a candidate type 2 diabetes (T2D) gene in the genomic profiling of muscle from a rodent model of T2D and identifying the human orthologs of genes localized in T2D susceptibility regions. Circulating and S100A9 expressions in muscle and adipose tissue, isolated fat cells, and mouse models were evaluated. A common 5′-upstream single-nucleotide polymorphism (SNP; rs3014866) for S100A9 was analyzed, as well as the effects of weight loss and treatments in vitro with recombinant S100A9. S100a9 expression was increased in muscle of diabetic mice (1.6-fold, p?=?0.002), and in muscle from subjects with impaired glucose tolerance (～4-fold, p?=?0.028; n?=?34). The rs3014866 SNP was associated with circulating S100A9 and the risk of T2D, having TT carriers at 28 % (p?=?0.03) lower risk (n?=?1,450). Indeed, increased circulating S100A9 (～4-fold, p?=?0.03; n?=?206) and subcutaneous (2-fold, p?=?0.01) and omental (1.4-fold, p?=?0.04) S100A9 gene expressions (n?=?83) in TT carriers run in parallel to decreased fasting glucose and glycated hemoglobin. Accordingly, metformin led to increased S100A9 mRNA in ex vivo-treated adipose tissue explants (n?=?5/treatment). Otherwise, obese subjects showed a compensatory increase in circulating and S100A9 expressions in adipose (n?=?126), as further demonstrated by decreased levels after diet- (?34 %, p?=?0.002; n?=?20) and surgery-induced (?58 %, p?=?0.02; n?=?8) weight loss. Lipopolysaccharide led to increased S100A9 in adipose from mice (n?=?5/treatment) while recombinant S100A9 downregulated inflammation in adipocytes (n?=?3/treatment). Current findings support the strategy of testing differentially expressed genes in mice and human orthologs associated with T2D. The increased S100A9 reported for obesity and insulin resistance may be envisioned as a compensatory mechanism for inflammation.     

Use of PCR for Diagnosis of Post-Kala-Azar Dermal Leishmaniasis

Microscopy and PCR were compared for use in the diagnosis of post-kala-azar dermal leishmaniasis (PKDL) in 63 patients. Aspirates of lymph nodes (samples from 52 patients), skin (23 samples), and bone marrow (18 samples) were used. For 11 patients lymph node aspiration could be repeated 6 months after they recovered from PKDL. During active PKDL, PCR was positive for 42 of 52 (80.8%) lymph node aspirates and 19 of 23 (82.7%) skin aspirates, whereas microscopy was positive for only 9 of 52 (17.3%) lymph node aspirates and 7 of 23 (30.4%) skin aspirates. PCR was always positive when parasites were seen by microscopy. When the results obtained with lymph node and skin aspirates from the same patient (n = 16) were compared, there was complete agreement. Bone marrow samples were negative by microscopy and PCR for 16 patients and positive by both methods for 1 patient; for one sample only the PCR was positive. PCR confirmed the co-occurrence of visceral leishmaniasis and PKDL in one patient and confirmed the suspicion of this co-occurrence in the other patient. After recovery, no parasites were found by microscopy, but 2 of 11 (18.2%) samples were still positive by PCR. Thirty negative controls were all found to be PCR negative, and 15 positive controls were all PCR positive. Cross-reactions with Mycobacterium leprae could be ruled out. In conclusion, PCR with inguinal lymph node or skin aspirates is suitable for confirming the clinical diagnosis of PKDL. In some patients, lymph node aspirates are probably preferred because aspiration of material from the skin may leave scars.     

