Impairment of the hematological response and interleukin-1β production in protein-energy malnourished mice after endotoxemia with lipopolysaccharide

The objectives of this study were to determine if protein-energy malnutrition (PEM) could affect the hematologic response to lipopolysaccharide (LPS), the interleukin-1β (IL-1β) production, leukocyte migration, and blood leukocyte expression of CD11a/CD18. Two-month-old male Swiss mice were submitted to PEM (N = 30) with a low-protein diet (14 days) containing 4% protein, compared to 20% protein in the control group (N = 30). The total cellularity of blood, bone marrow, spleen, and bronchoalveolar lavage evaluated after the LPS stimulus indicated reduced number of total cells in all compartments studied and different kinetics of migration in malnourished animals. The in vitro migration assay showed reduced capacity of migration after the LPS stimulus in malnourished animals (45.7 ± 17.2 x 10(4) cells/mL) compared to control (69.6 ± 7.1 x 10(4) cells/mL, P ≤ 0.05), but there was no difference in CD11a/CD18 expression on the surface of blood leukocytes. In addition, the production of IL-1β in vivo after the LPS stimulus (180.7 pg·h-1·mL-1), and in vitro by bone marrow and spleen cells (41.6 ± 15.0 and 8.3 ± 4.0 pg/mL) was significantly lower in malnourished animals compared to control (591.1 pg·h-1·mL-1, 67.0 ± 23.0 and 17.5 ± 8.0 pg/mL, respectively, P ≤ 0.05). The reduced expression of IL-1β, together with the lower number of leukocytes in the central and peripheral compartments, different leukocyte kinetics, and reduced leukocyte migration capacity are factors that interfere with the capacity to mount an adequate immune response, being partly responsible for the immunodeficiency observed in PEM.

Impairment of the hematological response and interleukin-1ß production in protein-energy malnourished mice after endotoxemia with lipopolysaccharide.

The objectives of this study were to determine if protein-energy malnutrition (PEM) could affect the hematologic response to lipopolysaccharide (LPS), the interleukin-1ß (IL-1ß) production, leukocyte migration, and blood leukocyte expression of CD11a/CD18. Two-month-old male Swiss mice were submitted to PEM (N = 30) with a low-protein diet (14 days) containing 4% protein, compared to 20% protein in the control group (N = 30). The total cellularity of blood, bone marrow, spleen, and bronchoalveolar lavage evaluated after the LPS stimulus indicated reduced number of total cells in all compartments studied and different kinetics of migration in malnourished animals. The in vitro migration assay showed reduced capacity of migration after the LPS stimulus in malnourished animals (45.7 ± 17.2 x 10(4) cells/mL) compared to control (69.6 ± 7.1 x 10(4) cells/mL, P ≤ 0.05), but there was no difference in CD11a/CD18 expression on the surface of blood leukocytes. In addition, the production of IL-1ß in vivo after the LPS stimulus (180.7 pg·h-1·mL-1), and in vitro by bone marrow and spleen cells (41.6 ± 15.0 and 8.3 ± 4.0 pg/mL) was significantly lower in malnourished animals compared to control (591.1 pg·h-1·mL-1, 67.0 ± 23.0 and 17.5 ± 8.0 pg/mL, respectively, P ≤ 0.05). The reduced expression of IL-1ß, together with the lower number of leukocytes in the central and peripheral compartments, different leukocyte kinetics, and reduced leukocyte migration capacity are factors that interfere with the capacity to mount an adequate immune response, being partly responsible for the immunodeficiency observed in PEM.

Protein-energy malnutrition halts hemopoietic progenitor cells in the G0/G1 cell cycle stage, thereby altering cell production rates

