Endogenous and exogenously-induced immunomodulation of tumour-host responsiveness

In spite of the availability of multiple effector mechanisms of the immune system to combat tumour growth and metastases, their impairment frequently accompanies the appearance of cancer. Factors contributing to this impairment may be related to properties of the host and/or the tumour itself and may be with respect to their origin -endogenous or exogenour. Based on the unique biological behavior of prostate cancer (PCa), and its apparent escape from immune surveillance in the presence of tumour immuno genicity, continuing investigation of endogenous and exogenous factors thought to be relevant to its pathogenesis have been made. For this purpose further studies of the suggested role of human seminal plasma (SePl) and the synthetic oestrogen, diethylstiboestrol (DES), as representative endogenous and exogenous immunomodulatory factors (IMF) of tumour-host responsiveness, together with evaluation of human prostatic tissue extracts and leuprolide (the luteinizing-hormone-releasing-hormone proposed as an alternate to DES therapy) have been made by evaluating their effect on the lytic activity of natural killer (NK) cells. SePl and prostate extracts significantly suppressed NK cell lysis. Physicochemical studies suggest SePl and prostate IMF to be associated with high and low molecular weight macromolecules; and implicate the participation of transglutaminase and prostaglandins. Comparative study of therapeutic levels of DES vs. leuprolide on NK cell lysis demonstrated significant suppression by DES vs. a negligible effect of leuprolide. Metastases are highly prevalent in PCa, and contribute significantly to its morbidity and mortality. Further knowledge of the range of effects of endogenous and exogenous IMF on effector mechanisms of tumour-host responsiveness, to include suppression of NK cells, and elucidation of their nature, may contribute toward our understanding of the unique biological behavior of tumours of the prostate, in addition to improvement in their clinical management.

Development of natural products as drugs acting on central nervous system

We have recenty studied several natural product constituents which have effects on the CNS. (1) Tetrahydropalmatine (THP) and its analogues were isolated from Corydalis ambigua and various species of Stephania. (+)-THP and (-)-THP posses not only analgesic activity, but also exert sedative-tranquillizing and hypnotic actions. Results of receptor binding assay and their pre-and post-synaptic effects on dopaminergic system indicate that (-)-THP and (-)-stepholidine are dopamine receptor antagonists while (+)-THP is a selective dopamine depletor. (2) 3-Acetylaconitine (AAC) is an alkaloid isolated from Aconitum flavum. The relative potency of analgesic action of AAC was 5.1-35.6 and 1250-3912 times that of morphine and aspirin, respectively. The analgesic effect of AAC was antagonized by naloxone, but was eliminated by reserpine. In monkeys, after AAC was injected for 92 days, no abstinence syndrome was seen after sudden AAC withdrawal or when challenged with nalorphine. (3) Huperzine A (Hup-A) is an alkaloid isolated from Huperzia serrata which was found to be a selective ChE inhibitor and could improve learning and retrieval process. Preliminary clinical studies showed that Hup-A improve short-and long-term memory in patients of cerebral arteriosclerosis with memory impairment. (4) Ranamargarin is a new tetradecapeptide isolated from the skin of the Chines frog Rana margaratae. This peptide may mainly act on NK-1 receptor.

Some biological properties of the human amniotic membrane interferon

Human amniotic interferon was investigated to define the species specificity of its antiviral action and compare its anti-cellular and NK cell stimulating activities with those of other human interferons. The antiviral effect was titrated in bovine (RV-IAL) and monkey (VERO) cells. Amniotic interferon exhibited, in bovine cells, 5% of the activity seen in monkey cells, while alpha interferon displayed 200%. No effect was detected with either beta or gamma interferon in bovine cells. Daudi cells were exposed to different concentrations of various interferons and the cell numbers were determined. The anticellular effect of the amniotic interferon reached its peak on the third day of incubation. Results suggested a higher activity for alpha and gamma interferons and a lower activity for beta when compared to amniotic interferon. Using total mononuclear cells as effector cells and K 562 as target cell in a 51Cr release assay, it was demonstrated that low concentrations of amniotic interferon consistently stimulated NK cell activity in cells derived from several donors, the results indicating a higher level of activity with this interferon than with alpha and beta interferons.

A rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites using fluorescence flow cytometry

Fluorescence flow cytometry was employed to assess the potential of a vital dye, hydroethiedine, for use in the detection and monitoring of the viability of hemoparasites in infected erythrocytes, using Babesia bovis as a model parasite. The studies demonstrated that hydroethidine is taken up by B. bovis and metabolically converted to the DNA binding fluorochrone, ethidium. Following uptake of the dye, erythrocytes contamine viable parasites were readily distinguished and quantitated. Timed studies with the parasiticidal drug, Ganaseg, showed that it is possible to use the fluorochrome assay to monitor the effects of the drug on the rate of replication and viability of B. bovis in culture. The assay provides a rapid method for evaluation of the in vitro effect of drugs on hemoparasites and for analysis of the effect of various components of the immune response, such as lymphokines, monocyte products, antibodies, and effector cells (T, NK, LAK, ADCC) on the growth and viability of intraerythrocytic parasites.

Molecular Genetic Analysis of Multi-drug Resistance in Indian Isolates of Mycobacterium tuberculosis

A total of 116 isolates from patients attending the out-patient department at the All India Institute of Medical Sciences, New Delhi and the New Delhi Tuberculosis Centre, New Delhi, India were collected. They were analyzed for resistance to drugs prescribed in the treatment for tuberculosis. The drug resistance was initially determined by microbiological techniques. The Bactec 460TB system was employed to determine the type and level of resistance in each isolate. The isolates were further characterized at molecular level. The multi-drug loci corresponding to rpo b, gyr A, kat G were studied for mutation(s) by the polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) technique. The SSCP positive samples were sequenced to characterize the mutations in rpo b, and gyr A loci. While previously reported mutations in the gyr A and rpo b loci were found to be present, several novel mutations were also scored in the rpo b locus. Interestingly, analysis of the gyr A locus showed the presence of point mutation(s) that could not be detected by PCR-SSCP. Furthermore, rifampicin resistance was found to be an important marker for checking multi-drug resistance (MDR) in clinical isolates of Mycobacterium tuberculosis. This is the first report on molecular genetic analysis of MDR tuberculosis one from India, highlights the increasing incidence of MDR in the Indian isolates of M. tuberculosis.

The spleen is an important site of T cell activation during human hepatosplenic schistosomiasis

We have undertaken a comparative immunephenotypic study of spleen cells from hepatosplenic patients (HS) and uninfected individuals (NOR) using flow cytometry. Our data did not show any significant differences in the mean percentage of T-cells and B-cells between the two groups. Analysis of activated T-cells demonstrated that HS present an increased percentage of CD3+HLA-DR+ splenocytes in comparison to NOR. Analysis of T-cell subsets demonstrated a significant increase on the percentage of both activated CD4+ T-splenocytes and CD8+ cells in HS. We did not find any difference in the mean percentage of CD28+ T-cells. Analysis of the B-cell compartment did not show any difference on the percentage of B1-splenocytes. However, the spleen seems to be an important reservoir/source for B1 lymphocytes during hepatosplenic disease, since after splenectomy we found a decreased the percentage of circulating B1-lymphocytes. We observed an increase on the percentage of CD2+CD3- lymphocytes in the spleen of HS suggesting that the loss of CD3 by activated T-cells or the expansion of NK-cells might play a role in the development/maintenance of splenomegaly.

Evaluation of the natural killer cytotoxicity and the levels of cytokines in rats with type I diabetes mellitus

Type I diabetes mellitus (insulin-dependent DM = IDDM) is a chronic disease characterized by specific destruction of pancreatic beta cells, resulting in an absolute lack of insulin. Immune mechanisms, genetic susceptibility, and environmental factors are all implicated in the pathogenesis of Type 1 diabetes. This study was aimed at determining the efficiency of cytokines, natural killer (NK) cells in the pathophysiology of IDDM. Therefore, we evaluated the plasma levels of cytokines by specific enzyme-linked immunosorbent assay (ELISA) and the cytotoxicity activity of NK cells by anti-candididal index in rats with type I diabetes. We found that the cytotoxicity activity of NK cells in IDDM groups significantly decreased compared to the control groups. The levels of interferon-g (IFN-g) in IDDM groups were slightly higher than in healthy controls. These results indicate that the changes of T H1 type cytokines such as IFN-g and NK cell activity can play a role in the etiology of IDDM. The data may provide new strategies for the treatment of IDDM.

