Mycobacterium abscessus subsp. massiliense expressing bacterioferritin have improved resistance to stressful conditions.

AIMS: The importance of bacterioferritin in the virulence and pathogenicity of the genus Mycobacterium is still unclear. The aim of this study was to analyse if the expression of a recombinant bacterioferritin from M. tuberculosis (Mtb) by Mycma could improve the capacity of this bacillus to resist the host defence mechanisms. METHODS AND RESULTS: Recombinant Mycma, expressing bacterioferritin (Rv1876) from Mtb, was developed by transformation with pMIP12_Rv1876. To determine bacterioferritin influence on Mycma physiology and virulence, the mycobacteria growth was analysed in vitro and in vivo. It was observed that the expression of bacterioferritin improved the growth rate of recombinant Mycma_BfrA under iron excess and oxidative stress, as compared to the wild type. Furthermore, in the murine model of infection, it was observed that Mycma_BfrA-infected mice had higher bacillary load and a more pronounced lesion in the lungs when compared with the wild type. CONCLUSION: This study showed that bacterioferritin confers additional resistance to stress conditions, resulting in increased pathogenicity of Mycma during mice infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new insights about the importance of bacterioferritin in the virulence and pathogenicity of the Mycobacterium genus.

Distribution of the HLA class II frequency alleles in patients with leprosy from the mid-west of Brazil.

In an attempt to clarify the issue of genetic predisposition to leprosy, we examined the distribution of class II human leucocyte antigen variants (DR and DQ) in 70 patients from around the city of Goiânia, Brazil. Only two of the patients presented the tuberculoid form of the disease, whereas 17 fell into the lepromatous category; 51 were intermediate. The allele frequencies found were compared with those in a group of 77 healthy controls. We found an increased frequency of the HLA-DRB1*11 allele in patients with lepromatous leprosy compared with healthy controls (P=0.0132; RR=4.130, 95% Cl: 1.338 to 12.747). These results suggest that the DRB1*11 allele could be related with susceptibility to lepromatous leprosy in Brazil.

Differential infiltration of CD8+ and NK cells in lip and oral cavity squamous cell carcinoma.

BACKGROUND: CD8+ and natural killer (NK) cells have been considered the most effective cells in the combat of cancer, contributing to better prognosis and longer survival. METHODS: The aim of this study was to evaluate the population of CD8+ and NK cells, by immunohistochemistry, in samples of oral cavity squamous cell carcinoma (OCSCC) and lip squamous cell carcinoma (LSCC), leukoplakia, actinic cheilitis, and healthy oral mucosa (control). The relationship of CD8+ and NK cells with survival data, lymph node metastasis, tumor size, and proliferative index was also evaluated. RESULTS: The number of peritumoral and intratumoral CD8+ and NK cells was significantly higher in LSCC, when compared with control, pre-malignant lesions, and OCSCC. A higher proportion of peritumoral CD8+ cells demonstrated correlation with a lower neoplastic proliferative index. Moreover, patients with OCSCC with a high density of peritumoral CD8+ cells showed a tendency towards a longer survival time. CONCLUSIONS: The differential CD8+ and NK cells infiltration in oral SCC might reflect a distinctive tumor microenvironment with a favorable local cytotoxic immune response against neoplastic cells.

Genetic heterogeneity of Escherichia coli strains isolated from raw milk, Minas Frescal cheese, and food handlers / Heterogeneidade genética de cepas de Escherichia coli isoladas de leite cru, queijo Minas Frescal e manipuladores

From February 2004 to March 2005, 140 samples of food handlers - hands and nostrils - (92), raw milk (24), and minas frescal cheese (24) were analyzed for the presence of Escherichia coli in a dairy processing plant of Goiás State. Forty-seven E. coli strains were obtained and compared by DNA macrorestriction patterns obtained from pulsed-field gel electrophoresis following XbaI restriction in order to investigate the possible sources of cheese contaminations. Based on PFGE genotyping, one strain isolated from food the hands of a handler and five strains isolated from raw milk were identical or closely related to six strains from cheese suggesting, in these cases, the probable source of E. coli contamination in cheeses. No strain isolated from the nostrils was related to those found in cheeses or milk strains. The results showed high diversity among the strains, demonstrating a lack of predominance of an endemic clone in the dairy plant. This paper highlights the usefulness of PFGE as an epidemiological tool for determining the source of E. coli contamination in the food industry.

