Rearing Africanized honey bee (Apis mellifera L.) brood under laboratory conditions

We developed a method for rearing larvae of Africanized bees under laboratory conditions to determine the amount of diet needed during larval development to obtain a worker bee. We started with larvae 18-24 h old, which were transferred to polyethylene cell cups and fed for five days. We found that the amount of diet needed for successful larval development was: 4, 15, 25, 50, and 70 mu L during the first to fifth days, respectively. The survival rate to the adult stage was 88.6% when the larvae received the daily amount of diet divided into two feedings, and 80% when they received only one feeding per day. The adult weight obtained in the laboratory, when the larvae received the daily amount of diet in a single dose, did not differ from those that were developed under field conditions (our control). All adults that we obtained in laboratory appeared to be normal. This technique has the potential to facilitate studies on brood pathogens, resistance mechanisms to diseases and also might be useful to test the impacts of transgenic products on honey bee brood.

Immunomodulatory effects of recombinant BCG expressing pertussis toxin on TNF-alpha and IL-10 in a bladder cancer model

Background: Since successful treatment of superficial bladder cancer with BCG requires proper induction of Th1 immunity, we have developed a rBCG-S1PT strain that induced a stronger cellular immune response than BCG. This preclinical study was designed to compare the modulatory effects of BCG and rBCG-S1PT on bladder TNF-alpha and IL-10 expression and to evaluate antitumour activity. Methods: For Experiment I, the MB49 bladder cancer cell line was used in C57BL/6 mice. Chemical cauterization of the bladder was performed to promote intravesical tumor implantation. Mice were treated by intravesical instillation with BCG, rBCG-S1PT or PBS once a week for four weeks. After 35 days the bladders were removed and weighed. TNF-

Endoscopic Ultrasound Facilitates Histological Diagnosis of Renal Cell Cancer

Over two-thirds of renal masses noted incidentally on abdominal CT scans performed for nonurological indications are most likely renal cell carcinoma (RCC). The prognosis in these instances is often quite favorable, as early diagnosis offers the best hope of a complete cure. We report the identification of a RCC incidentally at endoscopic ultrasound (EUS) while an earlier CT scan of abdomen was negative. The mass was biopsied resulting in a histological confirmation of RCC and successful surgical resection.

Laparoscopic nephrectomy outside gerota fascia for management of inflammatory kidney

Background and Purpose: A nonfunctioning inflammatory kidney is a challenging surgical condition for urologists. Some investigators recommend open surgery because of the surgical difficulties caused by the inflammatory process, whereas others try to apply the advantages of a ""simple"" non-hand-assisted laparoscopic approach. We report our experience with simple laparoscopic nephrectomy for inflammatory kidney management. Patients and Methods: From July 2002 through December 2006, 50 pure laparoscopic nephrectomies were performed for inflammatory kidney ( 43 because of pyelonephritis, 5 for xanthogranulomatous pyelonephritis (XGP), and 2 for pyonephrosis). Histopathologic analysis was the criterion used for inflammatory kidney diagnosis. Pain or recurrent urinary tract infection associated with a nonfunctioning excluded kidney was the eligibility criterion for the procedure. Preoperatively, all patients underwent complete image and functional renal assessment. Morcellation was used to remove surgical specimens. Conversion index, surgical difficulties, operative time, and postoperative complications were evaluated. Results: Conversion was performed in 14 of 50 (28%) patients, including two with XGP and one with pyonephrosis. Adhesions, vascular (two inferior vena cava) lesions, and intestinal lesions (two colon) were the main causes of conversion. Acute pancreatitis developed in one patient, and one patient had a wound infection. Reoperations were unnecessary, and no deaths occurred. Conclusion: Pure laparoscopic nephrectomy was successful in 72% of patients with inflammatory kidneys. The laparoscopic dissection was useful even in those cases converted to open surgery. This is a high-risk procedure, however, and both surgeon and patient must be aware of that before the decision is made for this approach.

