quarta-feira, 29 de junho de 2011

Investimento no Brasil - Finantial Times





In Brazil, the chances are that if you eat a burger, go to college or travel to Miami, you will be putting money into the pockets of private equity firms.
A boom in the industry in Latin America over the past few years has led to a host of new deals, with a Brazilian private equity firm, Vinci Partners, holding the local franchise for Burger King, and others, such as Advent International, targeting Brazil’s fast-growing education sector.

Carlyle’s South American buy-out team, meanwhile, made its first investment in the region last year in CVC Brasil Operadora e Agência de Viagens, the region’s biggest tour operator.
Most of the large companies have already set up offices in Brazil, or like Kohlberg Kravis Roberts and TPG, are looking to do so.
“The number of managers able to write $100m cheques here have multiplied by five,” said R. Duncan Littlejohn, managing director at Paul Capital in São Paulo.
The big private equity companies had been among the last to come back to the country because of their memory of previous hardships.
While the last five years have been upbeat – comanies raised $8.1bn for Latin America as a whole last year and are expected to have $10bn-$11bn available for investment in Brazil alone by the end of 2011 – it has not always been this good.
During the last boom in the 1990s, they raised about $6bn to invest in Brazil, according to Advent International. Then the internet bubble imploded, Brazil suffered a currency crisis and the leftist presidential candidate Luiz Inácio Lula da Silva came to power.
During the early 2000s, the market plunged and the downturn was so severe that some funds returned their money to investors and turned their backs on Brazil.
Patrice Nogueira Baptista Etlin, managing partner in São Paulo for Advent International, which opened in Brazil in 1997, said: “The market was abandoned in what we call in our industry the ‘nuclear winter’” .
But Mr Lula da Silva followed through on economic reforms started by his predecessor, Fernando Henrique Cardoso, including passing laws that reformed Brazil’s stock market and improved corporate governance among listed companies.
This paved the way for an increase in initial public offerings, that in turn provided an important platform for private equity’s return.
IPOs in Brazil increased from only about six between 1994 and 2004 to about 200 since then.

The industry received a scare again in 2009 when the global financial crisis hit Brazil. But the economy recovered quickly, leading to a rush of private equity funds into the country last year.
High-profile investments included JPMorgan’s acquisition of control of Gávea Investimentos in Rio, and Blackstone’s buying a stake in another local fund, Pátria Investimentos.
The increase in private equity money chasing deals in Brazil has led to concern that the market is becoming saturated. But Brazil’s private equity environment remains relatively under-developed compared with the US.
Personal relationships between companies and individual entrepreneurs or business families remain key and there are fewer open auctions of companies of the kind seen for most deals in the US.
Advent’s Mr Etlin says there are an estimated 50,000 companies in Brazil with revenue of R$50m ($31m) or more a year. “It’s a huge aquarium,” he said.
Brazil also has the advantage of being the most culturally similar emerging market for US and European funds, says Cate Ambrose, president of Latin American Venture Capital Association.
She said: “Brazil is much closer to the European and US business culture of working with financial investors, building up the company and eventually selling it”.
To be sure, Brazil has important differences with developed markets. Its high interest rates make highly leveraged transactions less attractive. Brazil’s taxes on financial transactions also make it expensive to import capital.
Its smaller and medium-sized family-run companies have also traditionally been suspicious of outside investors.
Yet this is changing as they seek growth capital, says Marcelo Di Lorenzo, managing director at 3i Brasil. Family businesses are becoming more ambitious about realising value.

terça-feira, 28 de junho de 2011

vacina para tuberculose - Science


TUBERCULOSIS
Immunogenicity of the Tuberculosis Vaccine MVA85A Is Reduced by Coadministration with EPI Vaccines in a Randomized Controlled Trial in Gambian Infants
Martin O. C. Ota1,*, Aderonke A. Odutola1, Patrick K. Owiafe1, Simon Donkor1, Olumuyiwa A. Owolabi1, Nathaniel J. Brittain2, Nicola Williams3, Sarah Rowland-Jones1, Adrian V. S. Hill2, Richard A. Adegbola4 and Helen McShane2
+ Author Affiliations

1Bacterial Diseases Programme, Medical Research Council Unit, P. O. Box 273, Banjul, The Gambia.
2Jenner Institute, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK.
3Centre for Statistics in Medicine, Wolfson College, Linton Road, Oxford OX2, UK.
4Bill & Melinda Gates Foundation, Seattle, WA 98102, USA.
↵*To whom correspondence should be addressed. E-mail: mota@mrc.gm
New tuberculosis vaccines are urgently needed to curtail the current epidemic. MVA85A is a subunit vaccine that could enhance immunity from BCG vaccination. To determine MVA85A safety and immunogenicity as well as interactions with other routine vaccines administered in infancy, we randomized healthy 4-month-old infants who had received Bacille Calmette-Guérin at birth to receive Expanded Program on Immunization (EPI) vaccines alone, EPI and MVA85A simultaneously, or MVA85A alone. Adverse events were monitored throughout. Blood samples obtained before vaccination and at 1, 4, and 20 weeks after vaccination were used to assess safety and immunogenicity. The safety profile of both low and standard doses was comparable, but the standard dose was more immunogenic and therefore was selected for the second stage of the study. In total, 72 (first stage) and 142 (second stage) infants were enrolled. MVA85A was safe and well tolerated and induced a potent cellular immune response. Coadministration of MVA85A with EPI vaccines was associated with a significant reduction in MVA85A immunogenicity, but did not affect humoral responses to the EPI vaccines. These results provide important information regarding timing of immunizations, which is required for the design of infant efficacy trials with MVA85A, and suggest that modifications to the standard EPI schedule may be required to incorporate a new generation of T cell–inducing vaccines.

INTRODUCTION

Globally, tuberculosis (TB) continues to be a disease of major public health importance (1) despite widespread coverage by Bacille Calmette-Guérin (BCG) vaccination; the persistent TB disease burden underlines the inadequacy of the BCG vaccine to protect against TB in adults. As such, there is an urgent need for a more effective vaccine (2). However, BCG protects against severe forms of TB in childhood and has nontargeted beneficial effects, including improving child survival, increasing responses to hepatitis B and oral polio vaccines administered in infancy, preventing allergic disorders, and protecting against leprosy (3–5). Consequently, it is logical to deploy a vaccination strategy against TB that includes BCG.

MVA85A is a TB vaccine designed to enhance BCG (6, 7). Modified vaccinia virus Ankara (MVA) is a strain of vaccinia virus that is replication-incompetent in human cells. MVA85A is a recombinant MVA strain that encodes the immunodominant mycobacterial antigen 85A (8). When administered after BCG, MVA85A can provide enhanced protection against TB challenge in animal models (9). MVA85A has been shown to be safe and highly immunogenic in clinical trials in adults in the UK, Gambia, and South Africa (6, 7, 10, 11).

The prevalence of TB in infants, children, and adolescents in most developing countries means that any effective TB vaccine should be given early in life, which coincides with the period that other childhood vaccines are given according to the Expanded Program on Immunization (EPI) schedule. The EPI program has been highly successful at increasing the coverage of existing licensed vaccines across the developing world. Integrating a new TB vaccine within the EPI schedule would facilitate deployment and uptake. However, before any vaccines can be coadministered, it is important to evaluate potential interference between the vaccines that could affect the induction of a protective immune response (12) because coadministration with EPI vaccines may diminish the potency of novel vaccine candidates (12). Here, we assessed the safety and immunogenicity of MVA85A in 4-month-old infants and, in parallel, evaluated potential interference with EPI schedule vaccines routinely administered at this age.

RESULTS

Study profile
We screened 350 infants in total (Fig. 1). Of these, 136 (39%) failed screening. Reasons for exclusion are listed in table S1. The first 36 infants were enrolled in the low-dose group (stage 1a), and the next 36 infants were enrolled in the standard-dose group (stage 1b). After safety and immunogenicity analyses of the stage 1 data, the standard dose was selected for the remainder of the study (stage 2), in which 142 infants were enrolled. All enrolled infants in each stage were randomized to one of the three study groups (Fig. 1). Demographic details, age at BCG vaccination, body weight, age at enrollment, and sex ratio were comparable across the three study groups (table S2). Stage 3 was initially planned in the protocol as an expansion of stage 2, if necessary. However, because the interference results from stage 2 were clear, the protocol was amended and stage 3 was not performed. Study duration was from October 2006 to October 2009.
Fig. 1
Consort flowchart. A consort diagram of the study from initial discussion: progressing from low dose to standard dose listing the number of participants for each stage and group of the study. During stage 2 of the study, 47 infants were randomized to group 2, but 48 received treatment because 1 infant inadvertently received EPI and MVA85A vaccines simultaneously. Therefore, in group 3, 48 were randomized but 47 received treatment.

Stage I safety results
To select a dose to be used for the rest of the study, we compared the safety profile between the low-dose (n = 24, excluding EPI-only controls) and the standard-dose (n = 24) MVA85A recipients in stage 1a and stage 1b, respectively (tables S3 to S5). Two days after vaccination, a significantly higher proportion of the standard-dose recipients had redness and pain, but not desquamation or swelling, at the site of vaccination in group 3 (MVA85A alone) but not group 2 (EPI + MVA85A) (table S3). None of the MVA85A recipients had pain that was greater than grade 1. Seven days after vaccination, a significantly higher proportion of subjects had redness in group 3 and desquamation in groups 2 and 3. Pain and swelling at this time point were comparable. There were no differences after vaccination in systemic adverse events (AEs) or hematological or biochemical values between stage 1a and 1b (tables S4 and S5), except for alanine transaminase, which was significantly higher in stage 1b than 1a in group 2 but not group 3, even though these values were still within normal limits (P = 0.018; table S5). There was no difference between all three groups when stages 1b and 2 were combined (table S5).

MVA85A dose response
In stage 1, the area under the curve (AUC) [interquartile range (IQR)] for the Ag85A immune response induced by low-dose MVA85A (stage 1a) was 223 (54 to 421) in group 2 and 1191 (129 to 1807) in group 3 (Table 1). The standard-dose group (stage 1b) induced significantly higher responses than low dose in group 2, as depicted by an AUC (IQR) of 1302 (742 to 3401) (P = 0.0005), and group 3, AUC of 4515 (3118 to 5543) (P = 0.0012 by Mann-Whitney U test). Responses in group 1 (EPI alone) did not differ across the three time points measured (Table

Because higher immunogenicity and a comparable safety profile were seen when results from stage 1a and 1b were compared, a decision to continue to use the standard dose in stage 2 was made and was approved by the ethical committees. Safety and immunogenicity data from stages 1b and 2 were therefore combined for the further data analyses.

