Magnitude of Gene Mutations Conferring Drug Resistance in Mycobacterium Tuberculosis Isolates from Lymph Node Aspirates in Ethiopia

Objective: Resistance to drugs is due to particular genomic mutations in the specific genes of Mycobacterium tuberculosis. Timely genetic characterization will allow identification of resistance mutations that will optimize an effective antibiotic treatment regimen. We determine the magnitude of gene mutations conferring resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among tuberculosis (TB) lymphadenitis patients.Methods: A cross sectional prospective study was conducted among 226 M.tuberculosis isolates from culture positive lymph node aspirates collected from TB lymphadenitis patients between April 2012 and May 2012. Detection of mutations conferring resistance to drugs was carried out using GenoType^® MTBDRplus and GenoType® MTBDRsl assay.Results: Out of the 226 strains, mutations conferring resistance to INH, RMP, multidrug resistance tuberculosis (MDR-TB) and EMB were 8, 3, 2 and 2 isolates, respectively. There was no isolated strain that showed mutation in the inhA promoter region gene. All INH resistant strains had mutations in the katG gene at codon 315 with amino acid change of S315T1. Among rifampicin resistant strains, two isolates displayed mutations at codon 531 in the rpoB gene with amino acid change of S531L and one isolate was by omission of wild type probes at Q513L. According to mutations associated with ethambutol resistance, all of the isolates had mutations in the embB gene with aminoacid change of M306I. All isolates resistant to INH, RMP and MDR using BacT/AlerT 3D system were correctly identified by GenoType® MTBDRplus assay.Conclusion: We observed mutations conferring resistance to INH at S315T1 of the katG gene, RMP at S531L and Q513L in the rpoB genes and EMB at M306I of the embB gene. In the absence of conventional drug susceptibility testing, the effort to develop easy, rapid and cost effective molecular assays for drug resistance TB monitoring is definitely desirable and the GenoType® MTBDRplus assay was found to be a useful method for diagnosis of resistance to INH, RMP and MDR from lymph node aspirates. Further molecular cluster analysis to determine transmission dynamics of mutated strain is required.     

Development of an in-house ELISA for detection of antibodies against Enterococcus cecorum in Pekin ducks

ABSTRACT

Enterococcus cecorum (EC) is known to cause skeletal lesions in broiler chickens and also systemic infections in Pekin ducks. Despite the importance of the pathogen, there is still a lack of serological diagnostic tools for the detection of EC infections. Here we describe the development of an in-house indirect enzyme-linked immunosorbent assay (ELISA) for the detection of EC-specific antibodies and its application by examination of 67 sera from experimentally infected Pekin ducks, 710 field samples from four Pekin duck breeder flocks previously vaccinated with inactivated vaccines, and 80 samples from commercial Pekin ducks coming from vaccinated parent flocks. All groups that had been experimentally inoculated via the air sac route were positive in the new ELISA, with significantly (P?≤?0.05) increased mean sample/positive (S/P) ratios of 0.71–2.70 at days 7, 14 and 21 post-infection, while orally inoculated ducks and the EC-free control group remained negative with mean S/P ratios of 0.0–0.15. Antibodies were also detected in each of four vaccinated Pekin duck breeder flocks; 67.8% of the samples were antibody positive. The highest S/P ratios were found between 16 and 26 weeks (median S/P ratios from 0.15 to 1.03), but antibodies were still detected in some serum samples in weeks 61–67 post-hatch. No antibodies were detected in the commercial Pekin ducks. Antibody development in the ducks may be influenced by the composition of the inactivated vaccine. The new ELISA provides a useful tool for investigations of response to EC infections and vaccinations.     

Detection of the phosphorylcholine epitope in streptococci,Haemophilusand pathogenicNeisseriaeby immunoblotting

The phosphorylcholine (PC) determinant inStreptococcus pneumoniaeis known to be linked to the cell wall polysaccharides (C-Ps) and to the lipoteichoic acid (LTA) (Forssman antigen) of the plasma membrane. Western blotting with two PC specific murine monoclonal antibodies (MAbs) designated 145,F-2 (IgM) and 147,A-1 (IgA) showed a similar ladder-like pattern for all examined strains ofS. pneumoniaeandStreptococcus mitis. Purified antigens run in parallel indicated that this ladder pattern is due to the PC of LTA. Unlike other techniques, Western blotting thus enables the identification of only one of the streptococcal structures carrying the PC epitope. Gram-negative organisms were also examined, and six of 11Haemophilus influenzaestrains reacted with the MAbs. For this species, unlike the streptococci, only one fast moving band was detected. Analyses by thin-layer chromatography (TLC) detected the PC epitope in lipopolysaccharide (LPS) fraction fromH. influenzae. Some strains of theNeisseriaceaefamily were also positive by Western blotting, but TLC and immunostaining did not detect the PC determinant in LPS.     