Combined Retrograde Flexible Ureteroscopic Lithotripsy with Holmium YAG Laser for Renal Calculi Associated with Ipsilateral Ureteral Stones

Purpose: The purpose of this study was to evaluate the effectiveness of combined ureteroscopic holmium YAG lithotripsy for renal calculi associated with ipsilateral ureteral stones. Materials and Methods: Between August 2002 and March 2007, retrograde flexible ureteroscopic stone treatment was attempted in 351 cases. Indication for treatment was concurrent symptomatic ureteral stones in 63 patients (group I). Additional operative time and perioperative complication rates were compared to a group of 39 patients submitted to ureteroscopic treatment for ureteral calculi exclusively (group II). Results: Mean ureteral stone size was 8.0 +/- 2.6 mm and 8.1 +/- 3.4 mm for groups I and II, respectively. Mean operative time for group I was 67.9 +/- 29.5 minutes and for group 2 was 49.3 +/- 13.2 minutes (p < 0.001). Flexible ureteroscopic therapy for renal calculi increased 18 minutes in the mean operative time. The overall complication rate was 3.1% and 2.5% for groups I and II, respectively (p = 0.87). Mean renal stone size was 10.7 +/- 6.4 mm, overall stone free rate in group I was 81%. However, considering only patients with renal stones smaller than 15 mm, the stone free rate was 88%. Successful treatment occurred in 81% of patients presenting lower pole stones, but only 76% of patients with multiple renal stones became stone free. As expected, stone free rate showed a significant negative correlation with renal stone size (p = 0.03; r = -0.36). Logistic regression model indicated an independent association of renal stones smaller than 15 mm and stone free rate (OR = 13.5; p = 0.01). Conclusion: Combined ureteroscopic treatment for ureteral and ipsilateral renal calculi is a safe and attractive option for patients presenting for symptomatic ureteral stone and ipsilateral renal calculi smaller than 15 mm.

Unexpected Diversity of Cellular Immune Responses against Nef and Vif in HIV-1-Infected Patients Who Spontaneously Control Viral Replication

Background: HIV-1-infected individuals who spontaneously control viral replication represent an example of successful containment of the AIDS virus. Understanding the anti-viral immune responses in these individuals may help in vaccine design. However, immune responses against HIV-1 are normally analyzed using HIV-1 consensus B 15-mers that overlap by 11 amino acids. Unfortunately, this method may underestimate the real breadth of the cellular immune responses against the autologous sequence of the infecting virus. Methodology and Principal Findings: Here we compared cellular immune responses against nef and vif-encoded consensus B 15-mer peptides to responses against HLA class I-predicted minimal optimal epitopes from consensus B and autologous sequences in six patients who have controlled HIV-1 replication. Interestingly, our analysis revealed that three of our patients had broader cellular immune responses against HLA class I-predicted minimal optimal epitopes from either autologous viruses or from the HIV-1 consensus B sequence, when compared to responses against the 15-mer HIV-1 type B consensus peptides. Conclusion and Significance: This suggests that the cellular immune responses against HIV-1 in controller patients may be broader than we had previously anticipated.

Cost risk benefit analysis to support chemoprophylaxis policy for travellers to malaria endemic countries

Background: In a number of malaria endemic regions, tourists and travellers face a declining risk of travel associated malaria, in part due to successful malaria control. Many millions of visitors to these regions are recommended, via national and international policy, to use chemoprophylaxis which has a well recognized morbidity profile. To evaluate whether current malaria chemo-prophylactic policy for travellers is cost effective when adjusted for endemic transmission risk and duration of exposure. a framework, based on partial cost-benefit analysis was used Methods: Using a three component model combining a probability component, a cost component and a malaria risk component, the study estimated health costs avoided through use of chemoprophylaxis and costs of disease prevention (including adverse events and pre-travel advice for visits to five popular high and low malaria endemic regions) and malaria transmission risk using imported malaria cases and numbers of travellers to malarious countries. By calculating the minimal threshold malaria risk below which the economic costs of chemoprophylaxis are greater than the avoided health costs we were able to identify the point at which chemoprophylaxis would be economically rational. Results: The threshold incidence at which malaria chemoprophylaxis policy becomes cost effective for UK travellers is an accumulated risk of 1.13% assuming a given set of cost parameters. The period a travellers need to remain exposed to achieve this accumulated risk varied from 30 to more than 365 days, depending on the regions intensity of malaria transmission. Conclusions: The cost-benefit analysis identified that chemoprophylaxis use was not a cost-effective policy for travellers to Thailand or the Amazon region of Brazil, but was cost-effective for travel to West Africa and for those staying longer than 45 days in India and Indonesia.