MVA85A safety data
Safety data were collected from all infants (tables S3 to S5). MVA85A was safe and well tolerated. Consistent with previous data, most subjects developed mild local and systemic AEs (6, 10, 13) (tables S3 and S4). Two days after vaccination, there was a significant increase in the number of infants in group 2 with excessive crying and vomiting (P = 0.035 for both, table S4 by χ2 test). Seven days after vaccination, all systemic AEs had resolved to nonsignificant differences among all three groups. There were no significant differences in the biochemical and hematological indices among the three groups, either at baseline or 1 week after vaccination (table S5). Infants’ mean weights at 9 and 12 months after vaccination were comparable across the three groups (table S2).

Seven serious adverse events (SAEs) occurred during the study. The frequency of SAEs was comparable across the three groups. The three SAEs in group 1 were malaria, urinary tract infection, and bronchopneumonia; the two SAEs in group 2 were both bronchopneumonia; and the two SAEs in group 3 were diarrhea and bronchopneumonia. None of these SAEs was judged to be related to study vaccine.

Effect of EPI vaccines on MVA85A immunogenicity
To evaluate potential interference between EPI vaccines and MVA85A, we compared the ex vivo interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) response to the Ag85A peptide pool between groups 2 and 3 (Fig. 2). Baseline immune responses were comparable. There were significantly higher responses at 1 (P = 0.0138), 4 (P = 0.0127), and 20 (P = 0.0093) weeks after vaccination in group 3 than in group 2 with the Mann-Whitney U test. Comparing the median (range) of the AUC, group 3 was significantly higher than group 2 [median difference, 1374; 95% confidence interval (CI), 499 to 2354; P = 0.0010], indicating that the average response was higher over the entire period of study. Despite the effect of the EPI vaccines, the cellular immune response to MVA85A remained significantly higher 20 weeks after vaccination compared to baseline in both group 2 (P < 0.0001) and group 3 (P < 0.0001). Fig. 2 Immunogenicity of MVA85A. Immunogenicity was measured by ex vivo IFN-γ ELISPOT assay to a single pool of antigen 85A peptides. The figure compares responses in group 2 (EPI + MVA85A, filled circles) and group 3 (MVA85A alone, open circles) at baseline and 1, 4, and 20 weeks after vaccination. The horizontal lines indicate median values. The baseline median (IQR) of purified protein derivative (PPD) responses was 75 (45 to 193), 90 (25 to 181.8), and 90 (42.5 to 212.5) for groups 1, 2, and 3, respectively, and was not significantly different from each other. The median (IQR) difference AUC for the PPD responses over the 5 months after MVA85A vaccination (groups 2 and 3) or not (group 1) was 467 (−50 to 1039; P = 0.073) between groups 1 and 2, 737 (158 to 1643; P = 0.010) between groups 1 and 3, and 345 (−275 to 1088; P = 0.265) between groups 2 and 3 by Mann-Whitney U test. Interference of MVA85A vaccines with antibody responses to EPI vaccines The concentrations of antibodies specific to each vaccine component in the DTwP-Hib (diphtheria, tetanus, whole-cell pertussis, and Haemophilus influenzae type b) and hepatitis B regimen were compared between groups 2 and 1. The geometric mean (GM) or median antibody titer to each of the antigens was not significantly different between the groups (Table 2). Further analysis with antibody titers categorized according to threshold values also showed no significant difference in the proportions achieving protective levels between the two groups. View this table: In this window In a new window Table 2 Antibody level to EPI vaccines measured at the age of 5 months after three doses of DTwP-Hib and hepatitis B at ages 2, 3, and 4 months according to study groups. The table shows geometric means (GM) with 95% confidence interval (CI). Hepatitis B surface antigen and pertussis toxoid data were not normally distributed after log transformation and are therefore presented as medians and ranges. Subanalysis categorizes antibody titers according to known protective threshold levels. There are no antibody data from the infant randomized to group 3 but treated as in group 2. Note that the assay for hepatitis B had a maximum cutoff of 1000 mIU/ml. Effect is the back-transformed coefficient unless where medians were used, and it represents the ratio of the effect on antibody titer of having EPI with MVA85A concomitantly compared to EPI vaccines only. DISCUSSION This clinical trial evaluates the administration of an MVA-based TB vaccine in 4-month-old infants, as well as interactions between MVA85A and routine EPI schedule vaccines. The higher proportion of infants with local AEs in the standard-dose group compared to the low-dose group was consistent with previous studies using MVA85A and other recombinant MVAs, where there is a dose-response relationship between vector dose and local AE profile (13, 14). Most AEs were comparable between the two doses 7 days after vaccination. A significantly higher proportion of infants that received the EPI + MVA85A vaccines simultaneously (group 2) showed an increase in vomiting and crying when compared to the EPI-alone (group 1) or MVA85A-alone (group 3) groups. Because these events were not more frequent in the infants receiving MVA85A alone, compared to the group receiving EPI vaccines alone, this finding may be a result of inflammatory processes at two injection sites rather than one. It may also be due to the EPI + MVA85A group receiving a relatively higher antigenic load, because they received six vaccines compared to either five or one in the EPI and MVA85A groups, respectively (15, 16). These events were all mild and resolved by day 7 after vaccination. Both doses of MVA85A induced a significant antigen-specific immune response compared to unvaccinated controls. The peak response occurred 7 days after vaccination as previously described (6, 7, 10, 11). The cellular immune response to MVA85A was significantly lower when given simultaneously with the EPI vaccines compared to when administered alone. This difference may relate to interference between MVA85A and the EPI vaccines themselves or, more likely, the alum adjuvant they contain. Alum is the only licensed adjuvant for human use and is associated with the induction of a T helper type 2 (TH2)–dominated immune response (17, 18), which reciprocally inhibits TH1-type responses (19). The increased response to PPD in group 3 (MVA85A without EPI vaccines) is likely to be related to the enhanced response to antigen 85A seen in this group, because antigen 85A is a component of PPD. Because IFN-γ is a key effector molecule in protective immunity against intracellular organisms, including Mycobacterium tuberculosis (20, 21), our finding has implications for the use of MVA for vaccines against other pathogens, for viral vectors in general, and for TH1 type–inducing vaccines. We do not fully understand the mechanisms responsible for the interaction of these vaccines or the biological implications; however, this finding indicates that the internal milieu at the time of processing antigens might affect the magnitude of the response. The coadministration of EPI vaccines with the malaria vaccine candidate RTS,S/AS02, which contains a TH1-biased adjuvant, led to lower levels of antibody responses to the malaria antigen than in regimes that avoided coadministration (12). The benefits of introducing a new vaccine within the EPI schedule include reduced clinic visits, increased coverage, and possible enhanced immunogenicity of the vaccines (4). However, these favorable outcomes must be balanced against potential negative immunological interactions between simultaneously administered vaccines. At present, our data do not support concomitant administration of MVA85A with licensed EPI vaccines, and this finding has supported the assessment of MVA85A administered outside of the EPI schedule in an ongoing Phase IIb efficacy trial in infants (clinicaltrials.gov trial identifier NCT00953927). However, although we have demonstrated a statistically significant reduction in immunogenicity by IFN-γ response when MVA85A is co-administered with the EPI vaccines, the clinical significance of this finding is uncertain in the absence of a validated immunological correlate of protection. Further work is needed to evaluate whether such interference can be overcome by increasing vaccine dose, thus retaining the advantages of concomitant administration. This study has demonstrated that MVA85A is safe and well tolerated and induces an immune response considered protective against M. tuberculosis. It illustrates the importance of doing interference studies with new vaccines and evaluating bidirectional interference. Trials such as this are essential with new TB vaccines and T cell–inducing vaccines in general. They provide essential information needed for the design of efficacy trials. More generally, our findings highlight that modifications to the standard EPI regime may be needed to incorporate a new generation of T cell–inducing vaccines. MATERIALS AND METHODS Study site and recruitment of study participants This study was conducted at Sukuta Health Centre, Gambia. The study was approved by the Gambian government/Medical Research Council Joint Ethics Committee and Oxford Tropical Research Ethics Committee. Preliminary discussions about the trial were conducted with parents shortly after an infant’s birth, and information sheets were distributed. When these infants attended for their EPI schedule vaccines at age 3 months (visit 1), they were enrolled provided they fulfilled the following criteria: They were healthy, had received BCG within the first 4 weeks of life and had a typical BCG scar, had received their EPI schedule immunizations according to the national program (DTwP-Hib at 2 and 3 months, oral polio vaccine at birth and 2 and 3 months, hepatitis B at birth and 2 months), and their parents had given written informed consent. A blood sample was taken for baseline safety and immunogenicity. An independent statistician randomly assigned participants in a 1:1:1 ratio in blocks of six to one of the three study groups. A unique number for each participant detailing the treatment group was placed in a sealed opaque envelope and arranged serially. At 4 months of age (visit 2), when a child fulfilled the eligibility criteria, one of the study team selected the next envelope to determine study number and treatment group. The EPI vaccines given at this time point were the third doses of DTwP-Hib and hepatitis B. This trial was conducted according to the principles of the International Conference on Harmonisation–Good Clinical Practice guidelines, was externally monitored by an independent contract research organization, and is registered on clinicaltrials.gov (NCT00480454). Vaccines MVA85A was administered intradermally on the side ipsilateral to the BCG vaccination. All EPI vaccines were given intramuscularly apart from the oral polio vaccine. After MVA85A injection, the vaccination site was covered to minimize virus dissemination into the environment. Because this was the first trial with MVA85A in infants, we commenced this trial with low-dose MVA85A [2.5 × 107 plaque-forming units (PFUs)]. Once we had established safety with this dose in 24 infants, we then administered 5 × 107 PFU (the standard dose of MVA85A used previously) to the next 24 infants. A dose of 5 × 107 PFU was selected for the remainder of the study after analysis of the safety data. We obtained ethical approval before proceeding to each stage of the study. MVA85A was manufactured under Good Manufacturing Practice conditions by Impfstoffwerk Dessau-Tornau, Germany. Concomitant licensed EPI vaccines were obtained from the same source as the Ministry of Health of Gambia through the World Health Organization. Reactogenicity Each infant’s vital signs were monitored before vaccination and 30 and 60 min after vaccination. Thereafter, the dressing over the injection site was removed. Trained field workers visited the infants at home daily for the following 2 days to administer a reactogenicity questionnaire to the parents/guardians that included history of fever, vomiting, diarrhea, reduced feeding, and excessive crying. The field worker also examined the infant for expected local AEs (swelling, tenderness, redness, and desquamation at the site of injection for MVA85A) and fever. Pain at the injection site was graded on a scale of 0 to 3 (where 0 = no pain, 1 = painful to touch, 2 = partial restriction of activities, and 3 = unable to use the limb). Systemic and laboratory AEs were evaluated in all three study groups. Follow-up visits Subjects were reviewed 1 week after MVA85A administration (visit 3), corresponding to the time of peak MVA85A immune response reported previously (6, 7, 10, 11). Blood samples were obtained from all study participants before vaccination with the EPI vaccines deferred from visit 2 for group 3. Local and systemic reactogenicity in the first week after vaccination was recorded. Subsequent visits for immune responses were at the ages of 5 (visit 4) and 9 (visit 5) months. The protocol was amended to include additional visits at 12 (visit 6) and 18 (visit 7) months to monitor longevity of responses to MVA85A. Data from these visits will be reported in a separate manuscript. In all scheduled visits, solicited and unsolicited AEs were recorded and physical examinations were conducted. Blood samples were taken at each visit apart from visit 6. Parents and guardians were encouraged to take their infants to the trial clinic or Medical Research Council clinic with their study ID cards if their infant was unwell during the study. Any such visit was reported immediately to the trial team. LABORATORY METHODS MVA85A immunogenicity Immunogenicity of MVA85A was determined by measuring the frequency of IFN-γ spot-forming units (SFUs) by ex vivo ELISPOT assay (10). Briefly, peripheral blood mononuclear cells (PBMCs) suspended in 5% human AB serum in RPMI were seeded at 2 × 105 cells per 100 μl per well in duplicates and incubated in the presence of the antigen or controls. A pool of 15-mer peptides spanning the Ag85A protein (66 peptides that overlapped by 10 amino acids; 2 μg/ml each) and M. tuberculosis PPD (10 μg/ml) were used as recall antigens. Medium alone served as a negative control, whereas phytohemagglutinin (10 μg/ml) was the positive control. The number of IFN-γ SFU counted in each well was automatically entered into a database minus background (medium wells). When responses were too numerous to quantify, a maximum cutoff value of 200 spots per well or 1000 SFU per million PBMCs was assigned. EPI vaccine antibody measurement Antibody (immunoglobulin G) concentrations against diphtheria toxoid, tetanus toxoid, pertussis (toxoid and filamentous hemagglutinin), Haemophilus influenzae type b capsular polysaccharide, and hepatitis B surface antigen were measured by standard enzyme-linked immunosorbent assays (ELISAs) at the Health Protection Agency Immunoassay Laboratory, UK. For each antigen, sera were titrated against known concentrations of specific antibodies in standard sera samples. All laboratory assays were done blinded to the child’s study group. Data management and statistical analyses Data were double-entered into an Access database and manually validated. For categorical variables, data were summarized using numbers and percentages, and groups were compared with the χ2 test or, for small numbers, Fisher’s exact test. For continuous, normally distributed data, the mean and SD were used to summarize data, and groups were compared with an analysis of variance (for comparison of three groups) or a t test (for comparison of two groups). In the case of non-normally distributed data, a Kruskal-Wallis (for comparison of three groups) or Mann-Whitney U test (for comparison of two groups) was used. Comparisons between time points within a group were made with Wilcoxon matched-pairs signed-rank test. Such data were summarized with medians and 25th and 75th percentiles. In the case where a log transformation resulted in normally distributed data, the GM was presented and groups were compared with a t test. An AUC analysis was used to assess for overall differences in response over the follow-up period. Subjects were analyzed according to the treatment they received, rather than the group to which they were randomized. Analyses were done with Stata Release 10 statistical software (Stata). SUPPLEMENTARY MATERIAL www.sciencetranslationalmedicine.org/cgi/content/full/3/88/88ra56/DC1 Table S1. Reasons for exclusion of infants screened for participation. Table S2. Demographic and baseline characteristics of subjects by study group. Table S3. Comparison of number (percent) of local adverse events between study groups in stages 1a, 1b, and 2. Table S4. Comparison of number (percent) of systemic adverse events between study groups in stages 1a, 1b, and 2. Table S5. Comparison of biochemistry and hematology parameters [mean (SD)] between study groups in stages 1a, 1b, and 2. Citation: M. O. C. Ota, A. A. Odutola, P. K. Owiafe, S. Donkor, O. A. Owolabi, N. J. Brittain, N. Williams, S. Rowland-Jones, A. V. S. Hill, R. A. Adegbola, H. McShane, Immunogenicity of the Tuberculosis Vaccine MVA85A Is Reduced by Coadministration with EPI Vaccines in a Randomized Controlled Trial in Gambian Infants. Sci. Transl. Med. 3, 88ra56 (2011).
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↵ M. Walther, F. M. Thompson, S. Dunachie, S. Keating, S. Todryk, T. Berthoud, L. Andrews, R. F. Andersen, A. Moore, S. C. Gilbert, I. Poulton, F. Dubovsky, E. Tierney, S. Correa, A. Huntcooke, G. Butcher, J. Williams, R. E. Sinden, A. V. Hill, Safety, immunogenicity, and efficacy of prime-boost immunization with recombinant poxvirus FP9 and modified vaccinia virus Ankara encoding the full-length Plasmodium falciparum circumsporozoite protein. Infect. Immun. 74, 2706–2716 (2006). Abstract/FREE Full Text
↵ V. S. Moorthy, E. B. Imoukhuede, S. Keating, M. Pinder, D. Webster, M. A. Skinner, S. C. Gilbert, G. Walraven, A. V. Hill, Phase 1 evaluation of 3 highly immunogenic prime-boost regimens, including a 12-month reboosting vaccination, for malaria vaccination in Gambian men. J. Infect. Dis. 189, 2213–2219 (2004). Abstract/FREE Full Text
↵ B. Watson, A. Cawein, B. L. McKee, J. G. Hackell, Safety and immunogenicity of acellular pertussis vaccine, combined with diphtheria and tetanus as the Japanese commercial Takeda vaccine, compared with the Takeda acellular pertussis component combined with Lederle’s diphtheria and tetanus toxoids in two-, four- and six-month-old infants. Pediatr. Infect. Dis. J. 11, 930–935 (1992). MedlineWeb of Science
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↵ L. Bungener, F. Geeraedts, W. Ter Veer, J. Medema, J. Wilschut, A. Huckriede, Alum boosts TH2-type antibody responses to whole-inactivated virus influenza vaccine in mice but does not confer superior protection. Vaccine 26, 2350–2359 (2008). CrossRefMedlineWeb of Science
↵ D. Spazierer, H. Skvara, M. Dawid, N. Fallahi, K. Gruber, K. Rose, P. Lloyd, S. Heuerding, G. Stingl, T. Jung, T helper 2 biased de novo immune response to Keyhole Limpet Hemocyanin in humans. Clin. Exp. Allergy 39, 999–1008 (2009). CrossRefMedlineWeb of Science
↵ A. K. Abbas, K. M. Murphy, A. Sher, Functional diversity of helper T lymphocytes. Nature 383, 787–793 (1996). CrossRefMedline
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↵ M. J. Newport, C. M. Huxley, S. Huston, C. M. Hawrylowicz, B. A. Oostra, R. Williamson, M. Levin, A mutation in the interferon-γ–receptor gene and susceptibility to mycobacterial infection. N. Engl. J. Med. 335, 1941–1949 (1996). CrossRefMedlineWeb of Science