Circulating natural killer and γδ T cells decrease soon after infection of rhesus macaques with lymphocytic choriomeningitis virus

Rhesus macaques infected with the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) serve as a model for human infection with Lassa fever virus. To identify the earliest events of acute infection, rhesus macaques were monitored immediately after lethal infection for changes in peripheral blood mononuclear cells (PBMCs). Changes in CD3, CD4, CD8 and CD20 subsets did not vary outside the normal fluctuations of these blood cell populations; however, natural killer (NK) and γδ T cells increased slightly on day 1 and then decreased significantly after two days. The NK subsets responsible for the decrease were primarily CD3-CD8+ or CD3-CD16+ and not the NKT (primarily CD3+CD56+) subset. Macaques infected with a non-virulent arenavirus, LCMV-Armstrong, showed a similar drop in circulating NK and γδ T cells, indicating that this is not a pathogenic event. V³9 T cells, representing the majority of circulating γδ T cells in rhesus macaques, displayed significant apoptosis when incubated with LCMV in cell culture; however, the low amount of cell death for virus-co-cultured NK cells was insufficient to account for the observed disappearance of this subset. Our observations in primates are similar to those seen in LCMV-infected mice, where decreased circulating NK cells were attributed to margination and cell death. Thus, the disappearance of these cells during acute hemorrhagic fever in rhesus macaques may be a cytokine-induced lymphopenia common to many virus infections.

Vector density and the control of kala-azar in Bihar, India

Bihar, India has been in the grip of kala-azar for many years. Its rampant and severe spread has made life miserable in most parts of the state. Such conditions require a comprehensive understanding of this affliction. The numbers coming out of the districts prone to the disease in the north and south Ganges have provided us with several startling revelations, as there are striking uniformities on both sides, including similar vegetation, water storage facilities, house construction and little change in risk factors. The northern areas have been regularly sprayed with DDT since 1977, but eradication of the disease appears to be a distant dream. In 2007 alone, there were as many as 37,738 cases in that region. In contrast, the southern districts of Patna and Nalanda have never had the disease in its epidemic form and endemic disease has been present in only some pockets of the two districts. In those cases, two rounds of spraying with DDT had very positive results, with successful control and no new established foci. In addition, an eleven-year longitudinal study of the man hour density and house index for the vector Phlebotomus argentipes demonstrated that they were quite high in Patna and Nalanda and quite low in north Bihar. Given these facts, an attempt has been made to unravel the role of P. argentipes saliva (salivary gland) in the epidemiology of kala-azar. It was determined that patchy DDT spraying should be avoided for effective control of kala-azar.

Innate immunity and regulatory T-cells in human Chagas disease: what must be understood?

There is a general consensus that during chronic Trypanosoma cruzi infection, the host immune system induces complex processes to ensure the control of parasite growth while preserving the potential to mount and maintain a life-long controlled humoral and cellular immune response against the invading pathogen. This review summarises evidence in an attempt to elucidate "what must be understood" to further clarify the role of innate immunity in the development/maintenance of clinical Chagas disease and the impact of etiological treatment on host immunity, highlighting the contributions of the innate immunity and regulatory T (Treg) cells. Recently, increasing focus on innate immunity suggest that chronic T. cruzi infection may cause morbidity when innate effector functions, or the down-regulation of adaptive regulatory mechanisms are lacking. In this context, stable asymptomatic host-parasite interactions seem to be influenced by the effector/regulatory balance with the participation of macrophages, natural killer (NK) and CD8+ T cells in parallel with the establishment of regulatory mechanisms mediated by NKT and Treg cells. Moreover, a balanced innate immune activation state, apart from Treg cells, may play a role in controlling the adverse events triggered by the massive antigen release induced by trypanosomicidal agents during Chagas disease etiological treatment.

Utilização de uréia em misturas com sulfato de amônio ou com gesso na cultura de milho

