Aggregation Temperature of Escherichia coli Depends on Steepness of the Thermal Gradient.

Bacterial chemotaxis, the directed migration of bacteria in a gradient of chemoattractant, is one of the most well-studied and well-understood processes in cell biology. On the other hand, bacterial thermotaxis, the directed migration of bacteria in a gradient of temperature, is understood relatively poorly, with somewhat conflicting reports by different groups. One of the reasons for that is the relative technical difficulty of the generation of well-defined gradients of temperature that are sufficiently steep to elicit readily detectable thermotaxis. Here, we used a specially designed microfluidic device to study thermotaxis of Escherichia coli in a broad range of thermal gradients with a high rate of data collection. We found that in shallow temperature gradients with narrow temperature ranges, E. coli tended to aggregate near a sidewall of the gradient channel at either the lowest or the highest temperature. On the other hand, in sufficiently steep gradients with wide temperature ranges, E. coli aggregated at intermediate temperatures, with maximal cell concentrations found away from the sidewalls. We observed this intermediate temperature aggregation in a motility buffer that did not contain any major chemoattractants of E. coli, in contradiction to some previous reports, which suggested that this type of aggregation required the presence of at least one major chemoattractant in the medium. Even more surprisingly, the aggregation temperature strongly depended on the gradient steepness, decreasing by ∼10° as the steepness was increased from 27 to 53°C/mm. Our experiments also highlight the fact that assessments of thermal gradients by changes in fluorescence of temperature-sensitive fluorescent dyes need to account for thermophoresis of the dyes.

Mechanics dictate where and how freshwater planarians fission.

Asexual freshwater planarians reproduce by tearing themselves into two pieces by a process called binary fission. The resulting head and tail pieces regenerate within about a week, forming two new worms. Understanding this process of ripping oneself into two parts poses a challenging biomechanical problem. Because planarians stop "doing it" at the slightest disturbance, this remained a centuries-old puzzle. We focus on Dugesia japonica fission and show that it proceeds in three stages: a local constriction ("waist formation"), pulsation-which increases waist longitudinal stresses-and transverse rupture. We developed a linear mechanical model with a planarian represented by a thin shell. The model fully captures the pulsation dynamics leading to rupture and reproduces empirical time scales and stresses. It asserts that fission execution is a mechanical process. Furthermore, we show that the location of waist formation, and thus fission, is determined by physical constraints. Together, our results demonstrate that where and how a planarian rips itself apart during asexual reproduction can be fully explained through biomechanics.

Rigidity of silicone substrates controls cell spreading and stem cell differentiation.

The dependences of spreading and differentiation of stem cells plated on hydrogel and silicone gel substrates on the rigidity and porosity of the substrates have recently been a subject of some controversy. In experiments on human mesenchymal stem cells plated on soft, medium rigidity, and hard silicone gels we show that harder gels are more osteogenic, softer gels are more adipogenic, and cell spreading areas increase with the silicone gel substrate rigidity. The results of our study indicate that substrate rigidity induces some universal cellular responses independently of the porosity or topography of the substrate.

Environmental effects on protection against Mycobacterium tuberculosis after immunization with Ad85A.

Previously we have shown that intradermal (i.d.) immunization with a recombinant adenovirus expressing antigen 85A (Ad85A) induced a strong splenic CD8T cell response in BALB/c mice but a weak lung immune response and did not protect mice against challenge with Mycobacterium tuberculosis (Mtb). After moving to a new animal house, the same i.d. immunization induced a strong lung immune response and the mice were protected against Mtb challenge. Increased numbers of antigen 85A-specific CD8 cells were present in lung tissue but were not recoverable by bronchoalveolar lavage (BAL). Mycobacterial growth was inhibited 21 days after Mtb challenge. In contrast, the effects of intranasal (i.n.) immunization did not change between the animal houses; 85A-specific T cells were recovered by BAL and were able to inhibit Mtb growth early after challenge. The effect of alterations to the environment was investigated by administering BCG or Mycobacterium abscessus in the drinking water, which induced protection against Mtb challenge, while Mycobacterium smegmatis did not. However, when Ad85A was given i.d. at the same time as BCG or M. abscessus, but not M. smegmatis, the protection induced by Ad85A was abolished. Treatment of mice with a CD25 antibody during the challenge period, abolished the suppressive effect of oral mycobacterial administration, suggesting that regulatory T cells (T regs) were involved. These results showed that exposure to environmental microorganisms can alter the protective immune response to a parenterally administered subunit vaccine, a result with important implications for the use of such vaccines in humans.

Simultaneous immunization against tuberculosis.

