Altered composition and functional profile of high-density lipoprotein in leprosy patients.

The changes in host lipid metabolism during leprosy have been correlated to fatty acid alterations in serum and with high-density lipoprotein (HDL) dysfunctionality. This is most evident in multibacillary leprosy patients (Mb), who present an accumulation of host lipids in Schwann cells and macrophages. This accumulation in host peripheral tissues should be withdrawn by HDL, but it is unclear why this lipoprotein from Mb patients loses this function. To investigate HDL metabolism changes during the course of leprosy, HDL composition and functionality of Mb, Pb patients (paucibacillary) pre- or post-multidrug therapy (MDT) and HC (healthy controls) were analyzed. Mb pre-MDT patients presented lower levels of HDL-cholesterol compared to HC. Moreover, Ultra Performance Liquid Chromatography-Mass Spectrometry lipidomics of HDL showed an altered lipid profile of Mb pre-MDT compared to HC and Pb patients. In functional tests, HDL from Mb pre-MDT patients showed impaired anti-inflammatory and anti-oxidative stress activities and a lower cholesterol acceptor capacity compared to other groups. Mb pre-MDT showed lower concentrations of ApoA-I (apolipoprotein A-I), the major HDL protein, when compared to HC, with a post-MDT recovery. Changes in ApoA-I expression could also be observed in M. leprae-infected hepatic cells. The presence of bacilli in the liver of a Mb patient, along with cell damage, indicated hepatic involvement during leprosy, which may reflect on ApoA-I expression. Together, altered compositional and functional profiles observed on HDL of Mb patients can explain metabolic and physiological changes observed in Mb leprosy, contributing to a better understanding of its pathogenesis.

Phenotypic characterization of a pair of molecules in tissues confer to classical Mendelian or non Mendelian ratios.

Studies have reported a wide range of inflammatory responses in the nerve, skin and plasma of leprosy patients. The expression levels of each biomolecule was individualistic, however could be categorized as high and low based on their statistical mean level. Here we report for the first time, expression of a set of biomolecules relating with each other in a defined proportion. The hypothesis of this paper is that the segregation of high and low combinations of a set of biomolecules follows either classical Mendelian dihybrid ratio or epistatic ratios. This hypothesis was tested for 17 molecules in three tissues; nerve, skin and plasma and were confirmed to interact in 9:7, 9:3:4, 12:3:1, 13:3, 15:1 epistatic proportions. These findings suggest that there could be a significant role of networking of molecules in defined epistatic proportions and could be important in pathophysiology of peripheral nerve.

Sensory improvement of leprosy peripheral neuropathy in patients treated with perineural injection of platelet-rich plasma.

BACKGROUND: Leprosy (Hansen's disease) is a chronic granulomatous infection caused by Mycobacterium leprae with peripheral neuropathy as cutaneous and neurological manifestations. Peripheral nerve regeneration may be stimulated by vascular endothelial growth factor and other growth factors (GFs) that have important roles in extracellular matrix regeneration. All of those GFs can be found in platelet-rich plasma (PRP) preparation. The effect of PRP injection in leprosy peripheral neuropathy has never been reported. MATERIALS AND METHODS: A double-blind, randomized, control clinical trial was conducted among 60 patients with leprosy peripheral neuropathy. They were randomized to receive either a 1-ml injection of PRP as treatment or a 1-ml injection of platelet-poor plasma (PPP) as control. Skin sensibilities were measured by two-point discrimination test (TPDT) and visual analog scale (VAS), which were taken before and two weeks after treatment. RESULTS: Perineural injection of PRP was shown to be significantly more effective than PPP (P < 0.05) either in VAS or TPDT measurements. In both groups, the patients had a tingling sensation at the time of injection that disappeared shortly after. CONCLUSION: This study shows that perineural PRP injection could promote improvement of peripheral neuropathy sensibility in patients with leprosy. More research is needed to better determine the effects of PRP in nerve regeneration.

Metabonomics reveals drastic changes in anti-inflammatory/pro-resolving polyunsaturated fatty acids-derived lipid mediators in leprosy disease.

