Mitogen-activated protein kinases contribute to temperature-induced cardiac remodelling in rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) live in environments where water temperatures range between 4 °C and 20 °C. Laboratory studies demonstrate that cold and warm acclimations of male trout can have oppositional effects on cardiac hypertrophy and the collagen content of the heart. The cellular mechanisms behind temperature-induced cardiac remodelling are unclear, as is why this response differs between male and female fish. Studies with cultured trout cardiac fibroblasts suggests that collagen deposition is regulated, at least in part, by mitogen-activated protein kinase (MAPK) cell signalling pathways. We, therefore, hypothesized that temperature-dependent cardiac remodelling is regulated by these signalling pathways. To test this, male and female trout were acclimated to 18 °C (warm) in the summer and to 4 °C (cold) in the winter and the activation of MAPK pathways in the hearts were characterized and compared to that of control fish maintained at 12 °C. In addition, cardiac collagen content, cardiac morphology and the expression of gene transcripts for matrix metalloproteinases (MMP) -9, MMP-2, tissue inhibitor of matrix metalloproteinases and collagen 1α were characterized. p38 MAPK phosphorylation increased in the hearts of female fish with cold acclimation and the phosphorylation of extracellular signal-regulated kinase increased in the hearts of male fish with warm acclimation. However, there was no effect of thermal acclimation on cardiac morphology or collagen content in either male or female fish. These results indicate that thermal acclimation has transient and sex-specific effects on the phosphorylation of MAPKs but also how variable the response of the trout heart is to thermal acclimation.

How the expression of green fluorescent protein and human cardiac actin in the heart influences cardiac function and aerobic performance in zebrafish Danio rerio.

The present study examined how the expression of enhanced green fluorescent protein (eGFP) and human cardiac actin (ACTC) in zebrafish Danio rerio influences embryonic heart rate (RH ) and the swim performance and metabolic rate of adult fish. Experiments with the adults involved determining the critical swimming speed (Ucrit , the highest speed sustainable and measure of aerobic capacity) while measuring oxygen consumption. Two different transgenic D. rerio lines were examined: one expressed eGFP in the heart (tg(cmlc:egfp)), while the second expressed ACTC in the heart and eGFP throughout the body (tg(cmlc:actc,ba:egfp)). It was found that RH was significantly lower in the tg(cmlc:actc,ba:egfp) embryos 4 days post-fertilization compared to wild-type (WT) and tg(cmlc:egfp). The swim experiments demonstrated that there was no significant difference in Ucrit between the transgenic lines and the wild-type fish, but metabolic rate and cost of transport (oxygen used to travel a set distance) was nearly two-fold higher in the tg(cmlc:actc,ba:egfp) fish compared to WT at their respective Ucrit . These results suggest that the expression of ACTC in the D. rerio heart and the expression of eGFP throughout the animal, alters cardiac function in the embryo and reduces the aerobic efficiency of the animal at high levels of activity.

Data for iTRAQ-based quantification of the cardiac proteome of rainbow trout (Oncorhynchus mykiss) at rest and with exercise training.

This data article presents the first description of the rainbow trout cardiac ventricle at the level of the proteome, with more than 700 proteins identified and quantified using isobaric tags for relative and absolute quantitation (iTRAQ) and LC-MS/MS. The abundances of these proteins were compared across 4 durations of moderate exercise training (0, 4, 7, and 14 d), and a total of 107 proteins were differentially abundant during the course of the training program. The differentially abundant proteins are presented here grouped by functional classification. In the research article associated with this data [1], the temporal changes in the cardiac proteome are discussed in the context of cardiac remodelling and development of a trained heart phenotype.

CAG repeat expansion in Huntington disease determines age at onset in a fully dominant fashion.