Activity of the julocrotine, a glutarimide alkaloid from Croton pullei var. glabrior, on Leishmania (L.) amazonensis

The antiproliferative effect of julocrotine, an alkaloid isolated from Croton pullei var. glabrior (Euphorbiaceae), was studied in the macrophage amastigote and promastigote stages of the protozoan Leishmania (L.) amazonensis, which causes cutaneous leishmaniasis in the New World. Julocrotine showed a dose-dependent effect against the amastigote and promastigote forms, where 79 μM julocrotine inhibited promastigote growth by 54%, with an IC50 of 67 μM. To analyze the antiamastigote activity of the drug, murine peritoneal macrophages infected with L. amazonensis promastigotes were treated with different concentrations of julocrotine. An 80% inhibition of amastigote development was observed using 79 μM julocrotine for 72 h, with an IC50 of 19.8 μM. In addition, ultrastructural observation of the parasites showed a significant reduction in the number of amastigotes in the parasitophorous vacuoles and morphological changes in promastigotes, such as swelling of the mitochondrion, chromatin condensation, presence of membranous structures near the Golgi complex, and some vesicle bodies in the flagellar pocket. A colorimetric assay (MTT), which measures cytotoxic metabolic activity, showed that macrophages maintain their viability after treatment with the drug. These results suggest that julocrotine effectively inhibits the growth of parasites and does not have any cytototoxic effects on the host cell.     

Is the IL-10 −819 Polymorphism Associated with Visceral Leishmaniasis?

Previous investigations demonstrated that immune responses play critical roles in the defense against visceral leishmaniasis (VL). A key regulator of immune responses is the cytokine, IL-10 and polymorphisms within its promoter which could alter its expression. Thus, the aim of this study was to examine the correlation between polymorphism at the ?819 position of the IL-10 gene and VL in a selected Iranian population. This cross-sectional study was performed on 100 patients with clinical presentation of VL and seropositive for the leishmania (group 1), 62 patients without clinical presentation but seropositive (group 2), and 128 healthy controls (group 3). The IL-10 ?819 polymorphism was evaluated using the PCR-RFLP technique. The anti-leishmania antibody titration was assessed using an immunofluorescence assay. Our results showed that the polymorphism at IL-10 ?819 (C/T) position was significantly associated with VL, and C/T genotype was significantly higher in VL patients when compared to groups 2 and 3 (p?<?0.001). However, the results demonstrated that the C and T alleles were not associated with VL (p?=?0.855). The data presented here confirm the results of previous reports that polymorphisms at the ?819 position of the IL-10 gene can influence susceptibility to VL suggesting that the C/T genotype may be considered as a risk factor for the disease.     

High levels of plasma IL-10 and expression of IL-10 by keratinocytes during visceral leishmaniasis predict subsequent development of post-kala-azar dermal leishmaniasis

Some patients develop post-kala-azar dermal leishmaniasis (PKDL) after they have been treated for the systemic infection kala-azar (visceral leishmaniasis). It has been an enigma why the parasites cause skin symptoms after the patients have been successfully treated for the systemic disease. We report here that PKDL development can be predicted before treatment of visceral leishmaniasis, and that IL-10 is involved in the pathogenesis. Before treatment of visceral leishmaniasis, Leishmania parasites were present in skin which appeared normal on all patients. However, IL-10 was detected in the keratinocytes and/or sweat glands of all patients who later developed PKDL (group 1) and not in any of the patients who did not develop PKDL (group 2). Furthermore, the levels of IL-10 in plasma as well as in peripheral blood mononuclear cell culture supernatants were higher in group 1 than in group 2.     

Peri-Infarct Zone Characterized by Cardiac Magnetic Resonance Imaging is Directly Associated with the Inflammatory Activity During Acute Phase Myocardial Infarction

Enhanced systemic inflammatory activity (SIA) during myocardial infarction (MI) and the extent of the peri-infarct zone characterized by cardiac magnetic resonance imaging (CMRi) are both associated with increased risk of life-threatening arrhythmias and sudden cardiac death. The present study investigated the existence of association between these two phenomena in 98 patients (55?±?10 years) with ST segment elevation MI. Plasma levels of C-reactive protein (CRP), interleukin-2 (IL-2), and tumor necrosis factor (TNF) were measured on admission (D1) and on the fifth day post-MI (D5). CMRi was performed 2 weeks after MI to quantify peri-infarct zone (PIZ). Between D1 and D5, the increase in CRP (6.0 vs. 5.6 times; p?=?0.02), IL-2 (3.6 vs. 3.4 times; p?=?0.04) and tumor necrosis factor type α (TNF-α; 4.6 vs. 3.9 times; p?=?0.001) were higher in patients with PIZ above the median than in the counterparts. PIZ was correlated with CRP-D5 (r?=?0.69), delta-CRP (r?=?0.7), IL-2-D5 (r?=?0.5), delta-IL-2 (r?=?0.6), TNF-α (r?=?0.5), delta-TNF-α (r?=?0.4; p?=?0.0001). Enhanced activation of SIA during the acute phase of MI is directly related with generation of PIZ.     