Durante um ano, de fevereiro de 2004 a março de 2005, 140 amostras retiradas das mãos e das narinas de manipuladores de alimentos (92), do leite cru (24) e do queijo-de-minas frescal (24) foram analisadas para a presença de Escherichia coli, em um laticínio do Estado de Goiás. As 47 cepas obtidas foram comparadas por macrorrestrição do DNA com enzima XbaI, seguida de eletroforese em gel em campo pulsado (PFGE), a fim de investigar as possíveis fontes de contaminação do queijo. Baseado na genotipagem pelo PFGE, uma cepa obtida do leite cru e cinco cepas obtidas dos manipuladores mostraram similaridade maior que 80 por cento com seis cepas isoladas do queijo, denotando forte correlação genética entre elas e sugerindo, nestes casos, a fonte provável de contaminação do produto final. Nenhuma cepa isolada do nariz foi relacionada às isoladas do queijo ou do leite. Os resultados mostraram grande diversidade entre as cepas, demonstrando ausência de um clone endêmico no laticínio avaliado. Este estudo destaca a utilidade do PFGE como uma ferramenta importante em investigações epidemiológicas e na determinação de possíveis fontes de contaminação por E. coli na indústria de alimentos.

Allele frequencies for an interferon-gamma microsatellite in a population of Brazilian leprosy patients.

A group of Brazilian leprosy patients and controls were genotyped for a CA-repeat microsatellite polymorphism within the interferon (IFN)-gamma gene. A significantly higher frequency of alleles 5-7 was observed in this patient population, indicating that IFN-gamma gene polymorphism may contribute to the course of leprosy post-infection.

Allele frequencies for an interferon-y microsatellite in a population of Brazilian leprosy patients

A group of Brazilian leprosy patients and controls were genotyped for a CA-repeat microsatellite polymorphism within the interferon (IFN)-gamma gene. A significantly higher frequency of alleles 5-7 was observed in this patient population, indicating that IFN-gamma gene polymorphism may contribute to the course of leprosy post-infection.

Regulation of 36-kDa beta-1,3-glucanase synthesis in Trichoderma harzianum.

The effect of carbon sources on the level of beta-1,3-glucanases in the culture filtrates of Trichoderma harzianum (Tc) was investigated. Enzyme activity was detected in all carbon sources, but highest levels were found when laminarin and purified cell walls were used. Three isoforms of beta-1,3-glucanase were produced during growth of the fungus on purified cell walls. Two isoforms were produced on chitin, chitosan, N-acetylglucosamine and laminarin, while only one was detected when the fungus was grown on cellulose and glucose. A 36-kDa beta-1,3-glucanase (GLU36) was secreted from T. harzianum (Tc) grown on all carbon sources tested as demonstrated by Western blot analysis. We found that a significant increase in the level of GLU36 in the culture filtrate follows glucose exhaustion, suggesting that this enzyme is controlled by carbon catabolite repression.

Effects of levodopa and viscosity on the velocity and accuracy of visually guided tracking in Parkinson's disease.

Deficits in velocity generation and movement accuracy occur in Parkinson's disease and are postulated to contribute to the characteristic bradykinesia. In the present study, we attempted to clarify the relationship between the deficits in velocity generation and movement accuracy. Patients with Parkinson's disease and normal controls tracked visually displayed sinusoidal and step targets with the wrist. Performance was evaluated using measurements of velocity and error. Movement velocity was manipulated by two methods: (i) administration of levodopa; (ii) viscous loading. Dependencies of velocity and error on disease state, medication state and viscosity were examined. Visually guided pursuit tracking was characterized by intermittent and frequent velocity excursions in both the patients and controls. For sinusoidal tracking, levodopa significantly increased velocity in the severely affected parkinsonian patients. Prior to the administration of levodopa, step tracking velocity was significantly lower in all patients than in controls. The "on' state produced an increase in velocity to control levels. Error was significantly greater in the parkinsonian subjects than in controls, but was unchanged by levodopa for both tracking tasks. Manipulations of viscosity produced greater changes in velocity than did levodopa, yet a similar independence with respect to accuracy remained. Velocity significantly changed by 40-60% in the two tracking tasks from the viscous to antiviscous loads. Error did not change significantly in 12 out of 14 comparisons of subgroups based on disease and medication state. This contradicts the hypothesis that patients with Parkinson's disease primarily reduce velocity during tracking to maintain acceptable accuracy in the presence of a defective error correction system. Although parkinsonian subjects tracked with reduced accuracy, both normal and parkinsonian subjects were able to compensate for significant changes in velocity due to external loading. Thus a propulsion deficit exists in parkinsonism that may be alleviated with either antiviscosity or levodopa. An error correction deficit is also present in parkinsonism, but is not modified by antiviscosity or levodopa.