Protein energy malnutrition (PEM) is a syndrome that often results in immunodeficiency coupled with pancytopenia. Hemopoietic tissue requires a high nutrient supply and the proliferation, differentiation and maturation of cells occur in a constant and balanced manner, sensitive to the demands of specific cell lineages and dependent on the stem cell population. In the present study, we evaluated the effect of PEM on some aspects of hemopoiesis, analyzing the cell cycle of bone marrow cells and the percentage of progenitor cells in the bone marrow. Two-month-old male Swiss mice (N = 7-9 per group) were submitted to PEM with a low-protein diet (4 percent) or were fed a control diet (20 percent protein) ad libitum. When the experimental group had lost about 20 percent of their original body weight after 14 days, we collected blood and bone marrow cells to determine the percentage of progenitor cells and the number of cells in each phase of the cell cycle. Animals of both groups were stimulated with 5-fluorouracil. Blood analysis, bone marrow cell composition and cell cycle evaluation was performed after 10 days. Malnourished animals presented anemia, reticulocytopenia and leukopenia. Their bone marrow was hypocellular and depleted of progenitor cells. Malnourished animals also presented more cells than normal in phases G0 and G1 of the cell cycle. Thus, we conclude that PEM leads to the depletion of progenitor hemopoietic populations and changes in cellular development. We suggest that these changes are some of the primary causes of pancytopenia in cases of PEM.

Protein-energy malnutrition halts hemopoietic progenitor cells in the G0/G1 cell cycle stage, thereby altering cell production rates.

Protein energy malnutrition (PEM) is a syndrome that often results in immunodeficiency coupled with pancytopenia. Hemopoietic tissue requires a high nutrient supply and the proliferation, differentiation and maturation of cells occur in a constant and balanced manner, sensitive to the demands of specific cell lineages and dependent on the stem cell population. In the present study, we evaluated the effect of PEM on some aspects of hemopoiesis, analyzing the cell cycle of bone marrow cells and the percentage of progenitor cells in the bone marrow. Two-month-old male Swiss mice (N = 7-9 per group) were submitted to PEM with a low-protein diet (4%) or were fed a control diet (20% protein) ad libitum. When the experimental group had lost about 20% of their original body weight after 14 days, we collected blood and bone marrow cells to determine the percentage of progenitor cells and the number of cells in each phase of the cell cycle. Animals of both groups were stimulated with 5-fluorouracil. Blood analysis, bone marrow cell composition and cell cycle evaluation was performed after 10 days. Malnourished animals presented anemia, reticulocytopenia and leukopenia. Their bone marrow was hypocellular and depleted of progenitor cells. Malnourished animals also presented more cells than normal in phases G0 and G1 of the cell cycle. Thus, we conclude that PEM leads to the depletion of progenitor hemopoietic populations and changes in cellular development. We suggest that these changes are some of the primary causes of pancytopenia in cases of PEM.

Evaluation of methods for detection and identification of Mycobacterium species in patients suspected of having pulmonary tuberculosis / Avaliação de métodos para detecção e identificação de espécies de Mycobacterium em pacientes com suspeita de tuberculose pulmonar

Tuberculosis control is a priority for the Ministry of Health policies in Brazil. In the present work, the detection of Mycobacterium tuberculosis by the Polymerase Chain Reaction (PCR) was standardized, and the laboratory diagnosis of pulmonary tuberculosis was evaluated comparing baciloscopy, culture and PCR tests. The study was carried out with 117 sputum samples from different patients suspected of having pulmonary tuberculosis, for whom physicians had ordered a baciloscopy test. Baciloscopy was performed using the Ziehl-Neelsen method, and culture was performed by incubation of treated samples in Lowenstein-Jensen's medium at 37ºC for eight weeks. For PCR, DNA was amplified with a specific pair of primers to the M. tuberculosis complex, with a resulting product of 123 bp from the insertion element IS6110. Three (2.56 percent) samples presented a positive baciloscopy result and a positive PCR result (100 percent agreement), and nine (7.69 percent) presented Mycobacterium sp. growth in culture (P= 0.1384). Among six samples with positive results in culture, one was identified by PCR-RFLP as belonging to the M. tuberculosis complex and one was identified as a non-tuberculosis mycobacteria. Sensitivity and specificity of PCR compared to culture were 33.3 percent and 100 percent, respectively.