Concentrations of immunoglobulin free light chains in cerebrospinal fluid predict increased level of brain atrophy in multiple sclerosis

Recent studies showed that B cells play a major role in the pathogenesis of neurodegeneration in multiple sclerosis (MS). In this study, we aimed to determine the possible link between immunoglobulin free light chains (FLC) and brain atrophy in patients with MS. Ninety-two patients (32 males and 60 females) with MS were included. Kappa and lambda FLC concentrations in serum and cerebrospinal fluid (CSF) samples of MS patients were measured using ELISA assay. FLC quotients (Q-k and Q-λ, respectively) were calculated. In a cross-sectional group (n?=?92), the MRI data were acquired within 6 months from the date of the lumbar puncture. Twenty patients from this cohort performed a follow-up MRI after 1 year of observation. Brain volumes were calculated with SIENAX and the brain atrophy (percentage brain volume change (PBVC)) was assessed with SIENA. Spearman’s test was performed to assess correlations. We have shown statistically significant correlation of Expanded Disability Status Scale (EDSS) level with normalized brain volume (NBV, r?=???0.2721, p?=?0.0062), white matter volume (WMV, r?=???0.2425, p?=?0.015), and gray matter volume (GMV, r?=???0.216, p?=?0.0309). Multiple Sclerosis Severity Score (MSSS) score correlated with NBV (r?=???0.2521, p?=?0.0352) and WMV (r?=???0.315, p?=?0.0079). Neither EDSS, nor MSSS scores correlated with the age of patients and relapse rate during the first year and 5 years. In our study, we found statistically significant correlations of k-FLC in the CSF with NBV (r?=???0.311, p?=?0.003) and with GMV (r?=???0.213, p?=?0.0423). Q-k correlated only with NBV (r?=???0.340, p?=?0.006) and Q-λ were negatively correlated with WMV (r?=???0.366, p?=?0.003). We did not find correlations of k-FLC in CSF, λ-FLC in CSF, Q-k, and Q-λ with duration of MS course, EDSS, MSSS, number of relapses during the first year, and during the first 5 years of disease. Additionally, we subdivided the study population in accordance with level of k-FLC CSF, Q-k, and Q-λ on the 25th and 75th percentile subgroups (25-k-FLC_CSF/75-k-FLC_CSF; 25-λ-FLC_CSF/75-λ-FLC_CSF; 25-Q-k/75-Q-k; 25-Q-λ/75-Q-λ). We found statistically significant difference of NBV and GMV between 25-k-FLC_CSF and 75-k-FLC_CSF subgroups (p?=?0.0047, p?=?0.0297 respectively), NBV between 25-Q-k and 75-Q-k subgroups (p?=?0.038), and NBV and WMV between 25-Q-λ and 75-Q-λ subgroups (p?=?0.0446, p?=?0.0026 respectively). PBVC in the prospective group showed negative correlation with kappa FLC in the CSF (r?=???0.4853, p?=?0.0301) and Q-k (r?=???0.6132, p?=?0.0224), but not with other clinical, epidemiological data. In this study, we showed a strong negative correlation of k-FLC, Q-k, and Q-λ with brain atrophy in MS patients. Additionally, patients with high concentration of FLC had lower brain volumes. We did not find correlations of FLC with the relapse rate, age of patients, and MS time course. In the prospective group, the rate of atrophy was correlated with k-FLC and Q-k. We suggest that level of intrathecal production of FLC can be a good prognostic biomarker for MS.     

