Hernan Aviles

PCR DETECTION AND IDENTIFICATION OF LEISHMANIA PARASITES IN CLINICAL SPECIMENS IN ECUADOR: A COMPARISON WITH CLASSICAL DIAGNOSTIC METHODS

A simplified polymerase chain reaction (PCR)-based assay was used for detection and typing of Leishmania parasites in clinical specimens from patients suspected of cutaneous leishmaniasis. Using cultures as the reference standard, our PCR detection method was more sensitive (92%) than classical diagnostic techniques, including microscopy (42% sensitivity), histologic staining (33%), and IgG enzyme-linked immunosorbent (20%). The PCR assay was also 100% specific. Parasites in both lesion biopsies and isolates cultured from lesion aspirates were identified as Leishmania braziliensis by PCR. In this study, we have demonstrated the suitability of simplified PCR assays for the simultaneous diagnosis and typing of parasites causing cutaneous leishmaniasis in a developing country where leishmaniasis is endemic.

Field Trial of a Vaccine against New World Cutaneous Leishmaniasis in an At-Risk Child Population: Safety, Immunogenicity, and Efficacy during the First 12 Months of Follow-Up

The safety, immunogenicity, and efficacy of a vaccine against cutaneous leishmaniasis in rural Ecuadorian children was assessed in a randomized, controlled, double-blinded study. Vaccine group subjects received 2 intradermal doses of a whole, killed promastigote vaccine cocktail plus bacille Calmette-Guérin (BCG) adjuvant. Control subjects got 2 doses of BCG only. The subjects who received both vaccination doses, 438 in the vaccine group (79.3%) and 406 in the control group (83.4%), were followed for 12 months. No serious adverse side effects were identified in either group. Significantly more vaccine group subjects than controls converted to a positive Montenegro skin test (85.1% vs. 20.1%; chi2 = 279; P < .001). The incidence of cutaneous leishmaniasis was significantly reduced in the vaccine compared with the control group (2.1% vs. 7.6%; chi2 = 8.95; P < .003). The protective efficacy of the vaccine was 72.9% (95% confidence interval = 36.1%-88.5%).

Enhancement of in vitro growth of pathogenic bacteria by norepinephrine: importance of inoculum density and role of transferrin.

ABSTRACT Norepinephrine is a stress hormone that enhances bacterial growth. We examined the effects of a small inoculum on the norepinephrine-induced growth of species previously reported to be unaffected by norepinephrine. The results indicated that a reduced inoculum density is essential for observing norepinephrine-induced effects. Additional studies using serum-free media suggested that transferrin plays a role in norepinephrine-induced growth.

Catecholamines and in vitro growth of pathogenic bacteria: enhancement of growth varies greatly among bacterial species.

The purpose of this study was to examine the effects of catecholamines on in vitro growth of a range of bacterial species, including anaerobes. Bacteria tested included: Porphyromonas gingivalis, Bacteriodes fragilis, Shigella boydii, Shigella sonnie, Enterobacter Sp, and Salmonella choleraesuis. The results of the current study indicated that supplementation of bacterial cultures in minimal medium with norepinephrine or epinephrine did not result in increased growth of bacteria. Positive controls involving treatment of Escherichia coli with catecholamines did result in increased growth of that bacterial species. The results of the present study extend previous observations that showed differential capability of catecholamines to enhance bacterial growth in vitro.

Active hexose correlated compound activates immune function to decrease bacterial load in a murine model of intramuscular infection

BACKGROUND Infection is a serious, costly, and common complication of surgery and constitutes the principal cause of late death in patients undergoing surgery. The objective of this study was to clarify the mechanisms by which active hexose correlated compound (AHCC) increases survival in a murine model of intramuscular infection. METHODS Food-deprived mice receiving either AHCC or excipient were infected with bacteria. Kinetics of bacterial load, white blood cell counts, cytokine levels, and antibody levels were compared between groups. RESULTS AHCC-treated mice had reduced bacterial load at day 5 and cleared bacteria entirely at day 6. Levels of interleukin-12, tumor necrosis factor-alpha, and interleukin-6 peaked earlier in this group (day 3) compared with controls (day 5). Increased percentages of peripheral lymphocytes and monocytes and decreased numbers of polymorphonuclear cells were detected in the AHCC group. CONCLUSIONS AHCC appears to induce an early activation of the immune response, leading to an effective clearance of bacteria and rapid recovery.