Acute effects of levodopa on wrist movement in Parkinson's disease. Kinematics, volitional EMG modulation and reflex amplitude modulation.

Acute changes in motor performance due to levodopa were evaluated by a series of four motor tests unified by their focus on wrist flexion-extension movements. Subjects with idiopathic Parkinson's disease were evaluated with this battery of tests before (OFF) and after their usual morning dose of levodopa (ON). The test battery consisted of (i) repetitive self-paced movement in which velocity was to be maximized; (ii) visually guided tracking of a sinusoid and a square wave; and (iii) an assay of stretch reflex modulation during volitional sinusoidal tracking. The maximal wrist joint velocity of self-paced reciprocating flexion and extension movements increased after levodopa (ON), without significant changes in the movement period or amplitude. In the two tracking tasks, some subjects improved as evident by a lower root mean square (rms) error, but in similar numbers of subjects the rms error increased. Overall, the rms error, peak velocity or peak movement amplitude did not change after levodopa in either tracking task. Significant and consistent changes did occur after levodopa in an assay of reflex modulation during error-constrained tracking (Johnson et al., Brain 1991; 114: 443-60). The amplitude of volitional EMG increased after levodopa, with a concurrent reduction in reflex EMG. These changes are consistent with the noted increase in movement velocity. These results show that the effects of levodopa on movement velocity were not consistently translated into increased accuracy. The changes in the long latency reflex gain argue for a central control of this reflex, mediated by structures sensitive to levodopa. Finally, the results show that the quantitative evaluation of levodopa therapy cannot be unidimensional, but requires a battery of motor tests as undertaken in this study.

Independent control of reflex and volitional EMG modulation during sinusoidal pursuit tracking in humans.

It is well known that during volitional sinusoidal tracking the long-latency reflex modulates in parallel with the volitional EMG activity. In this study, a series of experiments are reported demonstrating several conditions in which an uncoupling of reflex from volitional activity occurs. The paradigm consists of a visually guided task in which the subject tracked a sinusoid with the wrist. The movement was perturbed by constant torque or controlled velocity perturbations at 45 degrees intervals of the tracking phase. Volitional and reflex-evoked EMG and wrist displacement as functions of the tracking phase were recorded. The relationship of both short-latency (30-60 ms) and longer-latency (60-100 ms) reflex components to the volitional EMG was evaluated. In reflex tracking, the peak reflex amplitude occurs at phases of tracking which correspond to a maximum of wrist joint angular velocity in the direction of homonymous muscle shortening and a minimum of wrist compliance. Uncoupling of the reflex and volitional EMG was observed in three situations. First, during passive movement of the wrist through the sinusoidal tracking cycle perturbation-evoked long-latency stretch reflex peak is modulated as for normal, volitional tracking. However, with passive joint movement the volitional EMG modulation is undetectable. Second, a subset of subjects demonstrate a normally modulated and positioned long-latency reflex with a single peak. However, these subjects have distinct bimodal peaks of volitional EMG. Third, the imposition of an anti-elastic load (positive position feedback) shifts the volitional EMG envelope by as much as 180 degrees along the tracking phase when compared with conventional elastic loading. Yet the long-latency reflex peak remains at its usual phase in the tracking cycle, corresponding to the maximal velocity in the direction of muscle shortening. Furthermore, comparison of the results from elastic and anti-elastic loads reveals a dissociation of short- and long-latency reflex activity, with the short-latency reflex shifting with the volitional EMG envelope. Comparable results were also obtained for controlled velocity perturbations used to control for changes in joint compliance. The uncoupling of the reflex and volitional EMG activity in the present series of experiments points to a flexible relationship between reflex and volitional control systems, altered by peripheral input and external load.