A tuberculose é um dos agravos prioritários para as políticas do Ministério da Saúde. No presente trabalho, o método de detecção de Mycobacterium tuberculosis pela Reação em Cadeia da Polimerase (PCR) em amostras de escarro foi padronizado e o diagnóstico laboratorial da tuberculose pulmonar foi avaliado, comparando-se as metodologias de baciloscopia, cultura e PCR. Foram analisadas 117 amostras de escarro de diferentes pacientes com suspeita de tuberculose pulmonar, com solicitação de baciloscopia. A baciloscopia foi realizada com a coloração de Ziehl-Neelsen e a cultura pela semeadura das amostras em meio de Lowenstein-Jensen, incubadas a 37ºC por oito semanas. Para realização da PCR, o DNA foi amplificado com um par de oligonucleotídeos específicos para o complexo M. tuberculosis, resultando em um produto de 123 pb do elemento de inserção IS6110. Das 117 amostras analisadas, três (2,56 por cento) apresentaram baciloscopia positiva e PCR positiva para M. tuberculosis (concordância de 100 por cento), e nove (7,69 por cento) tiveram crescimento de Mycobacterium sp. na cultura (P= 0,1384). Das seis amostras que tiveram resultado positivo somente por cultura, uma foi identificada ainda como pertencente ao complexo M. tuberculosis por PCR-RFLP, e outra foi identificada como micobactéria não tuberculosa. A sensibilidade e a especificidade da baciloscopia e da PCR em relação à cultura foram 33,3 por cento e 100 por cento, respectivamente.

Evaluation of methods for detection and identification of Mycobacterium species in patients suspected of having pulmonary tuberculosis.

Tuberculosis control is a priority for the Ministry of Health policies in Brazil. In the present work, the detection of Mycobacterium tuberculosis by the Polymerase Chain Reaction (PCR) was standardized, and the laboratory diagnosis of pulmonary tuberculosis was evaluated comparing baciloscopy, culture and PCR tests. The study was carried out with 117 sputum samples from different patients suspected of having pulmonary tuberculosis, for whom physicians had ordered a baciloscopy test. Baciloscopy was performed using the Ziehl-Neelsen method, and culture was performed by incubation of treated samples in Lowenstein-Jensen's medium at 37°C for eight weeks. For PCR, DNA was amplified with a specific pair of primers to the M. tuberculosis complex, with a resulting product of 123 bp from the insertion element IS6110. Three (2.56%) samples presented a positive baciloscopy result and a positive PCR result (100% agreement), and nine (7.69%) presented Mycobacterium sp. growth in culture (P= 0.1384). Among six samples with positive results in culture, one was identified by PCR-RFLP as belonging to the M. tuberculosis complex and one was identified as a non-tuberculosis mycobacteria. Sensitivity and specificity of PCR compared to culture were 33.3% and 100%, respectively.

Evaluation of methods for detection and identification of Mycobacterium species in patients suspected of having pulmonary tuberculosis

Tuberculosis control is a priority for the Ministry of Health policies in Brazil. In the present work, the detection of Mycobacterium tuberculosis by the Polymerase Chain Reaction (PCR) was standardized, and the laboratory diagnosis of pulmonary tuberculosis was evaluated comparing baciloscopy, culture and PCR tests. The study was carried out with 117 sputum samples from different patients suspected of having pulmonary tuberculosis, for whom physicians had ordered a baciloscopy test. Baciloscopy was performed using the Ziehl-Neelsen method, and culture was performed by incubation of treated samples in Lowenstein-Jensen's medium at 37ºC for eight weeks. For PCR, DNA was amplified with a specific pair of primers to the M. tuberculosis complex, with a resulting product of 123 bp from the insertion element IS6110. Three (2.56%) samples presented a positive baciloscopy result and a positive PCR result (100% agreement), and nine (7.69%) presented Mycobacterium sp. growth in culture (P= 0.1384). Among six samples with positive results in culture, one was identified by PCR-RFLP as belonging to the M. tuberculosis complex and one was identified as a non-tuberculosis mycobacteria. Sensitivity and specificity of PCR compared to culture were 33.3% and 100%, respectively.