Mechanism of Ca2+/calmodulin-dependent kinase II regulation of AMPA receptor gating

The function, trafficking and synaptic signaling of AMPA receptors are tightly regulated by phosphorylation. Ca(2+)/calmodulin-dependent kinase II (CaMKII) phosphorylates the GluA1 AMPA receptor subunit at Ser831 to increase single-channel conductance. We show that CaMKII increases the conductance of native heteromeric AMPA receptors in mouse hippocampal neurons through phosphorylation of Ser831. In addition, co-expression of transmembrane AMPA receptor regulatory proteins (TARPs) with recombinant receptors is required for phospho-Ser831 to increase conductance of heteromeric GluA1-GluA2 receptors. Finally, phosphorylation of Ser831 increases the efficiency with which each subunit can activate, independent of agonist efficacy, thereby increasing the likelihood that more receptor subunits will be simultaneously activated during gating. This underlies the observation that phospho-Ser831 increases the frequency of openings to larger conductances rather than altering unitary conductance. Together, these findings suggest that CaMKII phosphorylation of GluA1-Ser831 decreases the activation energy for an intrasubunit conformational change that regulates the conductance of the receptor when the channel pore opens.     

Islet amyloid polypeptide, islet amyloid, and diabetes mellitus

Islet amyloid polypeptide (IAPP, or amylin) is one of the major secretory products of β-cells of the pancreatic islets of Langerhans. It is a regulatory peptide with putative function both locally in the islets, where it inhibits insulin and glucagon secretion, and at distant targets. It has binding sites in the brain, possibly contributing also to satiety regulation and inhibits gastric emptying. Effects on several other organs have also been described. IAPP was discovered through its ability to aggregate into pancreatic islet amyloid deposits, which are seen particularly in association with type 2 diabetes in humans and with diabetes in a few other mammalian species, especially monkeys and cats. Aggregated IAPP has cytotoxic properties and is believed to be of critical importance for the loss of β-cells in type 2 diabetes and also in pancreatic islets transplanted into individuals with type 1 diabetes. This review deals both with physiological aspects of IAPP and with the pathophysiological role of aggregated forms of IAPP, including mechanisms whereby human IAPP forms toxic aggregates and amyloid fibrils.     

The Role of the Crista Terminalis in Atrial Flutter and Fibrillation: A Computer Modeling Study

Although atrial fibrillation is a common arrhythmia, the underlying mechanisms are incompletely understood. Recent studies have determined the role of the crista terminalis in the mechanisms of a simpler arrhythmia, atrial flutter. We hypothesize that as transverse coupling across the crista terminalis increases, the activation pattern that results is less like typical atrial flutter and more like atrial fibrillation. 6480 Van Capelle elements were coupled in an icosahedron, simulating the right atrium. Atrial simulations were created which incorporated no heterogeneity, heterogeneous coupling, heterogeneous effective refractory periods, and both heterogeneous coupling and effective refractory periods. When the entire crista terminalis was uncoupled, typical atrial flutter occurred. When transverse coupling allowed activation to propagate across the crista terminalis, the flutter cycle length decreased (p < 0.0001). In addition, when heterogeneity was present, both the coefficient of variation of cycle length and the number of activation wavelets increased (p < 0.0001). Thus, a more rapid reentrant circuit in the superior right atrium drove fibrillatory activity in the remainder of the atrium, as predicted by the mother wavelet hypothesis. While awaiting in vivo validation, our study indicates that transverse coupling along the crista terminalis may play an important role in the development of atrial fibrillation from atrial flutter. © 2000 Biomedical Engineering Society. PAC00: 8719Hh, 8710+e     

Onset and Dynamics of Osteosclerosis in Mice Induced by Reilly-Finkel-Biskis (RFB) Murine Leukemia Virus : Increase in Bone Mass Precedes Lymphomagenesis

Newborn NMRI strain mice were infected with Reilly-Finkel-Biskis (RFB) murine leukemia virus (MuLV), a murine leukemia virus that has been shown to induce lymphomas, osteosclerosis, and osteomas in susceptible strains of mice. Bone histomorphometry of the distal femoral metaphyses at 3-month intervals showed osteosclerosis 3 (100%), 6 (100%), and 9 (93%) months after infection. This was represented by significantly augmented cancellous bone mass and accompanied by distinct changes in bone architecture. High numbers of provirus copies were detected at 2-4 weeks in femora, humeri, and calvaria, and viral protein was highly expressed in trabecular and cortical bone cells, particularly in osteocytes. Infected mice showed enhanced bone formation and smaller numbers of osteoclasts relative to sex- and age-matched controls. Osteoclastic differentiation was significantly reduced in cocultures of spleen or bone marrow cells with RFB MuLV-infected osteoclastogenic, osteoblast-like cells. However, RFB MuLV did not impair the activity of mature osteoclasts. In infected mice lymphomas were only observed at 6 (22%) and 9 months (40%) of age. At 3 months, IgG gene and TCR-beta gene rearrangements were not detectable, and new proviruses showed a heterogeneous integration pattern, indicating the absence of lymphoma in early osteosclerotic mice. In contrast, lymphomas, which developed in 8- to 9-month-old infected mice, showed IgG rearrangements indicating development of B-cell lymphomas, together with mono- or oligoclonal expansion of distinct patterns of proviral integrations. These results indicate that RFB MuLV-induced osteosclerosis develops within 3 months after infection and precedes lymphomagenesis. It may therefore be considered an independent skeletal lesion in MuLV-infected mice.     