Survival and Neuronal Differentiation of Mesenchymal Stem Cells Transplanted into the Rodent Brain Are Dependent upon Microenvironment

Introduction: The successful integration of stem cells in adult brain has become a central issue in modern neuroscience. In this study we sought to test the hypothesis that survival and neurodifferentiation of mesenchymal stem cells (MSCs) may be dependent upon microenvironmental conditions according to the site of implant in the brain. Methods: MSCs were isolated from adult rats and labeled with enhanced-green fluorescent protein (eGFP) lentivirus. A cell suspension was implanted stereotactically into the brain of 50 young rats, into one neurogenic area (hippocampus), and into another nonneurogenic area (striatum). Animals were sacrificed 6 or 12 weeks after surgery, and brains were stained for mature neuronal markers. Cells coexpressing NeuN (neuronal specific nuclear protein) and GFP (green fluorescent protein) were counted stereologically at both targets. Results: The isolated cell population was able to generate neurons positive for microtubule-associated protein 2 (MAP2), neuronal-specific nuclear protein (NeuN), and neurofilament 200 (NF200) in vitro. Electrophysiology confirmed expression of voltage-gated ionic channels. Once implanted into the hippocampus, cells survived for up to 12 weeks, migrated away from the graft, and gave rise to mature neurons able to synthesize neurotransmitters. By contrast, massive cell degeneration was seen in the striatum, with no significant migration. Induction of neuronal differentiation with increased cyclic adenosine monophosphate in the culture medium before implantation favored differentiation in vivo. Conclusions: Our data demonstrated that survival and differentiation of MSCs is strongly dependent upon a permissive microenvironment. Identification of the pro-neurogenic factors present in the hippocampus could subsequently allow for the integration of stem cells into nonpermissive areas of the central nervous system.

Endometrial claudin-4 and leukemia inhibitory factor are associated with assisted reproduction outcome

Background: Claudin-4 (CLDN4) is one of several proteins that act as molecular mediators of embryo implantation. Recently, we examined immunolabeling of leukemia inhibitory factor (LIF) in the endometrial tissue of 52 IVF patients, and found that LIF staining intensity was strongly correlated with successful pregnancy initiation. In the same set of patients, we have now examined endometrial CLDN4 expression, to see how expression intensity may vary with LIF. We examined CLDN4 in the luteal phase of the menstrual cycle, immediately preceding IVF treatment. Our aim was to compare expression of LIF and CLDN4 in the luteal phase, and document these patterns as putative biomarkers for pregnancy. Methods: Endometrial tissue was collected from women undergoing IVF. Endometrial biopsies were obtained during the luteal phase preceding IVF, and were then used for tissue microarray (TMA) immunolabeling of CLDN4. Previously published LIF expression data were then combined with CLDN4 expression data, to determine CLDN4/LIF expression patterns. Associations between successful pregnancy after IVF and combined CLDN4/LIF expression patterns were evaluated. Results: Four patterns of immunolabeling were observed in the endometrial samples: 16% showed weak CLDN4 and strong LIF (CLDN4(-)/LIF(+)); 20% showed strong CLDN4 and strong LIF (LIF(+)/CLDN4(+)); 28% showed strong CLDN4 and weak LIF (CLDN4(+)/LIF(-)); and 36% showed weak CLDN4 and weak LIF (CLDN4(-)/LIF(-)). Successful implantation after IVF was associated with CLDN4(-)/LIF(+)(p = 0.003). Patients showing this endometrial CLDN4(-)/LIF(+) immunolabeling were also 6 times more likely to achieve pregnancy than patients with endometrial CLDN4(+)/LIF(-) immunolabeling (p = 0.007). Conclusion: The combined immunolabeling expression of CLDN4(-)/LIF(+) in endometrial tissue is a potential biomarker for predicting successful pregnancy in IVF candidates.