Acknowledgments: We thank the Gambian government, the EPI program of Gambia, and staff of Sukuta Health Centre for their collaboration. We thank K. Flanagan, J. Adetifa, and the Sukuta field team for assisting us in this trial. We appreciate M. Sonko, K. Manneh, and S. Sanneh for their field work and A. Bojang and J. Sutherland for laboratory assistance. We also thank K. Bojang for safety monitoring; V. Thomas and J. Mueller for internal monitoring and support; A. Lawrie for coordination; S. Vermaak for help with data analysis; and C. McKenna for study monitoring. We are grateful to the parents and guardians who allowed their infants to participate in this trial. Funding: The study was funded by the Medical Research Council (UK) and the European Commission (EU 6th Framework; TBVAC). Neither funder had any role in the design of the study or the preparation of the manuscript. H.M. is a Wellcome Trust Senior Clinical Research Fellow, and A.V.S.H. is a Wellcome Trust Principal Research Fellow. Author contributions: M.O.C.O., H.M., A.V.S.H., S.R.-J., and R.A.A. designed the study. A.A.O., O.A.O., M.O.C.O., R.A.A., and S.R.-J. provided clinical help. M.O.C.O., A.A.O., N.J.B., and H.M. managed the project. P.K.O., M.O.C.O., H.M., A.V.S.H., and R.A.A. performed laboratory assays and analysis. N.W., S.D., N.J.B., H.M., and M.O.C.O. performed the statistical analysis. M.O.C.O., H.M., N.J.B., A.A.O., O.A.O., A.V.S.H., S.D., N.W., S.R.-J., and R.A.A. wrote the manuscript. Competing interests: A.V.S.H. and H.M. are named inventors on a patent filing related to MVA85A (International patent number WO 2006/072787 A1: Compositions for immunising against mycobacteria), and are shareholders in a joint venture, Oxford-Emergent Tuberculosis Consortium, formed for the future development of this vaccine. The other authors declare that they have no competing interests.



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segunda-feira, 27 de junho de 2011

Caso Clinico New England Journal of Med

Presentation of Case

Dr. Ana A. Weil (Medicine): A 4-year-old Haitian boy was admitted to a hospital in Haiti affiliated with this hospital because of vomiting and diarrhea of 10 hours' duration.

The patient had been well until approximately midnight the night before admission, when vomiting and diarrhea developed. After approximately 6 hours of symptoms, his parents brought him to the hospital by motorcycle taxi, traveling for 4 hours. On arrival, 10 hours after the onset of symptoms, episodes of vomiting and diarrhea were too numerous to count.