Despite considerable efforts over the last decades, our understanding of leprosy pathogenesis remains limited. The complex interplay between pathogens and hosts has profound effects on host metabolism. To explore the metabolic perturbations associated with leprosy, we analyzed the serum metabolome of leprosy patients. Samples collected from lepromatous and tuberculoid patients before and immediately after the conclusion of multidrug therapy (MDT) were subjected to high-throughput metabolic profiling. Our results show marked metabolic alterations during leprosy that subside at the conclusion of MDT. Pathways showing the highest modulation were related to polyunsaturated fatty acid (PUFA) metabolism, with emphasis on anti-inflammatory, pro-resolving omega-3 fatty acids. These results were confirmed by eicosanoid measurements through enzyme-linked immunoassays. Corroborating the repertoire of metabolites altered in sera, metabonomic analysis of skin specimens revealed alterations in the levels of lipids derived from lipase activity, including PUFAs, suggesting a high lipid turnover in highly-infected lesions. Our data suggest that omega-6 and omega-3, PUFA-derived, pro-resolving lipid mediators contribute to reduced tissue damage irrespectively of pathogen burden during leprosy disease. Our results demonstrate the utility of a comprehensive metabonomic approach for identifying potential contributors to disease pathology that may facilitate the development of more targeted treatments for leprosy and other inflammatory diseases.

Plasma from women with severe pre-eclampsia increases microvascular permeability in an animal model in vivo.

Pre-eclampsia results in oedema, hypertension and proteinuria, and is associated with increased vascular permeability. A number of studies have pointed to the existence of a circulating macromolecule that induces this endothelial dysfunction. To test whether this circulating factor could increase vascular permeability, we have measured the effect of dialysed human plasma from pregnant women with mild or severe pre-eclampsia (pre-eclamptic toxaemia). Plasma was collected from patients with mild or severe pre-eclampsia and from normotensive women. Plasma was dialysed against frog Ringer's solution using a 12-14 kDa molecular-mass cut-off dialysis tubing. pi c (colloid osmotic pressure) was measured with a modified Hansen oncometer. Lp (hydraulic conductivity) and sigma (oncotic reflection coefficient) were measured in individually perfused frog mesenteric microvessels using the Landis-Michel technique during perfusion with dialysed plasma. Perfusion of vessels with normal plasma or plasma from patients with mild pre-eclampsia did not alter either Lp or sigma. However, plasma from patients with severe pre-eclampsia resulted in a 3.8+/-0.3-fold increase in Lp and a reduction in sigma from 0.96+/-0.03 to 0.80+/-0.11. There was a significant correlation between the change in sigma and the change in Lp, suggesting that the increase in permeability was due to an increase in pore size in these vessels. A circulating macromolecule in human plasma in severe pre-eclampsia is therefore able to increase vascular permeability in an animal model. The nature of the circulating macromolecule is not known, except that it is, or is bound to, a molecule greater than 12 kDa.

Cultivo de micobactérias em plasma humano / Culture of mycobacteria in human plasma

Partindo do conceito de que Mycobacterium leprae é tido como incultivável em meios artificiais e tem afinidades com o organismo humano, um de nós (R.N.M) teve a idéia de utilizar, como meio de cultura para o mesmo"in vitro", uma substância do próprio corpo humano. Foi escolhido o plasma de pessoa sadia, lepromino-negativa. Colhendo-se o sangue e separando-se o plasma, os germes obtidos de doente virchowiano virgem de tratamento säo nele inoculados, colocando-se o inóculo em estufa a 3-C e realizando-se exames bacterioscópicos cada 15 dias. Após 60 experiências obtiveram-se os seguintes resultados: 1. multiplicaçäo de bacilos após 45 dias de incubaçäo, em mais de 50% das experiências 2. os basilos apresentaram-se em bastonetes, globias e coraram-se positivamente pelos métodos de Ziehl e Gram. 3. para verificar se seriam eles o Mycobacterium leprae, procederam-se as provas de pridina e da DOPA, que foram confirmativas. 4. Trêss leprominas bacilares preparadas com culturas bem características, inoculadas intradermicamente em hansenianos, comportaram-se como a lepromina clássica; 5. Näo houve crescimento idêntico do inóculo em outros meios de cultura; 6. foi constatado o crescimento, no plasma assim obtido, de M. tuberculosis e micobactérias atípicas; 7. considera-se o plasma humano um meio favorável para o cultivo de micobactérias, no qual houve evidências da reproduçäo de Mycobacterium leprae; 8. Solicita-se a confirmaçäo destes resultados em outros centros de pesquisa

Plasma exchange in severe erythema nodosum leprosum.

Four patients with severe erythema nodosum leprosum were treated by plasma exchange and/or fresh frozen plasma infusions after failure of classical therapy. After the procedures, the patients improved rapidly; with a follow-up between 4 and 7 years after the last plasma exchange, no clinical relapse was noted. The replacement fluids were variable; the most beneficial procedure seemed to be plasma exchange replaced with fresh frozen plasma. Elimination of circulating immune complexes, replacement of a lacking plasma factor are possible mechanisms of action. Plasma exchange may also work like a regulator of immune mechanisms, since it has been shown that there is a depression of suppressor cells in erythema nodosum leprosum.

Depressive effect of plasma on lymphocyte transformation in nodular lepromatous leprosy.