Effects of space flight conditions on the function of the immune system and catecholamine production simulated in a rodent model of hindlimb unloading.

The rodent model of hindlimb unloading has been successfully used to simulate some of the effects of space flight conditions. Previous studies have indicated that mice exposed to hindlimb-unloading conditions have decreased resistance to infections compared to restrained and normally housed control mice. Objective: The purpose of this study was to clarify the mechanisms involved in resistance to infection in this model by examining the effects of hindlimb unloading on the function of the immune system and its impact on the production of catecholamines. Methods: Female Swiss Webster mice were hindlimb-unloaded during 48 h and the function of the immune system was assessed in spleen and peritoneal cells immediately after this period. In addition, the kinetics of catecholamine production was measured throughout the hindlimb-unloading period. Results: The function of the immune system was significantly suppressed in the hindlimb-unloaded group compared to restrained and normally housed control mice. Levels of catecholamines were increased in the hindlimb-unloaded group and peaked at 12 h following the commencement of unloading. Conclusion: These results suggest that physiological responses of mice are altered early after hindlimb unloading and that catecholamines may play a critical role in the modulation of the immune system. These changes may affect the ability of mice to resist infections.

Immune responses in adult female volunteers during the bed-rest model of spaceflight: Antibodies and cytokines

BACKGROUND It is unknown whether a prolonged period of bed rest will affect human immune responses, particularly in female subjects. OBJECTIVE We sought to measure immune responses in adult female subjects exposed to prolonged bed rest. METHODS Adult (25-40 years) female volunteers (n = 24) were maintained in a supine (6 degrees tilt) head-down bed-rest (HDBR) position for 60 days: 8 with HDBR only, 8 with HDBR and regular muscular exercise, and 8 with HDBR and dietary protein supplementation. Subjects were immunized with bacteriophage phiX-174. Antibody production and plasma cytokine levels were determined. RESULTS The rate of primary antibody production of the HDBR plus exercise group increased faster (P = .01) and to a higher level versus that of the HDBR-only group (P = .03) and that of the HDBR plus diet group (trend P = .08). The rates of secondary antibody production between the 3 groups were similar, but the level of antibody in the HDBR plus exercise group remained higher versus that in the HDBR-only group (P = .03). Both the HDBR (P = .001) and HDBR plus diet (P = .02) groups had time-related progressive increases in TNF-alpha receptor levels, but the HDBR plus exercise group remained at baseline. The HDBR plus exercise group experienced an acute increase in IL-1 receptor antagonist levels versus the HDBR (P = .02) and the HDBR plus diet (P = .02) groups, with similar increases in RANTES levels. CONCLUSIONS The exercise countermeasure accelerated primary antibody production and increased antibody levels to bacteriophage phiX-174 and also opposed the potentially harmful effects of increased TNF-alpha levels caused by prolonged bed rest, possibly by activating the anti-inflammatory cytokine IL-1 receptor antagonist and the chemotactic factor RANTES.

Effects of Cold Stress on Spleen Cell Proliferation and Cytokine Production during Chronic Toxoplasma gondii Infection

Background: Cell-mediated immunity is critical for controlling infection and preventing reactivation during the chronic phase of Toxoplasma gondii infection. In people suffering from AIDS, T. gondii is one of the major opportunistic infectious agents. Mechanisms regulating rapid development of clinical signs in previously asymptomatic patients remain unclear; however, cofactors such as stress are suspected to play a role in the susceptibility to opportunistic infections. Objective: This study examined the role of cold stress (CS) in splenocyte function during chronic T. gondii infection. Methods: Control mice and mice previously infected orally with T. gondii were subjected to CS during the chronic phase (CSchr), i.e. 90 days after infection, and in vitro cell proliferation and cytokine production were measured before (day 0) and 1, 15 and 25 days after CSchr. Splenocyte proliferation and cytokine production were measured after in vitro stimulation with concanavalin A (Con-A), anti-CD3 antibody (A-CD3) and Toxoplasma lysate antigen. Results: CSchr enhanced splenocyte proliferation in cells stimulated with Con-A and A-CD3, but it suppressed proliferation in cells stimulated with T. gondii antigens. Increased levels of interferon (IFN)-γ were detected independent of the type of stimulation after CSchr and remained high throughout the experiment. CS had similar results in noninfected animals. Conclusion: Although an overall increase in splenocyte function occurred after nonspecific stimulation, CS suppressed primed spleen cells from responding to T. gondii antigens which could lead to reactivation of latent infection. The increase in IFN-γ after CSchr could be a result of spleen cells being primed by released parasites by this stressor. IFN-γ is critical in the control of parasite reactivation.