The patient's parents said that he had not urinated for hours. He had reportedly previously been healthy. He lived in a small village in Haiti with his parents and sibling. His 8-year-old brother had had mild diarrhea the previous day.

On examination, the patient seemed irritable and was rapidly drinking offered liquids. The pulse was low volume, at a rate of 150 beats per minute; the respirations were shallow, without retractions, at a rate of 45 breaths per minute; and the skin was not hot to the touch. The blood pressure and temperature were not obtained because of lack of equipment. The weight was estimated at 15 kg. The eyes were sunken, skin recoil was less than 1 second but not instantaneous, capillary refill was 2 seconds, and the skin and mucous membranes were dry. The lungs were clear, and there was mild abdominal tenderness. During the examination, the patient passed a clear, watery stool. He was admitted to the hospital, where he shared a cot with a pediatric patient who had similar symptoms, including diarrhea.

A reduced-osmolarity oral rehydration solution (ORS) consisting of glucose, sodium chloride, potassium chloride, and trisodium citrate dihydrate (with 75 mmol of glucose per liter, 75 mmol of sodium per liter, 20 mmol of potassium per liter, 65 mmol of chloride per liter, and 10 mmol of citrate per liter), with a total osmolarity of 245 mmol per liter, was administered. During the next hour, two episodes of vomiting and numerous episodes of diarrhea occurred.

On reexamination 1 hour after the initiation of treatment, the patient had ingested less than 200 ml. He was combative and pushed away the ORS. The pulse was weak, and the hands and feet were cool and clammy. Simultaneous attempts at insertion of intravenous catheters in the antecubital region and the hand were unsuccessful; the patient became increasingly obtunded.

On the third attempt at intravenous access, a catheter was inserted into the saphenous vein of the foot. A bolus (500 ml) of isotonic crystalloid solution containing sodium chloride, sodium lactate, potassium chloride, and calcium chloride was administered, with manual pressure applied to the bag. The patient remained lethargic. Dextrose (30 ml of a 20% solution) was administered rapidly into the intravenous catheter, without improvement in mental status. A second intravenous catheter was placed in the right antecubital region. Another bolus (500 ml) of crystalloid solution was infused during a 30-minute period, with improvement in the level of consciousness, followed by a second liter of the solution during the next 2 hours.

Approximately 4 hours after presentation, episodes of diarrhea were occurring too often to count, the frequency of vomiting had decreased, and no urine output had occurred. On examination, the patient was eagerly drinking ORS, and his mental status was markedly improved. The eyes remained sunken, and skin turgor was slightly decreased from normal. Azithromycin (300 mg) was administered orally. His family was encouraged to have the patient consume 200 ml of ORS per stool produced. During the next 4 hours, he had at least six episodes of diarrhea and drank approximately 400 ml of ORS; 1 liter of the crystalloid solution was administered intravenously. Eight hours after presentation, the total intravenous intake was 3 liters, or approximately 200 ml per estimated kilogram of body weight. He had urinated twice. On examination, there were no signs of dehydration, the pulse was 100 beats per minute, and the respiratory rate was 30 breaths per minute, without rales or cough. During the remainder of the first day, an additional liter of intravenous solution was administered (a total of 4 liters during 24 hours, or approximately 267 ml per kilogram). Overnight, the frequency of diarrhea decreased, with an estimated 10 stools and no vomiting. Oral intake included less than 200 ml of ORS and some broth.

On the morning of the second day, the patient's parents reported that he had cramping in his legs. On examination, signs of dehydration were present, including sunken eyes and slightly decreased skin turgor, with mild abdominal distention and tenderness. A bolus (500 ml) of crystalloid was administered intravenously over a period of 4 hours, and an educator was assigned to assist his parents in understanding the importance of ORS intake. During the next 4 hours, he consumed approximately 800 ml of ORS without vomiting. Signs of dehydration resolved, and abdominal distention decreased. Infusions of intravenous fluid were decreased to minimal flow. His parents were instructed again to match stool output by administering approximately 200 ml of ORS per stool, and his diet was increased to include meals of chicken broth and mashed bananas.

During the second night, three episodes of diarrhea occurred, and another episode between 8 a.m. and 2 p.m. On the third morning, the patient successfully consumed meals of solid food and ORS. He was discharged after 2.5 days, with instructions to the parents about oral hydration, point-of-use water sterilization, and hand sanitation with soap. One week after discharge, a diagnostic test result was received.

Differential Diagnosis

Dr. Jason B. Harris: I participated in the care of this child who presented with acute watery diarrhea during the second week of a cholera epidemic in Haiti, which began in October 2010 and is ongoing. The patient was admitted to a cholera treatment center that was established the previous week and was providing care for more than 100 patients daily who had diarrhea and, in many cases, other concomitant illnesses. No laboratory facilities were available. Like the vast majority of patients with diarrhea in developing countries, no specific laboratory diagnosis was made in this case.

The patient presented with a common problem. Children in developing countries have a median of three episodes of diarrhea annually,1 and diarrheal illness is the second leading cause of death among children, resulting in 1.6 million to 2.1 million deaths annually.2 Before the recent cholera epidemic, an average of 1 of every 93 children born in Haiti died from diarrheal illness before reaching their fifth birthday.3 For this child, the focus is on empirical management of the acute watery diarrhea, not on extensive clinical or laboratory investigations. Algorithms, such as those developed by the World Health Organization (WHO),4 are helpful in managing diarrheal illness in children in resource-limited communities.

Differential Diagnosis of Diarrheal Illness

The first step in the care of this patient is to classify the type of diarrheal illness (Table 1). Diarrhea lasting for more than 14 days is classified as persistent diarrhea. Persistent diarrhea is caused by a distinct set of organisms and is associated with malnutrition and chronic enteropathy. Persistent diarrhea should raise suspicion for underlying infection with the human immunodeficiency virus (HIV); in HIV-infected persons, unexplained persistent diarrhea is a defining illness of the acquired immunodeficiency syndrome.5

Table 1


Classification and Common Causes of Childhood Diarrhea in Developing Countries.

Since our patient had acute diarrhea, the next step is to classify the diarrhea as invasive (bloody) or noninvasive (watery). Invasive diarrhea is defined by grossly bloody or melanotic stools. Most patients with invasive diarrhea have fever and mucus in the stool. Shigella species are the predominant cause of invasive diarrheal illness in children in developing countries,6,7 and empirical management of the illness should include antibiotics aimed at treating and preventing complications of shigellosis.

This child passed watery stools without blood and did not have a tactile fever. The most common causes of acute watery diarrhea are rotavirus in infants and enterotoxigenic Escherichia coli in children.6 There is increasing recognition of the role of caliciviruses in causing gastroenteritis in children and also in adults. Many acute systemic illnesses (e.g., measles, dengue fever, and malaria) may also present with diarrhea.

Cholera

In this case, cholera was suspected because the patient presented during a known epidemic. It is important to distinguish cholera from the other causes of noninvasive diarrhea. A rapid and simple laboratory test for Vibrio cholerae is dark-field microscopy, which, when positive, reveals characteristically darting bacteria. However, in resource-limited communities, the diagnosis of cholera is most often based on clinical suspicion that takes into account the local epidemiology of diarrheal illness. Although mild illness caused by V. cholerae is clinically indistinguishable from other causes of diarrhea, severe cholera is associated with greater losses of fluid and electrolytes than is seen with other causes of noninvasive diarrhea. Furthermore, patients with cholera benefit from the early administration of appropriate antibiotics. Finally, cholera can cause large epidemics.

A classic finding in cholera is “rice water” stool (Figure 1D), which may contain more than 1 billion (109) organisms per milliliter. Patients with severe cholera may shed more than 10 trillion (1013) organisms per day.8 Although many V. cholerae serogroups have been identified in the environment, only serogroups O1 and O139 have caused epidemic cholera. V. cholerae O1, biotype El Tor, is the cause of the current global pandemic of cholera, which began in 1961. Emerging strains, termed “hybrid” or “variant” V. cholerae O1 El Tor, are the causes of more recent epidemics, including the current epidemic in Haiti.9 These variant strains appear to combine the enhanced ability of the El Tor biotype to persist in the environment with the greater virulence associated with the previously circulating classical biotype.

Figure 1


Pathogenesis of Cholera.

This case illustrates the manifestations of severe cholera, or cholera gravis. Epidemic strains of V. cholerae produce cholera toxin — a toxin resulting from the ribosylation of adenosine diphosphate — which causes chloride secretion and the loss of sodium and water into the lumen of the small intestine (Figure 1C). Stool losses in cholera are typically isotonic, and the mean sodium concentration in the stool of children with cholera is double that seen in the diarrhea of children without cholera (Table 2).

Table 2


Chemical Composition of Diarrheal Stool and Therapeutic Solutions.

Dehydration and Rehydration

In cases of rapid fluid losses, the large intestine's capacity for reabsorption is overwhelmed and death may occur within hours. The WHO has provided guidelines for using the physical examination to estimate dehydration in children; laboratory tests provide little additional useful information.4 This patient had deeply sunken eyes, markedly decreased skin turgor, a weak pulse, and mental-status changes, suggesting a 10% loss of fluid per kilogram of body weight within 12 hours after the onset of symptoms. This is typical of severe cholera. During the first weeks of the cholera epidemic in Haiti, deaths occurred in the community a median of 12 hours after the onset of symptoms.11 Children with severe cholera typically present with 5 to 10% dehydration but have additional stool losses that may exceed 20% of their body weight during the first 48 hours after admission.12 Rehydration is the cornerstone of care for patients with cholera, but nutritional interventions, the appropriate use of antibiotics, and recognition of common complications and coexisting conditions are also important.

Rehydration requires the rapid replacement of the initial deficit and ongoing losses with isotonic fluids. Therapeutic fluids for patients with cholera are shown in Table 2. In the United States and other developed countries, a typical approach to a child with dehydration is to use hypotonic solutions to replace estimated fluid and electrolyte deficits slowly over a 24-hour period. In resource-limited locations such as Haiti, children with diarrheal illness often present later, with more severe dehydration, and require more rapid rehydration with isotonic solutions, particularly patients with cholera. With optimal fluid management, the mortality associated with severe cholera is less than 0.2%.10 However, case fatality rates are usually higher in epidemic cholera,13 especially during the early stages, when there are obstacles to providing appropriate clinical care.14 This case illustrates some of the barriers to providing optimal rehydration therapy. Ideally, oral rehydration therapy is initiated at the onset of illness, in the home or in the community.4 This requires the local availability, knowledge, and acceptance of oral rehydration therapy. In this case, had ORS been used at the onset of illness, instead of 10 hours after the onset of symptoms, it is unlikely that life-threatening shock would have occurred during the patient's hospitalization.