Lymphocytes from 20 patients with NLL were cultured stimulated with PHA in presence of autologous and homologous plasma; the same procedure was carried out with lymphocytes from 20 healthy people, utilized as controls. A diminished capacity of transformation was noticed when lymphocytes, both from patients and controls, were cultured in plasma from NLL patients, this suggesting the presence of a plasmatic inhibitor factor. This effect was particularly noticeable in 8 cases (40%) of the studied patients. Regarding the nature of the plasmatic factor, the concentration of C reactive protein, rheumatoid factor, alpha, beta and gamma globulins, did not show any correlation with the presence of the depressive activity. It seems that at least in some cases, the impaired lymphocyte transformation observed in NLL patients, can be due to an immune complex present as a plasma factor, rather to an intrinsic defect in lymphocytes.

Factors contributing to impairment of the mixed lymphocyte reaction in leprosy.

Whereas the mixed lymphocyte reaction was essentially normal in inactive lepromatous leprosy and tuberculoid leprosy, it was severely impaired in active lepromatous leprosy. The impairment was found to be contributed by certain unknown factors in their plasma and subnormal reactivity of their T lymphocytes. The plasma derived from active lepromatous leprosy patients depressed the reaction of normal cells and normal plasma enhanced the reaction of active lepromatous lymphocytes. The cellular factor was studied by using a one-way reaction in which one of the two lymphocyte preparations was inactivated with mitomycin C. The impairment of blastogenesis of active lepromatous lymphocytes was partially reversed by substituting inactivated normal cells for similarly treated leprous cells, and conversely the response of normal allogeneic lymphocytes was depressed by substituting inactivated leprous lymphocytes as the stimulator cells.

Colloid osmotic pressure changes of dog's blood exposed to different mixtures of CO2 and air.

Hansen's membrane manometer method for measuring plasma colloid osmotic pressure was used to obtain the osmolality changes of dogs breathing different levels of CO2. Osmotic pressure was converted to osmolality by calibration of the manometer with saline and plasma, using freezing point depression osmometry. The addition of 10 vol% of CO2 to tonometered blood caused about a 2.0 mosmol/kg H2O increase of osmolality, or 1.2% increase of red blood cell volume. The swelling of the red blood cells was probably due to osmosis caused by Cl- exchanged for the HCO3- which was produced rapidly by carbonic anhydrase present in the red blood cells. The change in colloid osmotic pressure accompanying a change in co2 tension was measured on blood obtained from dogs breathing different CO2 mixtures. It was approximately 0.14 mosmol/kg H2O per Torr Pco2. The corresponding change in red cell volume could not be calculated from this because water can exchange between the plasma and tissues.

Plasma factors in delayed-type hypersensitivity. Augmentation of lymphocyte responses in borderline leprosy reactions.

The phytohaemagglutinin-induced responses of lymphocytes were found to be inhibited by plasma from patients with leprosy when compared with their responses in pooled serum from healthy donors. When patients developed reversal reactions, the initial inhibitory effect of their plasma was replaced by an augmentary effect on the responses to phytohaemagglutinin. The period of augmentation coincided with that of the reversal reaction in patients with borderline tuberculoid leprosy, but was delayed in patients with borderline lepromatous leprosy. The plasma from each leprosy patient was also observed to have the same effect on lymphocytes from unrelated individuals, showing that the inhibition and augmentation were due to factors in the plasma and not to a change in lymphocyte receptors. It is possible that the normal stable state of leprosy results from the presence of factors in plasma which act as a control mechanism, and that delayed hypersensitivity reactions may be caused by a breakdown of this control.

Polymorphonuclear activation in leprosy. I. Spontaneous and endotoxin-stimulated reduction of nitroblue tetrazolium: effects of serum and plasma on endotoxin-induced activation.

Spontaneous nitroblue tetrazolium (NBT) reduction was evaluated in neutrophils from patients with the different types and forms of leprosy, and compared with reduction obtained form cells from normal controls. Leucocytes from the same subjects were stimulated in vitro by endotoxin, and the rise in percentage of cells reducing NBT was determined. Patients of all groups, with the exception of those with reactional lepromatous leprosy (RLL) had an essentially normal proportion of reducing cells. Neutrophils were normally activated by endotoxin. This indicates that while Mycobacterium leprae does not by itself stimulate leucocytes from leprosy patients, there is no overall anergy of neutrophils in lepromatous or other forms of leprosy. In RLL the proportion of reducing cells was significantly raised. Stimulation with endotoxin was able further to enhance this proportion, but not above levels reached by stimulation of normal cells. Neutrophil activation could not be reproduced by mixing serum from highly activated RLL patients with normal leucocytes. An inhibitory effect of serum and plasma over in vitro endotoxin activation of neutrophils was found.