BACKGROUND: BCG, the only licensed vaccine against tuberculosis, provides some protection against disseminated disease in infants but has little effect on prevention of adult pulmonary disease. Newer parenteral immunization prime boost regimes may provide improved protection in experimental animal models but are unproven in man so that there remains a need for new and improved immunization strategies. METHODS AND FINDINGS: Mice were immunized parenterally, intranasally or simultaneously by both routes with BCG or recombinant mycobacterial antigens plus appropriate adjuvants. They were challenged with Mycobacterium tuberculosis (Mtb) and the kinetics of Mtb growth in the lungs measured. We show that simultaneous immunization (SIM) of mice by the intranasal and parenteral routes is highly effective in increasing protection over parenteral BCG administration alone. Intranasal immunization induces local pulmonary immunity capable of inhibiting the growth of Mtb in the early phase (the first week) of infection, while parenteral immunization has a later effect on Mtb growth. Importantly, these two effects are additive and do not depend on priming and boosting the immune response. The best SIM regimes reduce lung Mtb load by up to 2 logs more than BCG given by either route alone. CONCLUSIONS: These data establish SIM as a novel and highly effective immunization strategy for Mtb that could be carried out at a single clinic visit. The efficacy of SIM does not depend on priming and boosting an immune response, but SIM is complementary to prime boost strategies and might be combined with them.

CXCR6 is a marker for protective antigen-specific cells in the lungs after intranasal immunization against Mycobacterium tuberculosis.

Convincing correlates of protective immunity against tuberculosis have been elusive. In BALB/c mice, intranasal immunization with a replication-deficient recombinant adenovirus expressing Mycobacterium tuberculosis antigen 85A (adenovirus-85A) induces protective lower respiratory tract immunity against pulmonary challenge with Mycobacterium tuberculosis, while intradermal immunization with adenovirus-85A does not. Here we report that intranasal immunization with adenovirus-85A induces expression of the chemokine receptor CXCR6 on lung CD8 T lymphocytes, which is maintained for at least 3 months. CXCR6-positive antigen-specific T cell numbers are increased among bronchoalveolar lavage-recoverable cells. Similarly, intranasal immunization with recombinant antigen 85A with adjuvant induces CXCR6 expression on lung CD4 cells in BALB/c and C57BL/6 mice, while a synthetic ESAT6(1-20) peptide with adjuvant induces CXCR6 expression in C57BL/6 mice. Parenteral immunization fails to do so. Upregulation of CXCR6 is accompanied by a transient elevation of serum CXCL16 after intranasal immunization, and lung cells cultured ex vivo from mice immunized intranasally show increased production of CXCL16. Administration of CXCL16 and cognate antigen intranasally to mice previously immunized parenterally increases the number of antigen-specific T lymphocytes in the bronchoalveolar lavage-recoverable population, which mediates inhibition of the early growth of Mycobacterium tuberculosis after challenge. We conclude that expression of CXCR6 on lung T lymphocytes is a correlate of local protective immunity against Mycobacterium tuberculosis after intranasal immunization and that CXCR6 and CXCL16 play an important role in the localization of T cells within lung tissue and the bronchoalveolar lavage-recoverable compartment.

Preclinical development of an in vivo BCG challenge model for testing candidate TB vaccine efficacy.

There is an urgent need for an immunological correlate of protection against tuberculosis (TB) with which to evaluate candidate TB vaccines in clinical trials. Development of a human challenge model of Mycobacterium tuberculosis (M.tb) could facilitate the detection of such correlate(s). Here we propose a novel in vivo Bacille Calmette-Guérin (BCG) challenge model using BCG immunization as a surrogate for M.tb infection. Culture and quantitative PCR methods have been developed to quantify BCG in the skin, using the mouse ear as a surrogate for human skin. Candidate TB vaccines have been evaluated for their ability to protect against a BCG skin challenge, using this model, and the results indicate that protection against a BCG skin challenge is predictive of BCG vaccine efficacy against aerosol M.tb challenge. Translation of these findings to a human BCG challenge model could enable more rapid assessment and down selection of candidate TB vaccines and ultimately the identification of an immune correlate of protection.

Chemokine gene expression in lung CD8 T cells correlates with protective immunity in mice immunized intra-nasally with Adenovirus-85A.