For patients with severe dehydration, intravenous fluids are required immediately. Lactated Ringer's solution is the best and most widely available commercial intravenous fluid for cholera. Ideally, the entire fluid deficit should be replaced within 3 to 4 hours after the initiation of therapy in both children and adults.4,15 This patient required more than 300 ml per kilogram of isotonic intravenous and oral fluids to restore euvolemia during the first 28 hours of therapy, which is indicative of a rate of purging that is consistent with severe cholera.

Providing adequate volumes of isotonic fluids to patients with such massive ongoing losses is also a challenge, especially for health care workers who are unfamiliar with the fluid requirements of patients with severe cholera. Cholera cots were assembled at this cholera treatment center and generally are useful for recording stool output (Figure 2). Because of space constraints, this patient shared a cholera cot and bucket with other patients; therefore, a reliable record of the patient's stool output was not made. However, ongoing losses can be estimated at 10 to 20 ml per kilogram per stool, and the volume of these losses can be added to the amount of fluids needed during the initial rehydration period. In this case, euvolemia was initially restored after 8 hours and approximately 200 ml per estimated weight in kilograms; however, had such rapid ongoing losses been factored in, the fluid could have been restored more rapidly, ideally within a 3-to-4-hour window.

Figure 2


Cholera Cot.

Recurrent dehydration, leg cramps, and abdominal distention developed in this patient approximately 12 hours after the initial correction of his fluid deficit. Hypokalemia was the most likely cause of the leg cramps and abdominal distention. Hypokalemia is an important cause of death in patients with diarrheal illness who die after initial rehydration therapy.16 In this case, the hypokalemia and the recurrent dehydration might have been prevented if oral rehydration therapy had been used to replace ongoing diarrheal losses. Oral rehydration therapy provides more potassium than intravenous lactated Ringer's solution and is preferred over intravenous therapy whenever possible. Similarly, the resumption of normal feeding should also begin as soon as possible, to prevent the sequelae of malnutrition and such complications as hypokalemia and hypoglycemia.

Antibiotic Therapy

This patient received azithromycin early during his hospitalization, which is an appropriate treatment for the V. cholerae strain that is circulating in Haiti. Antibiotics can lead to reductions of more than 50% in stool volume and in the duration of diarrhea, from more than 4 days to 2 days. Antibiotics also reduce the shedding of viable V. cholerae from more than 6 days to slightly more than 1 day. This treatment can be useful on a patient-by-patient basis and also facilitates more rapid discharge from cholera treatment centers, thus conserving resources for other patients.17

After rehydration and antibiotic therapy, the next tier of care is to provide nutritional support and to recognize common complications and coexisting conditions seen in patients with cholera. Zinc supplementation (10 mg per day for infants less than 6 months of age and 20 mg per day for 10 days for children 6 months to 5 years of age) should be provided to reduce the severity and duration of childhood diarrheal illness in countries, such as Haiti, where zinc deficiency is common.4,18 Zinc has the added benefit of reducing the incidence of subsequent episodes of diarrhea for several months. In developing countries, children with diarrhea, such as this patient, are also at high risk for vitamin A deficiency and should receive supplementation with vitamin A. Patients with clinical signs of vitamin A deficiency should receive a three-dose series of treatment (50,000 IU for infants <6 months of age, 100,000 IU for infants 6 to 12 months of age, and 200,000 IU for children >12 months of age). In this case, neither zinc nor vitamin A was available. In endemic areas, coexisting conditions, such as pneumonia and sepsis, are a leading cause of death in patients with cholera.19 Therefore, reevaluation for clinical signs of pneumonia and sepsis after rehydration is important. In this case no such conditions were identified on sequential examinations after rehydration.

Dr. Jason B. Harris's Diagnosis

Life-threatening diarrheal illness due to Vibrio cholerae.

Pathological Discussion

Dr. Mary Jane Ferraro: The specimen that we received in the laboratory was from a different child in Haiti who had a similar illness at the same time as this child's illness. The isolate that we obtained was identified in our laboratory as V. cholerae. Susceptibility testing was performed and showed that the isolate was susceptible to tetracycline and azithromycin and was resistant to sulfa drugs and nalidixic acid. This isolate is undoubtedly the same one that had infected our patient, and this result confirms the diagnosis of cholera. Dr. Harris, can you tell us about the additional molecular characterization that was performed on this isolate?

Dr. Harris: At the onset of the epidemic, the initial isolates were rapidly identified as V. cholerae O1, serotype Ogawa, by the National Public Health Laboratory in Haiti. The Centers for Disease Control and Prevention (CDC) subsequently reported that these were hybrid strains of V. cholerae O1, biotype El Tor.9 These are strains that produce the more virulent toxin that is associated with the previously circulating classical biotype of cholera. To determine the phylogeny of the organism, the isolate that Dr. Ferraro described underwent complete genome sequencing. Analysis of variable regions in the organism's genome placed the isolate in the context of other known isolates in V. cholerae and showed that this was a seventh pandemic strain of V. cholerae O1 El Tor and that it was most closely related to isolates obtained in Bangladesh in the past decade.20 These phylogenetic comparisons were based on a single nucleotide variation in selected genes and in the content of selected hypervariable regions in both V. cholerae chromosomes. The isolate was distantly related to strains that had circulated in South America in the early 1990s and strains that are known to cause sporadic V. cholerae in the U.S. Gulf Coast.

The Haitian Cholera Outbreak

Dr. Eric S. Rosenberg (Pathology): Dr. Louise Ivers is with us by telephone from Port-au-Prince, Haiti. Dr. Ivers, would you give us an update on the status of the epidemic there and the responses to the crisis?

Dr. Louise C. Ivers: This patient presented during the second week of the cholera epidemic in Haiti, which began in October 2010. We know now that the first cases came from the center of the country, but the cases that alerted authorities to the epidemic occurred in the large coastal town of Saint-Marc, 2 hours north of the capital city of Port-au-Prince. At the time of this conference, 4 months later, the epidemic continued to evolve. As of April 8, 2011, more than 248,657 cases of cholera had been treated and 4524 patients had died. There continue to be mini-peaks of cases reported, particularly in areas with poor road access and in rural isolated communities that traditionally have limited access to any health services. The number of new cases has decreased substantially since the early phase of the epidemic and now remains relatively stable. Thus, the epidemic has not ended yet, and as the rainy season approaches, the number of cases may increase.

Sanitation and Clean Water

In Haiti, access to clean water is lacking for the majority of the population, as it most likely was for this patient's family. Almost a decade ago, Haiti ranked the worst of 147 countries in terms of water resources, and little has happened since then to substantially improve services and infrastructure. Few households have access to a formal latrine. Housing and shelter, which were inadequate in rural Haiti before the earthquake of 2010, became even less sufficient when persons who were displaced by the earthquake moved to stay with family and friends in already overcrowded and often poorly constructed housing in the countryside. Many rural homes in Haiti are subject to flooding during the rainy season, even during moderate rainfall, and most have dirt floors, which increase the challenges of sanitation and hygiene.

Response to the Outbreak

The outbreak of cholera, the likes of which had never been seen by the current population of Haiti, caused huge pressure on what was already a weak public health infrastructure. Initially, the causes and methods of transmission of cholera were poorly understood by those at risk, and clean water and soap were not widely available in the areas affected; these two issues in the context of poor access to services led to high initial mortality rates. This patient exemplifies this problem: he did not have access to oral rehydration early enough to prevent severe complications. The learning curve for institutions and providers with no cholera experience was steep. In view of these challenges, the response to the outbreak was relatively fast. Cholera treatment centers were erected, and efforts were made to establish temporary solutions to the problem of the lack of potable water and to introduce hygiene measures. There has been a strong response from the government of Haiti and national and international partners, and there are now more than 400 cholera treatment facilities, such as the one that this patient entered and that saved his life.

As the months have passed, the training of service providers, access to services for patients, water-treatment education and supplies, and education in the community have all increased, contributing to increased survival and a reduction in the number of new cases of cholera. Institutional mortality nationwide, originally as high as 7 or 8%, has fallen to less than 2%. This patient's family was given instructions in water purification and hand hygiene at the time of the patient's discharge. They were also given supplies so they could put their education into practice. However, challenges remain. Rural isolated communities have poor access to health services in general; poor living conditions, lack of sanitation, and lack of access to clean water persist as a result of dire poverty.

Dr. Rosenberg: What is the likelihood that cholera will be eradicated from Haiti?

Dr. Edward T. Ryan (Infectious Diseases): We have learned from previous cholera outbreaks that once this organism gets a foothold in the water supply in impoverished areas, it is almost impossible to eradicate.

Anatomical Diagnosis

Vibrio cholerae O1 (toxigenic), serotype Ogawa (testing performed at the CDC).

Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

This case was presented at the Medicine Grand Rounds, February 17, 2011.

We thank Dr. Lawrence Ronan for helpful input.

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Source Information

From the Departments of Infectious Diseases (J.B.H.) and Pathology and Microbiology (M.J.F.), Massachusetts General Hospital; the Department of Medicine, Brigham and Women's Hospital (L.C.I.); and the Departments of Pediatrics (J.B.H.), Medicine (L.C.I.), and Pathology (M.J.F.), Harvard Medical School — all in Boston.

sábado, 25 de junho de 2011

Lições do Brasil Artigo publicado na Nature

NATURE | OUTLOOKprevious article
Perspective: Lessons from Brazil

Marcia Moraes
Nature 474, S25 (23 June 2011) doi:10.1038/474S025a
Published online 22 June 2011
Thirty five years of experience has taught one of the world's leading biofuels producers several essential lessons, which other countries should heed, says Marcia Moraes.