Effects of motion sickness on thermoregulatory responses in a thermoneutral air environment

Motion sickness (MS) has been identified as a non-thermal factor that can moderate autonomic thermoregulatory responses. It has been shown that MS exaggerates core cooling during immersion in cold (15°C) and luke-warm (28°C) water by attenuating cold-induced vasoconstriction. The aim of the present study was to investigate whether MS affects thermal balance in a thermoneutral air environment. Eleven subjects were exposed to rotation in two conditions, control (CN) and MS. In the CN condition subjects refrained from head movements, whereas in the MS condition they performed a sequence of maximal head movements (left, right, up, down) at 15-s intervals until they were very nauseous. Sweating rate, rectal temperature (T _re), the difference in temperature between the right forearm and tip of the second finger (T _ff) as an index of cutaneous vasomotor tone, perceived MS, thermal comfort and temperature perception were recorded before and during rotation, and during 90-min post-rotation. During the post-rotation period, T _re dropped and sweating rate increased in the MS but not in the CN condition. The T _ff response suggests that MS-induced peripheral vasodilatation which, together with the sweating resulted in increased heat loss. During rotation, subjects perceived temperature to be uncomfortably high, suggesting that MS may also affect thermoregulatory behaviour. It thus appears that also in a thermoneutral air environment MS may substantially affect thermal balance.     

Evidence for FHL1 as a novel disease gene for isolated hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. HCM is caused by mutations in sarcomeric genes, but in >40% of patients, the mutation is not yet identified. We hypothesized that FHL1, encoding four-and-a-half-LIM domains 1, could be another disease gene since it has been shown to cause distinct myopathies, sometimes associated with cardiomyopathy. We evaluated 121 HCM patients, devoid of a mutation in known disease genes. We identified three novel variants in FHL1 (c.134delA/K45Sfs, c.459C>A/C153X and c.827G>C/C276S). Whereas the c.459C>A variant was associated with muscle weakness in some patients, the c.134delA and c.827G>C variants were associated with isolated HCM. Gene transfer of the latter variants in C2C12 myoblasts and cardiac myocytes revealed reduced levels of FHL1 mutant proteins, which could be rescued by proteasome inhibition. Contractility measurements after adeno-associated virus transduction in rat-engineered heart tissue (EHT) showed: (i) higher and lower forces of contraction with K45Sfs and C276S, respectively, and (ii) prolonged contraction and relaxation with both mutants. All mutants except one activated the fetal hypertrophic gene program in EHT. In conclusion, this study provides evidence for FHL1 to be a novel gene for isolated HCM. These data, together with previous findings of proteasome impairment in HCM, suggest that FHL1 mutant proteins may act as poison peptides, leading to hypertrophy, diastolic dysfunction and/or altered contractility, all features of HCM.     

Neurogenic inflammation in stress-induced termination of murine hair growth is promoted by nerve growth factor

Recently, we have revealed the existence of a "brain-hair follicle axis" in murine skin and have identified the neuropeptide substance P (SP) as a key mediator of stress-induced hair growth inhibition in vivo. Published evidence suggests that increased numbers of SP-immunoreactive sensory fibers, as seen in the dermis of stressed mice in anagen-catagen transition, are a result of transient high levels of nerve growth factor (NGF). Thus, we now aimed at dissecting the role of NGF in stress-triggered hair growth termination in our murine model. By real time PCR and immunohistochemistry, stress-exposed mice showed an up-regulation of NGF and its low-affinity receptor p75NTR; the NGF high-affinity receptor TrkA was moderately down-regulated. On neutralization of NGF, premature onset of catagen, apoptosis, and increased number/activation of perifollicular mast cells and antigen-presenting cells, which reflects the skin response to stress, was significantly abrogated. Stress or subcutaneous injection of recombinant NGF (to mimic stress) resulted in an increased percentage of SP(+) neurons in dorsal root ganglia, as measured by retrograde tracing. Taken together, these data suggest that NGF is a central element in the perifollicular neurogenic inflammation that develops during the murine skin response to stress and antagonizing NGF may be a promising therapeutic approach to counter the negative effect of stress on hair growth.