Aspectos qualitativos e quantitativos devem ser considerados na adubação nitrogenada de cobertura na cultura de milho. Este estudo teve por objetivo avaliar, nos municípios de Votuporanga (SP) e Uberlândia (MG), o efeito de diferentes misturas de grânulos, contendo N e S, na produtividade de milho. Em um Argissolo Vermelho eutrófico A moderado textura arenosa (120 g kg-1 de argila) de Votuporanga, foram estimadas as perdas por volatilização de N-NH3, das misturas de grânulos constituídas por uréia (U) e sulfato de amônio (SA), ou U e gesso agrícola, aplicadas no primeiro parcelamento de cobertura nitrogenada. Em Uberlândia, em Latossolo Vermelho ácrico típico fase Cerrado subcaducifólio muito argiloso (720 g kg-1), foram determinados a distribuição de N-inorgânico e S-sulfato em profundidade, após a aplicação das misturas em cobertura, e os custos de aplicação dessas fontes. Os experimentos foram instalados em delineamento de blocos casualizados, com quatro repetições, e as misturas de grânulos aplicadas em superfície, na entre linha de cultivo. Em Votuporanga, foram acompanhados dois experimentos: safra 2005/2006 e safrinha 2006. Na safra de 2005/2006, foram comparados quatro tratamentos de cobertura: testemunha, sem N, uréia+sulfato de amônio farelado (U+SAfa), uréia+gesso granulado (U+Gesso gr) e uréia+gesso em pó (U+Gesso pó), aplicados em dois parcelamentos de 45 kg ha-1 de N cada, nos estádios de cinco a seis folhas e 12 a 13 folhas. As perdas de N-NH3 volatilizado em função do N aplicado foram de 45,9, 56,6 e 61,1 % das misturas U+SAfa, U+Gesso pó e U+Gesso gr, respectivamente. A produtividade de grãos não mostrou diferença significativa entre os tratamentos, sendo, em média, de 5.362 kg ha-1. Na safrinha, uréia+sulfato de amônio granulado (U+SAgr) foi incluído nos tratamentos. Neste experimento, as misturas de grânulos foram aplicadas em doses de 50 kg ha-1 de N cada, em cobertura, nos estádios de três a quatro folhas e seis a sete folhas. A produtividade de grãos foi similar entre as fontes de U+SA e U+Gesso, sendo em média de 5.332 kg ha-1. Em Uberlândia, foram comparados os cinco tratamentos testados no experimento - safrinha de Votuporanga. As misturas de grânulos foram aplicadas em dose única de 90 kg ha-1 de N, no estádio de quatro a cinco folhas. Os maiores teores de N-mineral total foram detectados na camada de 0 a 10 cm de profundidade, em todos os tratamentos (abaixo de 3 mg dm-3), diminuindo em profundidade, enquanto o S-sulfato concentrou-se entre as camadas de 10 a 60 cm. Os tratamentos de U+Gesso e U+SA apresentaram, em média, produtividades de 11.364 e 10.300 kg ha-1 de grãos, respectivamente. O custo de aplicação de U+Gesso gr foi 27,7 % superior aos custos médios de aplicação de U+SAgr e U+SAfa, devido a seu menor teor de N, e 7,8 % superior em relação aos custos diretos totais por hectare. A mistura NK com aplicação posterior de U+Gesso pó apresentou custo de aplicação similar ao da U+Gesso gr, devido à dupla operação de aplicação do primeiro. Os resultados em ambas as regiões permitem concluir que o milho respondeu de forma similar à aplicação em cobertura das fontes mistas, U+SA e U+Gesso, independentemente da granulometria dos produtos, e que os custos de aplicação das formas U+Gesso foram superiores aos das misturas U+SA.

Recovery of degraded pasture in Rondônia: macronutrients and productivity of brachiaria brizantha

Pasture is the main form of land use in Amazonia. Over time the pasture grass loses vigor and yields decrease, indicating a certain degree of degeneration. The main causes of degradation are lack of pasture maintenance and subsequent weed infestation, the choice of regionally unsuitable forage species and excessive grazing. The main purpose of this study was to evaluate the impact of different recovery managements on soil chemical properties and grass yield of a degraded pasture in Rondônia. For this purpose, an experiment was installed in October 2001, consisting of five treatments: C = control; HA = harrowing + NPK + micronutrients; HE = Herbicide + NK + micronutrients; R = No-tillage rice + NPK + micronutrients; and S = No-tillage soybean + PK + micronutrients. The following N, P and K sources were used: ammonium sulfate for N, calcined phosphate for P and potassium chloride for K. The experiment was arranged in a randomized block design with four replications. The shoot dry matter yield of the grass was analyzed as of the 35th month of experimentation, in a dry and a rainy period. Phosphorus fertilization resulted in significant increases in Ca2+ and Mg2+ and increasing trend of P in the topsoil in the initial months of the experiment in treatments HA and S and increases in Ca2+ and P (trend) in the treatment R. The cumulative production of Brachiaria brizantha, from Sep/2004 to Mar/2005, was 30,025, 28,267 and 27,735 kg ha-1 shoot dry matter in the treatments HA, R and S, respectively. These values differed significantly from treatments C and HE, with 17,040 and 17,057 kg ha-1, respectively. It was concluded that phosphorus fertilization associated to pasture reform was effective to raise the dry matter yield of Brachiaria brizantha. Rice or soybean under no-tillage is recommended as a practice of pasture recovery, due to the residual effect of fertilization.