OBJECTIVE: Age at onset of diagnostic motor manifestations in Huntington disease (HD) is strongly correlated with an expanded CAG trinucleotide repeat. The length of the normal CAG repeat allele has been reported also to influence age at onset, in interaction with the expanded allele. Due to profound implications for disease mechanism and modification, we tested whether the normal allele, interaction between the expanded and normal alleles, or presence of a second expanded allele affects age at onset of HD motor signs. METHODS: We modeled natural log-transformed age at onset as a function of CAG repeat lengths of expanded and normal alleles and their interaction by linear regression. RESULTS: An apparently significant effect of interaction on age at motor onset among 4,068 subjects was dependent on a single outlier data point. A rigorous statistical analysis with a well-behaved dataset that conformed to the fundamental assumptions of linear regression (e.g., constant variance and normally distributed error) revealed significance only for the expanded CAG repeat, with no effect of the normal CAG repeat. Ten subjects with 2 expanded alleles showed an age at motor onset consistent with the length of the larger expanded allele. CONCLUSIONS: Normal allele CAG length, interaction between expanded and normal alleles, and presence of a second expanded allele do not influence age at onset of motor manifestations, indicating that the rate of HD pathogenesis leading to motor diagnosis is determined by a completely dominant action of the longest expanded allele and as yet unidentified genetic or environmental factors.

Expression and characterization of recombinant interferon gamma (IFN-gamma) from the nine-banded armadillo (Dasypus novemcinctus) and its effect on Mycobacterium leprae-infected macrophages.

Armadillos (Dasypus novemcinctus) manifest the full histopathological spectrum of leprosy, and are hosts of choice for in vivo propagation of Mycobacterium leprae. Though potentially useful as a model of leprosy pathogenesis, few armadillo-specific reagents exist. We have identified a region of high homology to the interferon gamma (IFN-gamma) of other mammals within the recently published armadillo whole genomic sequence. cDNA was made from ConA-stimulated armadillo peripheral blood mononuclear cells (PBMC), amplified, and cloned into a pET expression vector for transformation and over-expression in Escherichia coli. The recombinant protein (rDnIFN-gamma) was characterized by western blot and its biological function confirmed with bioassays including intracellular killing of Toxoplasma gondii and induction of indoleamine 2, 3-dioxygenase activity. In using rIFN-gamma to activate macrophages from mice, humans or armadillos, similar to humans, rIFN-gamma-activated armadillo MPhi did not produce nitrite and or inhibit the viability of M. leprae in vitro. Conversely, murine rIFN-gamma-activated mouse MPhi produced high levels of nitrite and killed intracellular M. leprae in vitro. These data indicate that the response of armadillo MPhi to rDnIFN-gamma is similar to that which occurs in humans, and demonstrates a potentially important value of the armadillo as a model in leprosy research.

Development of leprosy and type 1 leprosy reactions after treatment with infliximab: a report of 2 cases.

Humanized monoclonal antibodies to tumor necrosis factor- alpha are valuable for the treatment of rheumatologic conditions, but they have been associated with the development of serious infections. We report the first 2 cases of leprosy developing after treatment with infliximab. After discontinuation of infliximab, both patients developed type 1 ("reversal") leprosy reactions.

The continuing challenges of leprosy.

Leprosy is best understood as two conjoined diseases. The first is a chronic mycobacterial infection that elicits an extraordinary range of cellular immune responses in humans. The second is a peripheral neuropathy that is initiated by the infection and the accompanying immunological events. The infection is curable but not preventable, and leprosy remains a major global health problem, especially in the developing world, publicity to the contrary notwithstanding. Mycobacterium leprae remains noncultivable, and for over a century leprosy has presented major challenges in the fields of microbiology, pathology, immunology, and genetics; it continues to do so today. This review focuses on recent advances in our understanding of M. leprae and the host response to it, especially concerning molecular identification of M. leprae, knowledge of its genome, transcriptome, and proteome, its mechanisms of microbial resistance, and recognition of strains by variable-number tandem repeat analysis. Advances in experimental models include studies in gene knockout mice and the development of molecular techniques to explore the armadillo model. In clinical studies, notable progress has been made concerning the immunology and immunopathology of leprosy, the genetics of human resistance, mechanisms of nerve injury, and chemotherapy. In nearly all of these areas, however, leprosy remains poorly understood compared to other major bacterial diseases.