Association Study of Common Genetic Variants in Pre-microRNAs in Patients with Ulcerative Colitis

Background

Common single-nucleotide polymorphisms (SNPs) in microRNAs (miRNA) have been shown to be associated with susceptibility to several human diseases. We evaluated the associations of three SNPs (rs11614913, rs2910164, and rs3746444) in pre-miRNAs (miR-196a2, miR-146a, and miR-499) with the risk of ulcerative colitis (UC) in a Japanese population.

Methods

The rs11614913 (T?>?C), rs2910164 (C?>?G), and rs3746444 (A?>?G) SNPs were genotyped in 170 UC and 403 control subjects.

Results

The rs3746444 AG genotype was significantly higher among the UC group (odds ratio (OR)?=?1.51, 95% CI?=?1.03?C2.21, p?=?0.037). The rs3746444 AG genotype was associated with onset at an older age (OR?=?1.70, 95% CI?=?1.04?C2.78, p?=?0.035), left-sided colitis and pancolitis (left-sided colitis, OR?=?2.10, 95% CI?=?1.12?C3.94, p?=?0.024; pancolitis, OR?=?1.81, 95% CI?=?1.09?C3.01, p?=?0.028, left-sided colitis?+?pancolitis, OR?=?1.91, 95% CI?=?1.26?C2.92, p?=?0.003), higher number of times hospitalized (OR?=?2.63, 95% CI?=?1.22?C5.69, p?=?0.017), steroid dependence (OR?=?2.63, 95% CI?=?1.27?C5.44, p?=?0.014), and refractory phenotypes (OR?=?2.76, 95% CI?=?1.46?C5.21, p?=?0.002) while the rs3746444 AA genotype was inversely associated with the number of times hospitalized (2??, OR?=?0.36, 95% CI?=?0.17?C0.79, p?=?0.012), steroid dependence (OR?=?0.42, 95% CI?=?0.21?C0.88, p?=?0.021), and refractory phenotypes (OR?=?0.38, 95% CI?=?0.20?C0.72, p?=?0.003). The rs1161913 TT genotype also held a significantly higher risk of refractory phenotype (T/T vs. T/C?+?C/C, OR?=?2.21, 95% CI?=?1.17?C4.18, p?=?0.016).

Conclusions

Our results provided the first evidence that rs3746444 SNP may influence the susceptibility to UC, and both rs3746444 and rs11614913 SNPs may influence the pathophysiological features of UC.     

Aberrations of MET are associated with copy number gain of EGFR and loss of PTEN and predict poor outcome in patients with salivary gland cancer

Hepatocyte growth factor receptor (MET) is a key driver of oncogenic transformation. Copy number gain and amplification of MET positively enhance tumour growth, invasiveness and metastasis in different cancer types. In the present study, 266 carcinomas of the major and minor salivary glands were investigated for genomic MET status by fluorescence in situ hybridization and for protein expression by immunohistochemistry. Results were matched with clinicopathological parameters, long-term survival and the status of epidermal growth factor receptor (EGFR) and phosphatase and tensin homologue (PTEN). Low polysomy (n?=?42), high polysomy (n?=?27), amplification (n?=?2) and deletion (n?=?18) were found as aberrations of genomic MET in certain subtypes. MET aberrations were associated with increased patient age (>70 years, p?=?0.003), male gender (p?=?0.01), increased tumour size (p?=?0.002), lymph node metastases (p?<?0.001), high-grade malignancy (p?<?0.001) and unfavourable overall survival (p?<?0.001). Both copy number gain (p?<?0.001) and deletion (p?=?0.031) of MET correlated with copy number gain of EGFR. Tumours with genomic loss of PTEN (n?=?48) concurrently presented aberration of genomic MET (p?<?0.001). MET gene status significantly correlated with protein status (p?=?0.038). In conclusion, gain but also loss of genomic MET activity correlates with aggressive tumour growth, nodal metastasis and worse overall survival in salivary gland cancer. Moreover, aberrations of MET are associated with EGFR and PTEN signalling and might possess relevance for targeted therapies of salivary gland carcinomas in the future.