Sistemas de manejo do solo na recuperação de uma pastagem degradada em Rondônia

Na região amazônica, pastagens formadas e conduzidas de forma inadequada perdem a produtividade durante os primeiros anos em razão de superpastejo, ausência de adubação e de manutenção e emprego de espécies inadequadas para as condições edafoflorísticas da região. O objetivo deste estudo foi quantificar as modificações ocasionadas por diferentes sistemas de manejo nos atributos físicos de um Latossolo Vermelho-Amarelo (LVA), sob pastagem degradada no Estado de Rondônia, RO. Os sistemas de manejo utilizados nos tratamentos foram: T = testemunha; G = gradagem + NPK + micronutrientes; H = herbicida + NK + micronutrientes; A = plantio direto de arroz + NPK + micronutrientes; e S = plantio direto de soja + NPK + micronutrientes. O delineamento experimental foi em blocos ao acaso com quatro repetições. Para avaliar os tratamentos, amostras com estrutura indeformada foram coletadas em três profundidades (0-0,10; 0,10-0,20 e 0,20-0,30 m) para determinar a curva de retenção da água no solo, densidade do solo, resistência do solo à penetração de raízes, macroporosidade, microporosidade, porosidade total, estimativa do intervalo hídrico ótimo (IHO), densidade máxima do solo (Dsmax) e densidade relativa do solo (Dsrel). Os sistemas de manejo do solo adotados na recuperação da pastagem não proporcionaram melhorias significativas nos atributos físicos do solo, 40 meses após a implantação dos tratamentos. Em todos os sistemas de manejo, foram encontrados valores de densidade do solo acima do considerado ideal (1,40 Mg m-3) e abaixo do crítico (1,75 Mg m-3). Todos os sistemas de manejo apresentaram valores de densidade do solo relativa (Dsrel) acima do valor adotado como crítico (Dsrel = 86 %), exceto no sistema em que foi realizado o preparo do solo na profundidade de 0-0,10 m. A qualidade física do solo, avaliada pelo IHO, diminuiu com o aumento da profundidade do solo.

Crescimento e absorção de nutrientes pelo feijoeiro adubado com cama de aves e fertilizantes minerais

O feijoeiro é tradicionalmente cultivado em pequenas propriedades, onde é comum o uso de dejetos animais para adubação das culturas. Como é uma cultura de ciclo curto, os nutrientes precisam estar disponíveis logo após a germinação, o que nem sempre acontece quando a fertilização ocorre a partir de fertilizantes orgânicos. Neste trabalho, objetivou-se avaliar a eficiência de diferentes camas de aves em relação aos fertilizantes minerais na produção de matéria seca e na liberação de nutrientes para o feijoeiro, em casa de vegetação. O experimento foi conduzido em 2010, com amostras de um Latossolo Vermelho distroférrico com 16 g kg-1 de matéria orgânica, 1,9 e 84 mg dm-3 de P e K, respectivamente, e pH 6,0. Adotou-se delineamento experimental de blocos casualizados com 10 tratamentos e cinco repetições. As unidades experimentais foram constituídas por vasos com 14 dm³ de solo e cinco plantas de feijão, do cultivar BRS Requinte, durante 60 dias. Os tratamentos consistiram de cinco camas de aves compostas pelos seguintes materiais: palha de milho, bagaço de cana-de-açúcar, palha de pastagem natural, areia ou acícula de Pinus, formulações de nutrientes (NPK, NP, PK e NK) e um testemunha, sem nenhum fertilizante. Os fertilizantes minerais com P proporcionaram maior produção de matéria seca da parte aérea (MSPA) e de raízes (MSRA) do feijoeiro do que as camas de aves, por causa da maior liberação para o solo de N e P disponíveis. Dentre as camas estudadas, aquela constituída por areia foi a que proporcionou os maiores valores de MSPA e de MSRA. As plantas fertilizadas com as camas de aves acumularam, em média, 58,6 % do N e 59,0 % do P, em relação às fertilizadas com os tratamentos que continham N e P minerais. A taxa de recuperação pelas plantas de N e K foi maior para os nutrientes aplicados na forma mineral do que na orgânica. As camas de aves podem ser utilizadas como fertilizantes para a cultura do feijoeiro desde que sejam suplementadas com N e P minerais.