A tuberculose é um dos agravos prioritários para as políticas do Ministério da Saúde. No presente trabalho, o método de detecção de Mycobacterium tuberculosis pela Reação em Cadeia da Polimerase (PCR) em amostras de escarro foi padronizado e o diagnóstico laboratorial da tuberculose pulmonar foi avaliado, comparando-se as metodologias de baciloscopia, cultura e PCR. Foram analisadas 117 amostras de escarro de diferentes pacientes com suspeita de tuberculose pulmonar, com solicitação de baciloscopia. A baciloscopia foi realizada com a coloração de Ziehl-Neelsen e a cultura pela semeadura das amostras em meio de Lowenstein-Jensen, incubadas a 37ºC por oito semanas. Para realização da PCR, o DNA foi amplificado com um par de oligonucleotídeos específicos para o complexo M. tuberculosis, resultando em um produto de 123 pb do elemento de inserção IS6110. Das 117 amostras analisadas, três (2,56%) apresentaram baciloscopia positiva e PCR positiva para M. tuberculosis (concordância de 100%), e nove (7,69%) tiveram crescimento de Mycobacterium sp. na cultura (P= 0,1384). Das seis amostras que tiveram resultado positivo somente por cultura, uma foi identificada ainda como pertencente ao complexo M. tuberculosis por PCR-RFLP, e outra foi identificada como micobactéria não tuberculosa. A sensibilidade e a especificidade da baciloscopia e da PCR em relação à cultura foram 33,3% e 100%, respectivamente.

Experimental anxiety and the reinforcing effects of ethanol in rats.

In order to examine the relationship between anxiety and reinforcing effects of alcohol, drug-naive male Wistar rats weighing 250-300 g were classified as "anxious" and "non-anxious" in the elevated plusmaze test. A conditioned place preference test was then used to investigate the reinforcing effects of ethanol (EtOH) on these animals. On 2 alternate days, groups of "anxious", "non-anxious" and "normal" rats received intraperitoneal (i.p.) injections of EtOH (0.5, 1.0 or 1.5 g/kg) immediately before a 15-min confinement to the white compartment. On the 2 intervening days the same rats received i.p. injections of saline before confinement to the opposite compartment. On day 5, a 15-min free-choice test was carried out with no injections. Rats classified as "anxious" showed a significant, though not dose-dependent preference for all doses of ethanol compared to saline-treated animals. These data demonstrate that rats regarded as "anxious" are more sensitive to the reinforcing effects of EtOH than "non-anxious" and "normal" Wistar rats and emphasize the relevance of the basal levels of anxiety of rats when trying to detect the reinforcing effects of EtOH.

Experimental anxiety and the reinforcing effects of ethanol in rats

In order to examine the relationship between anxiety and reinforcing effects of alcohol, drug-naive male Wistar rats weighing 250-300 g were classified as "anxious" and "non-anxious" in the elevated plus-maze test. A conditioned place preference test was then used to investigate the reinforcing effects of ethanol (EtOH) on these animals. On 2 alternate days, groups of "anxious", "non-anxious" and "normal" rats received intraperitoneal (ip) injections of EtOH (0.5, 1.0 or 1.5 g/kg) immediately before a 15-min confinement to the white compartment. On the 2 intervening days the same rats received ip injections of saline before confinement to the opposite compartment. On day 5, a 15-min free-choice test was carried out with no injections. Rats classified as "anxious" showed a significant, though not dose-dependent preference for all doses of ethanol compared to saline-treated animals. These data demonstrate that rats regarded as "anxious" are more sensitive to the reinforcing effects of EtOH than "non-anxious" and "normal" Wistar rats and emphasize the relevance of the basal levels of anxiety of rats when trying to detect the reinforcing effects of EtOH.

Memory-impairing effects of local anesthetics in an elevated plus-maze test in mice.

Post-training intracerebroventricular administration of procaine (20 micrograms/microliter) and dimethocaine (10 or 20 micrograms/microliter), local anesthetics of the ester class, prolonged the latency (s) in the retention test of male and female 3-month-old Swiss albino mice (25-35 g body weight; N = 140) in the elevated plus-maze (mean +/- SEM for 10 male mice: control = 41.2 +/- 8.1; procaine = 78.5 +/- 10.3; 10 micrograms/microliter dimethocaine = 58.7 +/- 12.3; 20 micrograms/microliter dimethocaine = 109.6 +/- 5.73; for 10 female mice: control = 34.8 +/- 5.8; procaine = 55.3 +/- 13.4; 10 micrograms/microliter dimethocaine = 59.9 +/- 12.3 and 20 micrograms/microliter dimethocaine = 61.3 +/- 11.1). However, lidocaine (10 or 20 micrograms/microliter), an amide class type of local anesthetic, failed to influence this parameter. Local anesthetics at the dose range used did not affect the motor coordination of mice exposed to the rota-rod test. These results suggest that procaine and dimethocaine impair some memory process(es) in the plus-maze test. These findings are interpreted in terms of non-anesthetic mechanisms of action of these drugs on memory impairment and also confirm the validity of the elevated plus-maze for the evaluation of drugs affecting learning and memory in mice.