Development of leprosy and type 1 leprosy reactions after treatment with infliximab: a report of 2 cases

Humanized monoclonal antibodies to tumor necrosis factor- alpha are valuable for the treatment of rheumatologic conditions, but they have been associated with the development of serious infections. We report the first 2 cases of leprosy developing after treatment with infliximab. After discontinuation of infliximab, both patients developed type 1 ([quot ]reversal[quot ]) leprosy reactions.

The continuing challenges of leprosy

Leprosy is best understood as two conjoined diseases. The first is a chronic mycobacterial infection that elicits an extraordinary range of cellular immune responses in humans. The second is a peripheral neuropathy that is initiated by the infection and the accompanying immunological events. The infection is curable but not preventable, and leprosy remains a major global health problem, especially in the developing world, publicity to the contrary notwithstanding. Mycobacterium leprae remains noncultivable, and for over a century leprosy has presented major challenges in the fields of microbiology, pathology, immunology, and genetics; it continues to do so today. This review focuses on recent advances in our understanding of M. leprae and the host response to it, especially concerning molecular identification of M. leprae, knowledge of its genome, transcriptome, and proteome, its mechanisms of microbial resistance, and recognition of strains by variable-number tandem repeat analysis. Advances in experimental models include studies in gene knockout mice and the development of molecular techniques to explore the armadillo model. In clinical studies, notable progress has been made concerning the immunology and immunopathology of leprosy, the genetics of human resistance, mechanisms of nerve injury, and chemotherapy. In nearly all of these areas, however, leprosy remains poorly understood compared to other major bacterial diseases.

Expression of nine-banded armadillo (Dasypus novemcinctus) interleukin-2 in E. coli.

The nine-banded armadillo (Dasypus novemcinctus) is the only immunologically intact animal that regularly develops lepromatous-type leprosy when inoculated with Mycobacterium leprae. However, the ability to exploit this model for understanding the pathogenesis of leprosy has been limited by a lack of suitable immunological reagents. Recently, efforts began to sequence the entire armadillo genome, and this sequence information will help make possible the development of a wide array of new immunological reagents suitable for use with armadillos. Using the available sequence data, a region of high homology to interleukin-2 of other mammals was identified. Primers were designed to amplify the coding region corresponding to the mature peptide and its exact sequence was confirmed. cDNA was made from ConA-stimulated armadillo PBMC. The amplified coding region was sub-cloned into a pET expression vector and transformed into Escherichia coli for over-expression. The subsequent product was characterized by SDS-PAGE and bioassays. Tritiated thymidine incorporation by CTLL-2 and armadillo lymphoblasts confirmed functionality of the recombinant product. The advent of the D. novemcinctus genome sequence and subsequent generation of immunological tools will assist in advancing the armadillo as a translational model for leprosy.

Rehabilitation following bone marrow transplantation.

Bone marrow transplantation and stem cell transplantation are increasingly used to treat hematologic malignancies and some solid tumors. The treatment entails bone marrow-ablative therapies and intensive medical support to sustain the patient through pancytopenia and other complications of the disease, transplantation process, or drug side effects. Patients who develop graft-versus-host disease are the most difficult subset of transplant recipients to manage. Most transplant recipients perform at normal or near-normal functional levels at the inception of the transplantation process but are at high risk for developing functional deficits as a result of cumulative impairments. These impairments arise from their disease, their prior cancer treatment, transplant induction, graft-versus-host disease, immobility, infection, steroid-related side effects, and other sequelae of transplantation. Preventive and preemptive rehabilitation interventions can minimize functional loss and facilitate recovery, but the transplantation team must be sensitive to and regularly assess for early functional declines in these patients. The physiatrist and the other members of the rehabilitation team must be thoroughly acquainted with the unique needs and challenges of the bone marrow transplantation population in order to design and modify treatment programs effectively and safely. Outcome research has shown that some patients have continued limitations in function despite successful transplantation. Few evidence-based data are available that addresses factors correlating with poor functional outcomes other than graft-versus-host disease. However, this disease has not been investigated utilizing objective functional instruments. Future research should more clearly elucidate the functional impact of allogeneic and autologous transplants by using standardized physical performance measures as well as thorough function-based symptomatology questionnaires.

Simultaneous detection of Mycobacterium leprae and its susceptibility to dapsone using DNA heteroduplex analysis.