A Vaccine Encoding Conserved Promiscuous HIV CD4 Epitopes Induces Broad T Cell Responses in Mice Transgenic to Multiple Common HLA Class II Molecules

Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, ""promiscuous"" ( multiple HLA-DR-binding) B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II-transgenic mice (-DR2, -DR4, -DQ6 and -DQ8). Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.

Near full-length genome analysis of low prevalent human immunodeficiency virus type 1 subclade F1 in Sao Paulo, Brazil

Background: The genetic diversity of the human immunodeficiency virus type 1 (HIV-1) is critical to lay the groundwork for the design of successful drugs or vaccine. In this study we aimed to characterize and define the molecular prevalence of HIV-1 subclade F1 currently circulating in Sao Paulo, Brazil. Methods: A total of 36 samples were selected from 888 adult patients residing in Sao Paulo who had previously been diagnosed in two independent studies in our laboratory as being infected with subclade F1 based on pol subgenomic fragment sequencing. Proviral DNA was amplified from the purified genomic DNA of all 36 blood samples by 5 fragments overlapping PCR followed by direct sequencing. Sequence data were obtained from the 5 fragments of pure subclade F1 and phylogenetic trees were constructed and compared with previously published sequences. Subclades F1 that exhibited mosaic structure with other subtypes were omitted from any further analysis Results: Our methods of fragment amplification and sequencing confirmed that only 5 sequences inferred from pol region as subclade F1 also holds true for the genome as a whole and, thus, estimated the true prevalence at 0.56%. The results also showed a single phylogenetic cluster of the Brazilian subclade F1 along with non-Brazilian South American isolates in both subgenomic and the full-length genomes analysis with an overall intrasubtype nucleotide divergence of 6.9%. The nucleotide differences within the South American and Central African F1 strains, in the C2-C3 env, were 8.5% and 12.3%, respectively. Conclusion: All together, our findings showed a surprisingly low prevalence rate of subclade F1 in Brazil and suggest that these isolates originated in Central Africa and subsequently introduced to South America.

Effect of Low-Level Laser Therapy on Malondialdehyde Concentration in Random Cutaneous Flap Viability

Objective: The aim of this study was to assess the effects of 830 and 670 nm laser on malondialdehyde (MDA) concentration in random skin-flap survival. Background Data: Low-level laser therapy (LLLT) has been reported to be successful in stimulating the formation of new blood vessels and activating superoxide-dismutase delivery, thus helping the inhibition of free-radical action and consequently reducing necrosis. Materials and Methods: Thirty Wistar rats were used and divided into three groups, with 10 rats in each one. A random skin flap was raised on the dorsum of each animal. Group 1 was the control group; group 2 received 830 nm laser radiation; and group 3 was submitted to 670 nm laser radiation. The animals underwent laser therapy with 36 J/cm(2) energy density immediately after surgery and on the 4 days subsequent to surgery. The application site of the laser radiation was 1 point, 2.5 cm from the flap's cranial base. The percentage of the skin-flap necrosis area was calculated 7 days postoperative using the paper-template method, and a skin sample was collected immediately after as a way of determining the MDA concentration. Results: Statistically significant differences were found between the necrosis percentages, with higher values seen in group 1 compared with groups 2 and 3. Groups 2 and 3 did not present statistically significant differences (p > 0.05). Group 3 had a lower concentration of MDA values compared to the control group (p < 0.05). Conclusion: LLLT was effective in increasing the random skin-flap viability in rats, and the 670 nm laser was efficient in reducing the MDA concentration.