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There was nothing inevitable about the path Brazil took to where it is today — a country with a vibrant biofuel industry that annually turns half of its sugarcane harvest into 24 billion litres of ethanol to power 12.5 million vehicles. In 2009, nearly 1.2 million jobs in Brazil stemmed from the sugarcane, sugar and ethanol sectors. About half were held by poorly educated workers on sugarcane plantations. Sugarcane workers are among the best-paid in Brazilian agriculture, and their children have better socioeconomic outcomes than the children of other agricultural workers. Here are some of the lessons that Brazil has learnt as it has built its biofuels industry:


NEWSCOM/PHOTOSHOT

Moema sugar mill in Orindiuva helps supply one of 25,000 biofuel filling stations across the country.

Lesson 1: Provide stable government oversight
In 1975, there was no market for sugarcane ethanol. The Brazilian government had to intervene to create this market, and for decades tightly controlled it. Clear and stable rules allowed companies to make the long-term investments needed to achieve their goals.

The late 1990s saw a sharp reduction of state interference in the biofuel industry. Production quotas were abolished, as was the ethanol subsidy. Sugar and ethanol prices were left to the market. Sugarcane producers are now remunerated by processors based on sugar and ethanol prices; and ethanol producers and distributors are free to negotiate prices and quantities. The free market brought considerable efficiency gains. But planning to meet the market demand is tricky. Unlike corn and soy, sugarcane is a perennial crop, going through a six-year cycle (planting in the first year and harvesting in the next five years). Planning the supply of sugarcane means considering the estimated demand for the next five years, for both ethanol and sugar. A market economy is not always up to this task.

Lesson 2: Plan for maximum flexibility
From the outset, Brazil chose to produce two types of ethanol: anhydrous ethanol, which is blended with petrol (gasoline), and hydrated ethanol, used in vehicles that run only on ethanol and also in dual-fuel (flexible fuel) cars. State intervention was essential for the creation of the necessary infrastructure for both hydrated and anhydrous ethanol, including the installation of hydrous ethanol pumps at more than 25,000 filling stations. Today, government intervention is basically limited to determining the proportion of the anhydrous ethanol blend, setting the tax rate on sugar exports and enforcing the existing environmental and labour rules. The focus on anhydrous ethanol certainly reduces the necessary investments, aids the planning of the supply and allows the co-existence of fossil and renewable fuel — simply blending anhydrous ethanol with petrol (gasoline) in different proportions yields whatever grade fuel is desired.

Lesson 3: Establish clear rules about ethanol stocks
Ethanol is produced from an agricultural product, subject to weather conditions and the length of the growing season. So although sugarcane harvesting and ethanol production last for 6–8 months every year, ethanol is sold year round. There must be a policy for buffering stocks to avoid shortages or sharp price oscillations throughout the year.

Lesson 4: Get the scale right
When Brazil launched its biofuel initiative in 1975, the Programa Nacional do Álcool encouraged the establishment of small-scale distilleries. These distilleries lacked economies of scale and most closed through economic inefficiency.

Lesson 5: Protect the environment
Minimizing environmental impact is imperative. Brazil regulates issues such as where sugarcane may be grown1 — for example prohibiting production in the sensitive biomes of Amazonia and Pantanal. The government regulates use of water, use of the by-product vinasse as a fertilizer, preservation of forests and burning of sugarcane stalks as a method of straw removal. Even without mandates, biofuel producers have seen the virtue of minimizing waste. Biorefineries burn the fibrous sugarcane bagasse to generate electricity for the ethanol plant and for sale.

Lesson 6: Welcome private investment
Brazil has greatly expanded its sugar and ethanol production in recent years, relying on both domestic and foreign private investment. External capital has enabled the industry to introduce new technologies and management standards. Investment from other countries in particular has helped give Brazilian biofuel producers access to foreign markets that may be shielded by protectionist policies.

No two countries will face exactly the same circumstances. But the strategies that have guided this nation of almost 200 million people should show the rest of the world that the future of energy need not depend on oil.

References
Author information

quinta-feira, 23 de junho de 2011

Vacina contra Tb

* MVA85A TB vaccine less effective when given with others

* New TB shots urgently needed, data pose timing questions

By Kate Kelland

LONDON, June 22 (Reuters) - A new vaccine designed to fight tuberculosis is less effective when given alongside shots for other diseases, a study has found, suggesting child immunisation programmes in developing countries may need a rethink.

Data from clinical trials of the vaccine, called MVA85A, in babies in Gambia showed it was safe, but the immune response it prompted was lower in babies who got it with other infant immunisations than in those who got it on its own.

Martin Ota of the Medical Research Council Laboratories in Banjul, Gambia, who led the study, said the data should help doctors work out the best way to integrate the MVA85A into infant immunisation programmes in the future.

"We have a real opportunity to make sure that children are protected ... against tuberculosis by introducing effective and well-timed immunisation programmes," he said in a statement about the study. "This can only be achieved with robust information gathered from well-conducted clinical trials."

Standard childhood vaccinations are routinely given in developing countries as part of a plan known as the Expanded Programme on Immunisation (EPI).

It includes vaccines for diphtheria, tetanus and whooping cough, as well as the current vaccine for TB, Bacille Calmette-Guerin (BCG). The plan helps boost vaccine coverage by cutting the need for repeated visits to health clinics, which are often difficult to get to in poor, rural areas.

Although BCG protects against severe forms of TB in childhood, increasing rates of the disease in adults suggest its effect is not long-lasting.

TB is currently a worldwide pandemic that kills around 1.7 million people a year. The infection is caused by the bacterium Mycobacterium tuberculosis and destroys patients' lung tissue, causing them to cough up the bacteria, which then spread through the air and can be inhaled by others.

Experts say there is an urgent need for more effective TB vaccines and MVA85A -- being developed and trialled by Emergent BioSolutions in a joint venture with Britain's Oxford University -- is one of the most advanced potential candidates.

It has already been shown to be safe and capable of eliciting powerful immune responses in clinical trials in adults in Britain, Gambia and South Africa.

This study, published in the journal Science Translational Medicine on Wednesday, was the first trial to evaluate the safety of the vaccine in babies. It involved 214 healthy four-month-old infants who had already received BCG at birth.

The children were given either EPI alone, MVA85A alone, or MVA85A with EPI, and their immune responses were monitored.

Overall, Ota's team reported, MVA85A was deemed to be safe, well tolerated and induced a strong immune response. And importantly, the responses to the standard EPI vaccines were not affected by giving MVA85A at the same time. But the immune response prompted by MVA85A was lower in infants who received it with EPI vaccines, compared with those who got it alone.

"It's reassuring to see that MVA85A does not affect immunity to the other vaccines," said Helen McShane of Oxford University, who helped develop the new shot. But she said scientists would now need to find the best way to integrate MVA85A into infant immunisation plans in future without limiting its effect. (Editing by Andrew Heavens)

domingo, 19 de junho de 2011

agora já é possível dosar citocinas em uma única célula








A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells




Design rationale and detection limit of the SCBC

The SCBC system consists of four modules (Fig. 1): microchannels that contain cells, control valves that isolate cells into microchambers, inlet and outlet ports for the introduction and depletion of reagents and cells and a barcode-encoded glass substrate for protein detection. The chip itself consists of two polydimethylsiloxane (PDMS) layers and fits onto a microscope slide (Fig. 1a). The bottom PDMS layer has inlets for the loading of reagents and cells that branch into 80 microchannels of 100 μm × 17 μm cross-sectional size. Thirteen sets of vertical valves on the top PDMS layer divide those microchannels into 1,040-nl–volume microchambers. For a microchamber containing one to ten cells, the cell density is 0.3 × 106–3 × 106 cells ml−1, falling into the normal range for culture conditions and physiological environments18.
Figure 1: Design of the SCBC for single-cell protein secretome analysis.

Design of the SCBC for single-cell protein secretome analysis.
(a) Image of an SCBC in which flow channels are shown in red and the control channels are shown in blue. Input and output ports are labeled. Ab, antibody. (b) An optical micrograph showing cells loaded and isolated within the microchambers, overlaid with the fluorescence micrograph of the developed assay barcode for those same microchambers. Numbers of cells per microchamber are indicated by the yellow numbers. (c) Drawing of the multiplex DEAL primary antibody barcode array used for capture of secreted proteins from single cells and then developed for the detection of those proteins. SA, Streptavidin. (d) Scanned fluorescent images used for the antibody barcode calibration measurements using spiked recombinant proteins. The protein concentrations (in numbers of molecules per chamber) are given to the left of each row of images. The plot at the top is a line profile of the top row of images and represents the reproducibility of the barcodes across the antibody array of an SCBC. (e) Recombinant protein calibration curves for TNF-α, IL-1β, IL-6, IL-10 and GM-CSF. Individual measurements (red) are shown for IL-1β. Other proteins measurements are represented by average intensity values and s.d.


The barcode array is a pattern of parallel stripes, each coated with a distinctive antibody. The stripe width is 25 μm at a pitch of 50 μm. To achieve high and consistent antibody loading, and to prevent antibody denaturation during microfluidics assembly, we used the DNA-encoded antibody library (DEAL) approach18, coupled with microchannel-guided flow patterning19 (Supplementary Fig. 1). The chemistry and reproducibility of the DNA barcode patterning process has been previously described192021. The SCBC barcodes contain 13 stripes, 12 for assaying a dozen different proteins and one as a control and spatial reference. Two sets of barcodes are included per microchamber. Cells are randomly loaded so that each SCBC microchamber contains zero to a few cells, following a Gamma Poisson distribution (goodness-of-fit test: P > 0.8.Supplementary Fig. 2). The numbers of cells in each chamber are counted via imaging (Fig. 1b).
We determined the dynamic range and detection limit of our design by performing on-chip immunoassays with recombinant proteins (Fig. 1c). The barcode array initially consists of 13 uniquely designed orthogonal DNA strands labeled in order as A through M (Supplementary Table 1). Before loading of recombinant proteins, a cocktail containing all capture antibodies conjugated to different complementary DNA strands (A′—L′) is used to transform, via DNA hybridization, the DNA barcode into an antibody array (Fig. 1c). As few as 100–1,000 copies (1 × 10−22–1 × 10−21 mol) could be detected (3 s.d.), with a dynamic range of three to four orders of magnitude (Fig. 1e and Supplementary Fig. 3), which is compatible with single-cell secretion measurements18. Antibody loading, and thus assay sensitivity, was uniform across the whole chip (coefficient of variation (CV) <10%) (Fig. 1d,e).