Currently recommended control measures for treating leprosy with multidrug therapy should control the spread of drug-resistant strains; however, dapsone (DDS) resistance continues to be reported. Comprehensive estimates of drug-resistant leprosy are difficult to obtain due to the cumbersome nature of the conventional drug susceptibility testing method using mouse footpad inoculation, which requires at least 6 months to obtain results. Recently, it has been determined that DDS-resistant strains contain missense mutations in codon 53 or 55 of the folP1 gene of Mycobacterium leprae, and definitive evidence linking these mutations with DDS resistance in M. leprae has been obtained. Based on these mutations, a heteroduplex DDS M. leprae (HD-DDS-ML) assay was developed for the simultaneous detection of M. leprae and of its susceptibility to DDS. The assay relies on the PCR amplification of an M. leprae-specific 231-bp fragment of folP1 containing codons 53 and 55. The PCR products are allowed to anneal to a universal heteroduplex generator, and the separation of the resultant DNA duplexes is accomplished by polyacrylamide gel electrophoresis. M. leprae was detected in crude cell lysates of skin biopsy specimen homogenates from eight leprosy patients and from M. leprae-infected mouse or armadillo tissues infected with 14 separate strains using the HD-DDS-ML assay. The assay was specific for M. leprae in a comparison with results obtained from 14 species of mycobacteria other than M. leprae and four bacterial species known to colonize human skin. The HD-DDS-ML assay detected as few as 100 M. leprae organisms present in homogenates of human skin and demonstrated a 93% correlation with DDS susceptibility as determined by both DNA sequencing of folP1 and mouse footpad susceptibility testing. The HD-DDS-ML assay provides a new tool for the simultaneous detection of M. leprae and of its susceptibility to DDS from a single specimen. The assay should prove useful for drug resistance surveillance in leprosy control programs when combined with similar molecular tests developed for other drug resistance markers.

Cardiopulmonary rehabilitation and cancer rehabilitation. 4. Oncologic rehabilitation.

UNLABELLED: This self-directed learning module highlights the treatment and rehabilitation of patients with cancer by means of a case study format. It is part of the chapter on cardiac, pulmonary, and cancer rehabilitation in the Self-Directed Physiatric Education Program for practitioners and trainees in physical medicine and rehabilitation. This article reviews medical and rehabilitation issues in patients with various types of cancer. Cases were selected to cover problems seen in both younger and older patient populations. Identification of common sequelae of cancer and cancer treatments, associated rehabilitation challenges, and appropriate interventions are included. OVERALL ARTICLE OBJECTIVE: To summarize the medical and rehabilitation issues in patients with various types of cancer.

Ca(2+) binding to cardiac troponin C: effects of temperature and pH on mammalian and salmonid isoforms.

A reduction in temperature lowers the Ca(2+) sensitivity of skinned cardiac myofilaments but this effect is attenuated when native cardiac troponin C (cTnC) is replaced with skeletal TnC. This suggests that conformational differences between the two isoforms mediate the influence of temperature on contractility. To investigate this phenomenon, the functional characteristics of bovine cTnC (BcTnC) and that from rainbow trout, Oncorhynchus mykiss, a cold water salmonid (ScTnC), have been compared. Rainbow trout maintain cardiac function at temperatures cardioplegic to mammals. To determine whether ScTnC is more sensitive to Ca(2+) than BcTnC, F27W mutants were used to measure changes in fluorescence with in vitro Ca(2+) titrations of site II, the activation site. When measured under identical conditions, ScTnC was more sensitive to Ca(2+) than BcTnC. At 21 degrees C, pH 7.0, as indicated by K(1/2) (-log[Ca] at half-maximal fluorescence, where [Ca] is calcium concentration), ScTnC was 2.29-fold more sensitive to Ca(2+) than BcTnC. When pH was kept constant (7.0) and temperature was lowered from 37.0 to 21.0 degrees C and then to 7.0 degrees C, the K(1/2) of BcTnC decreased by 0.13 and 0.32, respectively, whereas the K(1/2) of ScTnC decreased by 0.76 and 0.42, respectively. Increasing pH from 7.0 to 7.3 at 21.0 degrees C increased the K(1/2) of both BcTnC and ScTnC by 0.14, whereas the K(1/2) of both isoforms was increased by 1.35 when pH was raised from 7.0 to 7.6 at 7.0 degrees C.