Challenges and opportunities for primary, secondary, and tertiary prevention of Chagas' disease

A century after its discovery, Chagas' disease still represents a major public health challenge in Latin America. Moreover, because of growing population movements, an increasing number of cases of imported Chagas' disease have now been detected in non-endemic areas, such as North America and some European countries. This parasitic zoonosis, caused by Trypanosoma cruzi, is transmitted to humans by infected Triatominae insects, or occasionally by non-vectorial mechanisms, such as blood transfusion, mother to fetus, or oral ingestion of materials contaminated with parasites. Following the acute phase of the infection, untreated individuals enter a chronic phase that is initially asymptomatic or clinically unapparent. Usually, a few decades later, 40-50% of patients develop progressive cardiomyopathy and/or motility disturbances of the oesophagus and colon. In the last decades several interventions targeting primary, secondary and tertiary prevention of Chagas' disease have been attempted. While control of both vectorial and blood transfusion transmission of T cruzi (primary prevention) has been successful in many regions of Latin America, early detection and aetiological treatment of asymptomatic subjects with Chagas' disease (secondary prevention) have been largely underutilised. At the same time, in patients with established chronic disease, several pharmacological and non-pharmacological interventions are currently available and have been increasingly used with the intention of preventing or delaying complications of the disease (tertiary prevention). In this review we discuss in detail each of these issues.

Vaccination of Mice with Salmonella Expressing VapA: Mucosal and Systemic Th1 Responses Provide Protection against Rhodococcus equi Infection

Conventional vaccines to prevent the pneumonia caused by Rhodococcus equi have not been successful. We have recently demonstrated that immunization with Salmonella enterica Typhimurium expressing the VapA antigen protects mice against R. equi infection. We now report that oral vaccination of mice with this recombinant strain results in high and persistent fecal levels of antigen-specific IgA, and specific proliferation of the spleen cells of immunized mice in response to the in vitro stimulation with R. equi antigen. After in vitro stimulation, spleen cells of immunized mice produce high levels of Th1 cytokines and show a prominent mRNA expression of the Th1 transcription factor T-bet, in detriment of the Th2 transcription factor GATA-3. Following R. equi challenge, a high H(2)O(2), NO, IL-12, and IFN-gamma content is detected in the organs of immunized mice. On the other hand, TNF-alpha and IL-4 levels are markedly lower in the organs of vaccinated mice, compared with the non-vaccinated ones. The IL-10 content and the mRNA transcription level of TGF-beta are also higher in the organs of immunized mice. A greater incidence of CD4(+) and CD8(+) T cells and B lymphocytes is verified in vaccinated mice. However, there is no difference between vaccinated and non-vaccinated mice in terms of the frequency of CD4(+)CD25(+)Foxp3(+) T cells. Finally, we show that the vaccination confers a long-term protection against R. equi infection. Altogether, these data indicate that the oral vaccination of mice with S. enterica Typhimurium expressing VapA induces specific and long-lasting humoral and cellular responses against the pathogen, which are appropriately regulated and allow tissue integrity after challenge.

First Report of the Isolation and Molecular Characterization of Rickettsia amblyommii and Rickettsia felis in Central America

During 2010, 15 adult ticks, identified as Amblyomma cajennense, were collected from horses in Cahuita and Turrialba districts, whereas 7 fleas, identified as Ctenocephalides felis, were collected from a dog in San Jose city, Costa Rica. In the laboratory, three A. cajennense specimens, two from Cahuita and one from Turrialba, were individually processed for rickettsial isolation in cell culture, as was a pool of seven fleas. Rickettsiae were successfully isolated and established in Vero cell culture from the three ticks and from a pool of seven fleas in C6/36 cell culture. The three tick isolates were genotypically identified as Rickettsia amblyommii, and the flea isolate was identified as Rickettsia felis through DNA sequencing of portions of the rickettsial genes gltA, ompA, and ompB of each isolate. In addition, other seven ticks were shown to contain rickettsial DNA. Polymerase chain reaction products of at least two of these ticks were sequenced and also showed to correspond to R. amblyommii. Overall, 66.7% (10/15) of the A. cajennense adult ticks were found to be infected with rickettsiae. This is the first report of a successful isolation in cell culture of R. amblyommii and R. felis from Central America.