Analysis of cytokine production by LPS-stimulated macrophages

We validated the SCBC by using the THP-1 human monocyte cell line. We differentiated the THP-1 cells into cytokine-producing macrophages using phorbol 12-myristate 13-acetate (PMA). Before loading into the device, we added LPS to activate Toll-like receptor 4 (TLR4) signaling2223, a process that mimics the innate immune response to Gram-negative bacteria (Supplementary Fig. 4). For these experiments, we designed the antibody barcode to measure 12 proteins: tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-1α, IL-1β, IL-6, IL-10, IL-12, granulocyte-macrophage colony–stimulating factor (GM-CSF), chemokine (C-C motif) ligand-2 (CCL-2), transforming growth factor-β (TGF-β) and prostate-specific antigen (PSA) (Supplementary Table 2).
The microchambers contained between 0 and 40 cells so that both single-cell behavior and signals from a population could be measured. Through an automated image processing algorithm, we quantified the fluorescence intensities for each protein from each microchamber. For chambers with cells centered between the two barcodes, measurements from the barcode replicates were consistent (CV < 15%, Fig. 2). Uncentered cells contributed to variance between replicates (CV up to 50%); however, the averages of the barcode replicates from chambers with centered and uncentered cells were indistinguishable (P > 0.2,Supplementary Fig. 5). Furthermore, measurements between chips showed good consistency (P > 0.2).
Figure 2: On chip secretion measurements of macrophage differentiated from THP-1 monocyte cell.

On chip secretion measurements of macrophage differentiated from THP-1 monocyte cell.
(a) Top, representative scanned images of barcode signals from individual chambers. Green bars represent microchamber boundaries, red bars are protein signals and the yellow numbers indicate the number of cells. Bottom, comparison of averaged intensity from SCBC measurements and bulk culture supernatant. Data are normalized so that references have the same intensity. (b) Scatter plots of measured levels of TNF-α and the reference control for individual microchambers containing n = 0,1,2,3,4 or 5 cells. P values are calculated by comparing neighboring columns (*P < 0.005; **P < 0.001; ***P < 0.0001; NS, not significant). (c) Scatter plots of TNF-α versus IL-1β derived from SCBC measurements (left) and from ICS coupled with flow cytometry (right). Histograms (frequency versus intensity) for the individual protein are provided at the tops and sides of the scatter plots. All y axis units are fluorescence intensity. For the SCBC data, each quadrant is labeled with numbers that reflect the percentage of single cells (red) and two to three cells (green) in that section. The gates separating cytokine-secreting and nonsecreting cells are determined from 0 cell microchamber (background) measurements (blue). s.d. from barcode replicates of the same chamber are provided for selected points (see Results for selection criteria). For the ICS measurements, the gates were determined by isotype control staining.


The intensities of the individual proteins, averaged over many individual SCBC microchamber measurements, agreed with measurements of those same proteins from cell culture supernatants (Fig. 2a). Secretome data, when binned according to the numbers of cells per chamber, yielded statistically distinct protein signals (P < 0.05, Fig. 2b and Supplementary Fig. 6). However, the reference signal was insensitive to the numbers of cells (P > 0.2, Fig. 2b and Supplementary Fig. 6). Most notably, single-cell protein signals could be clearly detected (P < 0.0001, Fig. 2b and Supplementary Fig. 6). There was also clear separation of secreting and nonsecreting cells, as visualized by multiple peaks in flow cytometry histograms (Fig. 2c).
We gated the fraction of cells detected to secrete a given protein using background signals from empty chambers. We measured the frequencies of cells producing TNF-α, IL-1β, IL-10 and GM-CSF to be similar by both SCBC and by ICS flow cytometry (Fig. 2c and Supplementary Fig. 6). Furthermore, the measured fraction of cells in each quadrant also showed a good similarity between the two techniques (Fig. 2c andSupplementary Fig. 6). Thus, the SCBC platform can efficiently provide a functional profiling of single immune cells. In general, these macrophages showed strongly coordinated functions24.

Analysis of polycytokine production by human CTLs

We next turned toward using the SCBC to functionally profile antigen-specific CTLs, which are the main effector cell type of an adaptive immune response targeting intracellular pathogens25. CTLs can show a great diversity of antigen specificities, phenotypic surface proteins and functions, and capturing this diversity is a major challenge. Most data suggest that the ability of CTLs to produce multiple cytokines (polyfunctionality) correlates with protective immune responses in vivo42627.
We assayed the functional diversity of healthy donor CD8+ T cells (n = 3), melanoma-associated antigen recognized by T cells 1 (MART-1)–specific T cell receptor (TCR)-transgenic cells collected from the peripheral blood of an individual with metastatic melanoma participating in an ACT immunotherapy clinical trial, and ex vivo–expanded tyrosinase-specific T cells. TCR engineering provides a means to generate large quantities of antigen-specific T cells amenable to use for the therapy of cancer28. The TCR-transgenic cells collected from the peripheral blood had been previously generated in vitro by retroviral vector transduction to insert the two chains of a TCR specific for MART-1 and then expanded ex vivo for 2 d followed by re-infusion into the patient after a lymphodepletion conditioning regimen. The patient then received three vaccinations with dendritic cells pulsed with a MART-1 peptide and a high dose of IL-2 to further activate and expand the cellsin vivo. Peripheral blood mononuclear cells (PBMCs) were collected on day 30 after re-infusion by leukapheresis, at a time when multiple metastatic melanoma lesions were responding to this therapy. The tyrosinase-specific T cell culture was generated ex vivo from a tyrosinase-specific cell culture obtained by peptide–human leukocyte antigen A0201 (HLA-A0201) tetramer–based selection followed by nonspecific expansion with CD3-specific antibody and IL-2 to obtain a population of nontransgenic but uniform antigen-specific T cells.
We enriched the PBMCs for CD3 marker by negative magnetic bead selection before sorting on DEAL-based CD8-specific antibody–coated or peptide–HLA-A0201 tetramer–coated microarrays29. Then we released CD3+CD8+ or CD3+tetramer+ cells from the microarrays. The released T cells underwent activation either by polyclonal TCR engagement of CD3 and CD28-specific antibody binding or by antigen-specific TCR engagement via tetramer and CD28-specific antibody. Activation and SCBC loading was completed within 5 min. To maximize the on-percentage of single-cell measurement to 25–40%, we loaded ~1 × 104 sorted T cells in 5 μl medium into the device. Protein heat maps and plots that compare and contrast these different T cell groups are presented in Figure 3. We included multiple markers that indicate functions such as cytoxicity (perforin), T cell growth and differentiation (IL-2), apoptosis promotion (TNF-α and IFN-γ), inflammation (IL-1β, IL-6 and TNF-β), anti-inflammation (IL-10) and the stimulation and recruitment of other immune compartments (CCL-2, CCL-3CCL-5 and GM-CSF)27 (Supplementary Table 3).
Figure 3: Single-cell secretion measurements of CTLs from individuals with melanoma and healthy donors.

Single-cell secretion measurements of CTLs from individuals with melanoma and healthy donors.
(a) Unsupervised clustering of CD8+ T cells from three healthy donors, presented as a heat map. Each row represents a measurement of 12 secreted cytokines from a single cell, with protein labels provided at the bottom. (b) Data from MART-1–specific TCR–transgenic CTL single-cell experiments, presented as a heat map organized via unsupervised clustering. The fluorescence intensity scale for all four heat maps is the same. (c) Phenotyping data from flow cytometry for MART-1–specific TCR–transgenic CTLs. Each bar shows the percentage of cells positive for the specific surface marker. (d) Fluorospot analysis of the MART-1–specific TCR–transgenic T cell population. The curves on top compare SCBC and Fluorospot measurements on fraction (or number) of cells secreting CCL-2, CCL-5, perforin, IL-1β, IL-6 and TNF-α. Representative Fluorospot images are provided at the bottom. (e) Univariate comparison of antigen-specific TCR–transgenic T cells from a subject with melanoma and healthy donor CD8+ T cell culture controls. The lines of the top plot represent the percentage of cytokine-producing cells from each sample. Blue area shows the range detected from healthy donor samples. The one-dimensional scatter plot on the bottom compares the signal intensities measured from the subject's MART-1–specific TCR–transgenic T cells and the CD8+ T cell background from one representative healthy donor.


More than 20% (median) of the healthy donor CD3+CD8+ T cells produced TNF-α, IL-6 and the chemokines CCL-2, CCL-3 and CCL-5. The MART-1–specific TCR–transgenic cells that were inducing an objective tumor response in vivo showed a wide range of positive functions demonstrated by the release of perforin, IL-1β, IL-10, IFN-γ, IL-2 and CCL-5 upon ex vivo antigen re-stimulation with the MART-1–HLA-A0201 tetramer. The functionality of the antigen-specific TCR-transgenic cells derived from the subject with melanoma was higher compared to the healthy donor lymphocytes in terms of both signal intensity and fraction of positive cells (Fig. 3a,b,e). The frequency of cytokine-producing cells among the subject-derived TCR transgenic cells was consistent with Fluorospot results (Fig. 3d). Phenotyping results identified by flow cytometry of surface markers of T cell phenotype (Fig. 3c) illustrated that the population of subject-derived TCR-transgenic, MART-1–specific cells was mostly homogeneous; the principal (>90%) population of these cells was CD8+ and had a phenotype consistent with effector CCR7CD45RA+ T cells at a late differentiation stage (Fig. 3c)15. In comparison, the ex vivo–expanded tyrosinase-specific T cells showed decreased production of CCL-3, IL-6 and TNF-α (Fig. 3e). Consistent with the notion that ex vivo expansion of T cells by IL-2 and CD3-specific antibody results in terminal differentiation of T cells, the tyrosinase-specific T cell clone had elevated release of perforin, IL-1β and IL-2, compared to healthy control samples, upon activation (Fig. 3e and Supplementary Fig. 7).

Evaluation of polyfunctionality

We analyzed the multivariate features of these T cell populations by studying the protein-protein correlations for two and three proteins. Pseudo–three-dimensional plots (Fig. 4a–c) of markers representing various functions revealed that certain functions of the MART-1–specific cell population were highly coordinated compared to the healthy donor cells. For example, 70% of IL-6+ cells produced CCL-5, whereas for CTLs from healthy donors the frequency was around 50%. TNF-α and IFN-γ production were anticorrelated in MART-1–specific cells (Fig. 4c), with >90% of the population expressing at most one of these effector molecules. However, for the small fraction of MART-1–specific TCR–transgenic cells that were TNF-α+IFN-γ+, secretion of IL-2 was often an additional function (75%, Fig. 4). A full set of protein-protein correlations is provided inSupplementary Figure 8.
Figure 4: Polyfunctional diversity analysis for CTLs from a subject with metastatic melanoma and from healthy donors.