Mycobacterium leprae typing by genomic diversity and global distribution of genotypes.

The genetic diversity and related global distribution of 51 Mycobacterium leprae isolates were studied. Isolates were obtained from leprosy patients from 12 geographically distinct regions of the world and two were obtained from nonhuman sources. Polymerase chain reaction (PCR) followed by DNA sequencing was performed targeting the rpoT gene of M. leprae. Isolates were classified into two groups based on the number of tandem repeats composed of 6 base pairs in the rpoT gene. Isolates from Japan (except Okinawa) and Korea belonged to one group, while those from Southeast Asian countries, Brazil, Haiti and Okinawa in Japan belonged to a second genotype. M. leprae obtained from two nonhuman sources (an armadillo and a mangabey monkey) revealed the latter genotype. These results demonstrate the genetic diversity of M. leprae and the related genotype-specific distribution in the world.

The effect of ultraviolet light radiation on Mycobacterium leprae.

Ultraviolet (UV) light is recognized as a potent sterilizing aid, but its relative effectiveness against Mycobacterium leprae has not been shown. We examined the influence of UV on the growth and metabolic activity of M. leprae harvested fresh from foot pads of nude mice. Temporary static suspensions were exposed to timed intervals of UV radiation generated from a fixed source to constitute dosages ranging from 0-12.64 x 10(4) erg/cm2. The metabolic activity of the bacilli was indexed by the oxidation of 14C-palmitate in BACTEC 12-B vials. The long-term effects of irradiation on cell division and growth were assessed by inoculation of BALB/c mouse foot pads. The metabolic activity in BACTEC showed an immediate dose-response-related decline to a maximum of 50% of the control activity after exposure to 6.3 x 10(4) erg/cm2. Mouse foot pad studies showed a similar dose-response pattern. Effective-dose determinations based on metabolic or foot pad data were similar. UV doses of 3.52 x 10(4) erg/cm2 resulted in an average 50% killing, and 7.73 x 10(4) erg/cm2 killed 84% of the M. leprae exposed. This UV sensitivity is similar to that reported for M. tuberculosis. UV sterilization and disinfection practices suitable for M. tuberculosis are likely to be equally effective for M. leprae.

Dihydropteroate synthase of Mycobacterium leprae and dapsone resistance.

Two Mycobacterium leprae genes, folP1 and folP2, encoding putative dihydropteroate synthases (DHPS), were studied for enzymatic activity and for the presence of mutations associated with dapsone resistance. Each gene was cloned and expressed in a folP knockout mutant of Escherichia coli (C600DeltafolP::Km(r)). Expression of M. leprae folP1 in C600DeltafolP::Km(r) conferred growth on a folate-deficient medium, and bacterial lysates exhibited DHPS activity. This recombinant displayed a 256-fold-greater sensitivity to dapsone (measured by the MIC) than wild-type E. coli C600, and 50-fold less dapsone was required to block (expressed as the 50% inhibitory concentration [IC(50)]) the DHPS activity of this recombinant. When the folP1 genes of several dapsone-resistant M. leprae clinical isolates were sequenced, two missense mutations were identified. One mutation occurred at codon 53, substituting an isoleucine for a threonine residue (T53I) in the DHPS-1, and a second mutation occurred in codon 55, substituting an arginine for a proline residue (P55R). Transformation of the C600DeltafolP::Km(r) knockout with plasmids carrying either the T53I or the P55R mutant allele did not substantially alter the DHPS activity compared to levels produced by recombinants containing wild-type M. leprae folP1. However, both mutations increased dapsone resistance, with P55R having the greatest affect on dapsone resistance by increasing the MIC 64-fold and the IC(50) 68-fold. These results prove that the folP1 of M. leprae encodes a functional DHPS and that mutations within this gene are associated with the development of dapsone resistance in clinical isolates of M. leprae. Transformants created with M. leprae folP2 did not confer growth on the C600DeltafolP::Km(r) knockout strain, and DNA sequences of folP2 from dapsone-susceptible and -resistant M. leprae strains were identical, indicating that this gene does not encode a functional DHPS and is not involved in dapsone resistance in M. leprae.