Cold stress-induced modulation of cell immunity during acute Toxoplasma gondii infection in mice.

Infection with Toxoplasma gondii in the acute phase results in nonspecific suppression of immunologic function in mice and humans. The present study examined the effects of a physical stressor, i.e., cold stress (CS), on macrophage function (nitrite production, parasite survival) and splenic blastogenesis in the acute phase of murine T. gondii infection. In our stress paradigm, female BALB/c mice were placed in cold water (1 +/- 0.5 C), 5 min each day for 8 days. Nitrite production and parasite survival were measured in cultured peritoneal macrophages obtained from mice subjected to CS after in vivo activation with interferon-gamma/lipopolysaccharide (CS + ACT), and in vitro infection with T. gondii tachyzoites. Peritoneal macrophages from CS + ACT mice showed decreased nitrite production compared to control but activated cells (ACT). Spleen cell proliferation to in vitro stimulation with the mitogens concanavalin A (Con A) and anti-CD3, and Toxoplasma lysate antigen (TLA) was measured in splenocytes obtained from BALB/c mice during the acute phase of infection with T. gondii. Mice subjected to CS and infection (CS + INF) had maximum splenocyte proliferation on days 8 and 15 followed by a subsequent decline on day 28 postinoculation (PI). In contrast, infected mice not subjected to stress (INF) showed decreased splenocyte proliferation on days 8 and 15 followed by an increase on day 28 PI. The rate of mortality was decreased in the CS + INF compared to the INF group during acute infection. These results suggest that CS may alter the pathogenesis of T. gondii infection by modulating acute-phase responses, provoking a state of transient disequilibrium between the host and parasite.

Cold stress-induced modulation of inflammatory responses and intracerebral cytokine mRNA expression in acute murine toxoplasmosis.

The effects of a physical stressor, cold water stress (CWS), within the central nervous system were investigated in the acute phase of infection with Toxoplasma gondii. Female BALB/c mice were subjected to CWS for 5 min each day for 8 days prior to oral infection with 20 cysts of the low virulent ME 49 strain. Animals were killed at 10-day intervals to detect inflammation, gliosis, and expression of intracerebral cytokine mRNAs. Zones of inflammation were detected by Nissl staining and gliosis by immunoreactivity to glial fibrillary acidic protein. Larger zones of inflammation and reactive astrogliosis were consistently observed in mice subjected to CWS and infected (CWS +INF) compared to control infected (INF) mice. Expression of interleukin (IL)-1beta, IL-2, IL-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and inducible nitric oxide synthase (iNOS) were decreased in CWS+INF mice at 10 days postinoculation (PI), followed by a gradual increase after day 20 PI. This was in contrast to increased expression of these cytokines at 10 days PI in INF mice with a gradual decline thereafter. Inflammation and astrogliosis in CWS+INF mice were associated with an increased expression of IL-1beta, IL-6, IL-12, and TNF-alpha between 20 and 30 days PI. These findings correlated with the continuous gene expression of tachyzoite surface antigen (SAG)-1 mRNA in CWS+INF mice compared to its sharp decline in INF mice after 20 days PI. These results suggest that CWS delays regulation and control of intracerebral Toxoplasma gondii during acute infection in BALB/c mice by decreasing the early expression of IFN-gamma, IL-2, TNF-alpha, iNOS, IL-1beta, and IL-12, while increasing the expression of IL-6, a counterregulatory cytokine.