The complement system of Calomys callosus, Rengger, 1830 (Rodentia, Cricetidae).

The complement system (C) of Calomys callosus, Rengger, 1830 (Rodentia, Cricetidae), a wild reservoir for several infectious agents in Latin America, was characterized. Sera from normal adult animals lysed sheep erythrocytes (Es) previously sensitized with rabbit serum anti-Es (Ar) in the presence of veronal-buffered saline containing 0.15 mM CaCl2 and 0.5 mM MgCl2, pH 7.4, or unsensitized rabbit erythrocytes (Er) in the presence of one-half isotonic strength veronal-buffered-saline containing 2.5% glucose, 2 mM MgCl2 and 10 mM EGTA, pH 7.4. Both hemolytic curves were sigmoidal in shape, with CH50 values of 30-40 for females and 20-30 for males. C5, determined hemolytically using the intermediate cells EsArClm4m2m3m, was approximately 4.5 x 10(8)/ml and 4.0 x 10(8)/ml for females and males, respectively. Immunochemical serum analyses by double immunodiffusion or by immunoblotting using polyclonal antisera against human C1s, C1q, C2, C3, C4, C5, C8 and factors B, I and H indicated that C. callosus C components factor B, C4 and C3 cross-reacted with the corresponding human C components. Thus, C. callosus was found to contain effective classical and alternative pathways (CP, AP) and common pathways, reasonable amounts of C5 and common epitopes in the key C components, factor B, C4 and C3, which were preserved during evolution.

The complement system of calomys callosus, rengger, 1830 (Rodentia, Cricettidae)

The complement system (C) of Calomys callosus, rengger, 1830 (Rodentia, Cricetidae) a wild reservoir for several infectious agents in Latin America, was characterized. Sera from normal adult animals lysed sheep erythrocytes (Es) previouusly sensitized with rabbit serum antii-Es (Ar) in the presence of veronal-buffered saline containing 0.15 mM CaCl2 and 0.5 mM MgCl2, pH 7.4, or unsensitized rabbit erythrocytes (Er) in the presence of one-half isotonic strength veronal-buffered-saline containing 2.5% glucose, 2mM MgCl2 and 10 mM EGTA, pH 7.4. Both hemolytic curves were sigmoidal in shape, withh CH50 values of 30-40 for females and 20-30 for males. C5, determined hemolytically using the intermediate cells EsArClm4m2m3m, was approximately 4.5 x 10 8/ml and 4.0 x 10 8/ml for females and males, respectivelyy. Immunochemical serum analyses by double immunodiffusion or by immunoblotting using polyclonal antisera against human C1s, C1q, C2, C3, C4, C5, C8 and factors B, I and H indicated that C. callosus was found to contain effective classical and alternative pathways (CP, AP) and common pathways, reasonable amounts of C5 and common epitopes in the key C components, factor B, C4 and C3, which were preserved during evolution

Modulation of the stretch reflex during volitional sinusoidal tracking in Parkinson's disease.

Sinusoidal visually-guided wrist tracking, in normal and parkinsonian subjects, was perturbed by torque transients every 90 degrees throughout the movement. Long-latency stretch reflex and volitional EMG amplitude modulations were assessed as functions of the tracking phase. Reflex modulation during tracking, both in wrist flexor and extensor muscles, was found to differ significantly between parkinsonian and normal subjects. In the parkinsonian group, the abnormality consisted of an increased reflex activity during tracking phases in which the muscle was lengthening. At these phases the reflex generated torque is opposite in direction to the volitionally generated torque and the tracking movement. No differences in the unperturbed volitional EMG modulation were observed between groups for this error constrained tracking paradigm. Significant correlations were found between ratings of bradykinesia and the amount of abnormal reflex modulation in the wrist flexor. These data suggest that a component of bradykinesia results from a defective coordination of supraspinal reflex and volitional control systems.