Single lesion paucibacillary leprosy: baseline profile of the Brazilian Multicenter Cohort Study.

In Brazil, there is little information about the clinical and epidemiological characteristics of paucibacillary, single skin lesion leprosy patients (SSL-PB). Only recently has the official notification system distinguished leprosy patients with a single lesion as a clinical entity, for whom the single-dose ROM (rifampin, ofloxacin and minocycline) regimen has been recommended. In this paper, we describe the baseline clinical features and the immunological background of a multicenter cohort of SSL-PB leprosy cases enrolled between December 1997-1998. Patients were recruited at health centers located in the following regions: Southeast = Rio de Janeiro; North = Amazon and Rondônia states and Center-West = Goiás state. Eligible cases were newly detected, untreated single-lesion leprosy patients without thickened nerve involvement, and were assessed by clinical, bacilloscopic and histopathological exams. The Mitsuda skin test and anti-PGL-I serology (ELISA) were also performed. Of the 299 SSL-PB leprosy patients, 259 (86.6%) fulfilled the criteria for single-dose ROM intervention. Our results showed that patients recruited from different sites had similar features, considering the clinical and immunological profiles. There was a predominance of adults (mean age 32.4; S.D. = 16.0), and a BCG scar was detected in 76.7% of the children (< or = 15 years old). Only 7 cases were diagnosed as the multibacillary type, representing less than 3% of the patients being misclassified. Our data indicate that in Brazil SSL-PB case ascertainment based on clinical and bacilloscopic criteria can be accurately defined under a routine control program; 75.0% of SSL-PB cases were Mitsuda positive (> or = 5 mm) and seropositivity for anti-PGL-I was detected in 17.3% of the patients. These data are compatible with effective cell-mediated immunity and low bacillary load, suggesting favorable clinical outcomes for most SSL-PB participants of this cohort.

Dapsone resistance in Mycobacterium leprae.

The folP1 gene of Mycobacterium leprae, which encodes dihydropteroate synthase (DHPS), was studied for the presence of mutations associated with resistance to dapsone (DDS). When the folP1 of several DDS-resistant clinical isolates of M. leprae were sequenced, two missense mutations were identified. One mutation occurred at codon 53, substituting isoleucine for threonine in DHPS-1, and a second mutation occurred in codon 55, substituting arginine for proline. DNA sequencing of strains of M. leprae resistant to 0.01 g% DDS in the mouse diet revealed that 13 of 14 strains contained either the 53 or 55 folP1 mutation. None of the susceptible strains and only one of five strains resistant to 0.001 g% DDS revealed a mutation in folP1, suggesting that only high-level DDS resistance is associated with the mutations identified in folP1. Development and application of simple molecular tests to assess drug-related mutations in M. leprae could establish current levels of drug resistance in leprosy as a reference point for future monitoring of drug resistance at the global level.

Dapsone resistance does not appear to be associated with a mutation in the dihydropteroate synthase-2 gene of Mycobacterium leprae.

Evidence suggests that resistance to dapsone (DDS) in Mycobacterium leprae is related to the enzyme dihydropteroate synthase (DHPS). Two M. leprae genes (folP-1 and folP-2) encoding DHPS-1 and DHPS-2, respectively, have been identified through the M. leprae genome project. We have studied DDS-susceptible and resistant strains of M. leprae to determine whether the DDS-resistant phenotype is associated with a mutation(s) in folP-2 and to establish the number of genomic copies of the gene encoding DHPS-2 (folP-2). RFLP analysis of genomic DNA from DDS-susceptible and resistant strains of M. leprae exhibited a unique 4.2 kb restriction fragment consistent with a single genomic copy of folP-2 in both phenotypes. DNA encoding folP-2 was amplified by PCR and sequenced from two susceptible and two resistant strains of M. leprae. The folP-2 sequences from these strains were identical indicating that resistance to DDS was not associated with mutation(s) in the gene encoding DHPS-2.