Memory-impairing effects of local anesthetics in an elevated plus-maze test in mice

Post-training intracerebroventricular administration of procaine (20 mug/mul) and dimethocaine (10 or 20 mug/mul), local anesthetics of the ester class, prolonged the latency(s) in the retention test of male and female 3-month-old Swiss albino mice (25-35 g body weight; N=140) in the elevated plus-maze (mean + SEM for 10 male mice: control= 41.2 + 8.1; procaine = 78.5 + 10.3; 10 mug/mul dimethocaine = 58.7 + 12.3; 20 mug/mul dimethocaine = 109.6 + 5.73; for 10 female mice: control = 34.8 + 5.8; procaine = 55.3 + 13.4; 10 mug/mul dimethocaine = 59.9 + 12.3 and 20 mug/mul dimethocaine = 61.3 + 11.1). However, lidocaine (10 or 20 mug/mul), an amide class type of local anesthetic, failed to influence this parameter. Local anesthetics at the dose range used did not affect the motor coordination of mice exposed to the rota-rod test. These results suggest that procaine and dimethocaine impair some memory process(es) in the plus-maze test. These findings are interpreted in terms of non-anesthetic mechanisms of action of these drugs on memory impairment and also confirm the validity of the elevated plusmaze for the evaluation of drugs effecting learning and memory in mice.

Serologic evaluation of human immunodeficiency virus type 1-infected individuals from Argentina and the United States indicates a similar distribution of subgroup B isolates.

Utilizing peptides based on the V3 region of gp120, we undertook a serologic examination of human immunodeficiency virus type 1 (HIV-1)-infected individuals from Argentina to determine if prevalent HIV-1 isolates could be identified in this population. Our findings suggest that a similar pool of HIV-1 subgroup B isolates exists in both Argentina and the United States.

PIP: This study examined serum specimens from HIV-1 infected individuals from Argentina (n = 50) and the United States (n = 38) for antibody reactivity to a panel of V3-based synthetic peptides. Serum specimens were further analyzed for the ability to neutralize laboratory and clinical isolates of HIV-1 in vitro. Patterns of antibody reactivity to these V3 peptides, together with neutralizing activity, indicated that infected individuals from both Argentina and the US have been exposed to HIV-1 isolates belonging to subgroup B. Serum specimens from the United States (37 males and 1 female) were obtained from military personnel and their dependents. Of these patients, 35 were asymptomatic and 3 were symptomatic. Specimens from Argentina were obtained from HIV-1-infected individuals examined in Buenos Aires, Argentina (37 males and 13 females). Half of the infected individuals from Argentina were symptomatic. Serum specimens were screened for antibody reactivity to HIV-1 gp160 synthetic peptides by an enzyme-linked immunosorbent assay. Examination of V3 peptide recognition indicated that a higher percentage of Argentinean serum specimens reacted with peptide RP189 than serum specimens from the United States (34% and 5%, respectively). A higher percentage of serum specimens from the United States reacted with peptide RP135 (LAI) than was observed with serum specimens from Argentina (47% vs. 16%, respectively). Neutralization assays again indicated a similar pattern of antibody reactivity with serum specimens from infected individuals from Argentina and the United States. Nucleotide sequence analysis of clinical isolates has demonstrated that the HIV-1 subgroup B is predominant in the United States. Serologic reactivity to V3-based peptides in this study suggests that isolates commonly found in the US (i.e., MN, SF2, and NY-5) are also frequently observed in Argentina. These results suggest that there is similar distribution of HIV-1 subgroup B isolates among infected individuals from Argentina and the United States.