Polyfunctional diversity analysis for CTLs from a subject with metastatic melanoma and from healthy donors.
(ac) Pseudo–three-dimensional scatter plots for representative single-cell cytokine measurements from CD8+ T cells. Top and bottom rows are correlations for perforin versus IL-6 and TNF-α versus IFN-γ, respectively. The third dimension (CCL-5 or IL-2) is projected to the two-dimensional plot by using red for positive cells and blue for negative cells. Numbers on the right show percentage in each quadrant. Samples are labeled on the left. (d,e) Functional diversity plots for the subject's MART-1–specific TCR–transgenic cells and CD8+ T cells from a representative healthy donor. Each major functional subset identified (with frequency >0.5%) is shown by an individual bar. The bar heights represent population percentages. Fifty-three major subsets were identified from the MART-1–specific TCR–transgenic cells; 23 were identified from the healthy donor. The number at the top of the plot is the number of functions associated with each subset. The matrix at the bottom provides the function detail (×: positive). The pie charts give the percentage of cells that fell into one of the major functional subsets. Asterisks above the blue bars denote those subsets within the most frequent 60% of the population.

We defined functional subsets of the various T cells by identifying groups of cells that secreted the same combination of proteins (Fig. 4d,e). There were at least 45 distinct subpopulations that accounted for 60% of the MART-1–specific TCR–transgenic cells (Fig. 4e and Table 1). A similar accounting for the healthy donor CD8+ T cells and the tyrosinase-specific T cells yields 4–17 subpopulations (Fig. 4d and Table 1). Furthermore, for the MART-1–specific TCR–transgenic cells, the major functional subsets averaged more than five active functions, whereas both healthy donor and tyrosinase-specific cells averaged only one or two functions (Table 1). This demonstrates the ability of SCBC to visualize and discriminate different levels of functional heterogeneity.

Table 1: Summary of functional diversity of assayed samplFull table

Discussion



The SCBC permits highly multiplexed (more than ten proteins) measurements of effector molecules from single cells by detecting the natural protein secretome from macrophages and T cells upon activation. The multiplex capacity can be further expanded beyond what we explored here. The ability to use small sample size (~1 × 104 cells) implies that the SCBC can be integrated with other upstream multiplexed analysis, such as flow cytometry or (as we show here) microarray sorting, to enable a detailed functional study of phenotypically defined sets of cells selected from heterogeneous populations. Analysis of signals from chambers containing different numbers of cells may also provide information relevant to cell-cell interactions.
The MART-1–specific TCR–transgenic CTLs had stronger perforin, IFN-γ and interleukin secretion and more functional heterogeneity compared with the healthy donor CTL controls. This functional status is also indicated by their identity as effector T cells (CD45RA+CCL7CD27CD28CD62L)153031. Previous vaccination studies identified that polyfunctional T cells are better cytokine producers and that the quality of a polyfunctional T cell response is a good predictor of clinical outcome432. We found that the MART-1–specific TCR–transgenic CTLs showed favorable features compared against CTLs from healthy donors (for example, polyfunctional subset frequency 62% versus 6–25%; Supplementary Fig. 9). These data are consistent with the observation that, at the time the CTLs were collected, there was active inflammation and the tumors were responding to the ACT therapy in this patient.
We observed signs of T cell terminal effector differentiation and exhaustion, as indicated by the anticorrelation of TNF-α and IFN-γ secretion, high IL-10+ cell frequency and high PD1 and CD127 expression15. However, the quality of the T cell response at a single time point within a vaccination trial may not provide an indicator of long-term vaccination or therapy response4. A similar multiparameter SCBC analysis carried out at multiple time points throughout the course of a cancer immunotherapy treatment is currently underway.
We saw a high level of functional heterogeneity within a population defined as relatively homogeneous by surface markers2533, and that heterogeneity was also focused. For example, 45 out of >4,000 possible functional subsets could account for 60% of all MART-1–specific TCR-transgenic cells. The observed high level of polyfunctionality (up to 12 functions per cell, with an average of more than five functions) exceeds current multiplexing capacity by most existing single-cell secretion assays. Moreover, none of the proteins being profiled were interchangeable with others within the panel (with R2 < 0.6, Supplementary Fig. 8). These findings indicate that a high dimensional analysis is in fact required for comprehensively profiling of T cell effector functions.
The SCBC provides a new platform for analyzing the functional activity of immune cells immediately after short-term ex vivo activation. This technology compares favorably to current cellular immunoassays in terms of sensitivity, multiplexing capacity, quantification, sample size, cost and infrastructure requirements and thus has potential for a thorough, cost-effective characterization of human immune cell responses.

Methods



Microchip fabrication.

The SCBCs were assembled from a DNA barcode microarray glass slide and a PDMS slab containing a microfluidic circuit. The DNA barcode array was created with microchannel-guided flow patterning (Supplementary Fig. 1). Each barcode was comprised of thirteen stripes of uniquely designed single-stranded DNA molecules. The PDMS microfluidic chip was fabricated using a two-layer soft lithography approach34(Supplementary Methods).

Human samples.

Human samples were obtained from individuals with metastatic melanoma (males) enrolled in a TCR-transgenic ACT protocol clinical trial (registration number NCT00910650). The studies using human samples were approved by the appropriate human use committees (UCLA Institutional Review Board 08-02-020, IND#13859), and informed consent was obtained from all individuals studied.

Isolation, purification and expansion of T cells.

PBMCs were collected from individuals receiving TCR ACT immunotherapy by leukapheresis and periodic peripheral blood draws as previously described35. Aliquots of cryopreserved PBMCs were thawed and immediately diluted with RPMI complete medium containing 5% human AB serum (Omega Scientific). Cells were washed and subjected to enzymatic treatment with DNase (Sigma) for 1 h at 37 °C, washed and rested overnight in a 5% CO2 incubator. Antigen-specific MART-1 T cells were purified sequentially by magnetic negative enrichment for CD3 (Stemcell) and by a MART-1–HLA-A0201 tetramer microarray that have been previously described29 (Supplementary Methods). Purified cells were collected, washed, stimulated with MART-1 tetramer and CD28-specific antibody and loaded into SCBC chip. PBMCs from healthy donors were negatively enriched in the same way and were further purified by CD8-specific antibody microarray, followed by stimulation with CD3/CD28–specific antibody. Sorted cells was checked to be >95% pure.

On-chip secretion profiling.

Before loading cells onto the chip, the DNA barcode array was transformed into an antibody microarray. The chip was then ready for cell loading. Chips with cells were incubated and then assays were developed with secondary antibodies and fluorescent markers (Supplementary Methods).

Intracellular cytokine staining of THP-1 cells.

Brefeldin A (eBioscience) was added in the presence of PMA and LPS at the recommended concentration in the final 4 h of stimulation. Standard intracellular staining was performed as described by the supplier's protocol (eBioscience) with additional blocking with human serum (Sigma) and washes. Cells were fixed and permeabilized by using a fixation and permeabilization kit (eBioscience) and then were stained intracellularly with antibody to TNF-α (MAb11), antibody to IL-1β (H1b-98), antibody to IL-10 (JES3-9D7) and antibody to GM-CSF (BVD2-21C11) (all from eBioscience). Isotype control staining was used as negative control, and 2 × 104 events were collected for each condition. Samples were analyzed on a FACSCalibur (BD Biosciences) machine with CellQuest Pro software (BD Biosciences).

Flow cytometry analysis of antigen specific T cells.

Cryopreserved PBMC samples from peripheral blood draws or leukapheresis were thawed and analyzed by HLA-A*0201 tetramer assay (Beckman Coulter) with flow cytometric analysis as previously described3536. In brief, PBMCs were resuspended in 100 μl of adult bovine serum (Omega Scientific) and stained for 15 min at room temperature (20 °C) using a cocktail of antibodies to the following proteins in replicate aliquots: CD3 (UCHT1, BD Bioscience), CD8 (3B5, Invitrogen), CD45RA (2H4LDHIILDB9, Beckman Coulter), CD62L (DREG56, Beckman Coulter), CCR7 (150503, BD Bioscience), CD27 (MT271, BD Bioscience), CD28 (CD28.2, BD Bioscience), CD127 (HIL-7R-M21, BD Bioscience) and PD1 (MIH4, BD Bioscience). For all flow cytometry experiments, anti-mouse IgK isotype control FBS compensation particles (BD Biosciences) were used for compensation purposes, and 5 × 105 to 1 × 106 lymphocytes were acquired for each condition. To correctly gate the flow cytometry data, the fluorescent minus one approach was used. Samples were acquired on an LSR II system (BD Biosciences), and data were analyzed using FlowJo software (TreeStar).

Fluorospot assay for sorted antigen-specific T cells.

Antigen specific T cells were captured by tetramer microarray. Primary cytokine capture antibody was co-localized on the same array. The captured cells were then incubated at 37 °C and 5% CO2 for 12 h in 10% FBS in RPMI 1640 medium. Phycoerythrin-labeled secondary antibody (eBioscience) was applied after incubation. Then slides were washed and imaged by the EZ-C1 confocal microscope system (Nikon).

Data and statistical analyses.

We used GenePix 4400 (Axon Instruments) to obtain the scanned fluorescent image for both Cy3 and Cy5 channels. All scans were performed at constant instrument settings: laser power 80% (635 nm) and 15% (532 nm), optical gains 600 (635 nm) and 450 (532 nm), brightness 80 and contrast 83 for T cell experiments; laser power 100% (635 nm) and 33% (532 nm), optical gains 800 (635 nm) and 700 (532 nm), brightness 87 and contrast 88 for macrophage experiments. All the barcodes were processed in PhotoShop (Adobe) and ImageJ software (US National Institutes of Health) to generate fluorescence line profiles. A home-developed Excel (MicroSoft) macro was employed for automatic extraction of average fluorescence signal for all bars in each set of barcode, and all the barcode profiles were compared to the number of cells by using the same program. On the basis of these data, heat maps were generated by using the software Cluster and Treeview37. Flow cytometry data were analyzed in FlowJo software. P values were calculated from two-tailed Student's t tests assuming unequal variance.