BACKGROUND: Immunization of BALB/c mice with a recombinant adenovirus expressing Mycobacterium tuberculosis (M. tuberculosis) antigen 85A (Ad85A) protects against aerosol challenge with M. tuberculosis only when it is administered intra-nasally (i.n.). Immunization with Ad85A induces a lung-resident population of activated CD8 T cells that is antigen dependent, highly activated and mediates protection by early inhibition of M. tuberculosis growth. In order to determine why the i.n. route is so effective compared to parenteral immunization, we used microarray analysis to compare gene expression profiles of pulmonary and splenic CD8 T cells after i.n. or intra-dermal (i.d.) immunization. METHOD: Total RNA from CD8 T cells was isolated from lungs or spleens of mice immunized with Ad85A by the i.n. or i.d. route. The gene profiles generated from each condition were compared. Statistically significant (p ≤ 0.05) differentially expressed genes were analyzed to determine if they mapped to particular molecular functions, biological processes or pathways using Gene Ontology and Panther DB mapping tools. RESULTS: CD8 T cells from lungs of i.n. immunized mice expressed a large number of chemokines chemotactic for resting and activated T cells as well as activation and survival genes. Lung lymphocytes from i.n. immunized mice also express the chemokine receptor gene Cxcr6, which is thought to aid long-term retention of antigen-responding T cells in the lungs. Expression of CXCR6 on CD8 T cells was confirmed by flow cytometry. CONCLUSIONS: Our microarray analysis represents the first ex vivo study comparing gene expression profiles of CD8 T cells isolated from distinct sites after immunization with an adenoviral vector by different routes. It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d. CD8 T cells. The sustained expression of chemokines and activation genes enables CD8 T cells to remain in the lungs for extended periods after i.n. immunization. This may account for the early inhibition of M. tuberculosis growth observed in Ad85A i.n. immunized mice and explain the effectiveness of i.n. compared to parenteral immunization with this viral vector.

Nasal associated lymphoid tissue (NALT) contributes little to protection against aerosol challenge with Mycobacterium tuberculosis after immunisation with a recombinant adenoviral vaccine.

Intra-nasal administration of a recombinant adenovirus expressing Mycobacterium tuberculosis antigen 85A (Ad85A) has been shown to provide protection against challenge with M. tuberculosis. However the role of the upper respiratory tract associated lymphoid tissue, specifically the nasal associated lymphoid tissue (NALT), in providing protection has yet to be elucidated. Here we administered Ad85A to BALB/c mice alone or following BCG priming, using intranasal inocula targeting the whole respiratory tract or only the NALT, to show that Ad85A induces an immune response in the NALT insufficient to provide protection. Rather, Ad85A delivered through the respiratory tract must induce a deep lung immune response in order to protect against M. tuberculosis.

Immunization of mice with a recombinant adenovirus vaccine inhibits the early growth of Mycobacterium tuberculosis after infection.

BACKGROUND: In pulmonary Mycobacterium tuberculosis (Mtb) infection, immune responses are delayed compared to other respiratory infections, so that antigen-specific cells are not detected in the lungs earlier than day 14. Even after parenteral immunization with Bacille Calmette Guerin (BCG) or a subunit vaccine, the immune response after Mtb challenge is only slightly accelerated and the kinetics of pulmonary Mtb growth do not differ between naïve and immunized animals up to day 14. METHODS AND FINDINGS: Mice were immunized intranasally with a recombinant adenovirus expressing mycobacterial antigen 85A (Ad85A), challenged by aerosol with Mtb and the kinetics of Mtb growth in the lungs measured. Intranasal immunization with Ad85A inhibits Mtb growth in the early phase of infection, up to day 8. Protection is sustained for at least 7 months and correlates with the presence of antigen-specific activated effector CD8 T cells in the lungs. Antigen 85A-specific T cells respond to antigen presenting cells from the lungs of mice immunized with Ad85A 23 weeks previously, demonstrating the persistence of antigen in the lungs. CONCLUSIONS/SIGNIFICANCE: Intranasal immunization with Ad85A can inhibit early growth of Mtb because it establishes a lung antigen depot and maintains an activated lung-resident lymphocyte population. We propose that an optimal immunization strategy for tuberculosis should aim to induce both lung and systemic immunity, targeting the early and late phases of Mtb growth.

Evaluating the impact of the humanities in medical education.

The inclusion of the humanities in medical education may offer significant potential benefits to individual future physicians and to the medical community as a whole. Debate remains, however, about the definition and precise role of the humanities in medical education, whether at the premedical, medical school, or postgraduate level. Recent trends have revealed an increasing presence of the humanities in medical training. This article reviews the literature on the impact of humanities education on the performance of medical students and residents and the challenges posed by the evaluation of the impact of humanities in medical education. Students who major in the humanities as college students perform just as well, if not better, than their peers with science backgrounds during medical school and in residency on objective measures of achievement such as National Board of Medical Examiners scores and academic grades. Although many humanities electives and courses are offered in premedical and medical school curricula, measuring and quantifying their impact has proven challenging because the courses are diverse in content and goals. Many of the published studies involve self-selected groups of students and seek to measure subjective outcomes which are difficult to measure, such as increases in empathy, professionalism, and self-care. Further research is needed to define the optimal role for humanities education in medical training; in particular, more quantitative studies are needed to examine the impact that it may have on physician performance beyond medical school and residency. Medical educators must consider what potential benefits humanities education can contribute to medical education, how its impact can be measured, and what ultimate outcomes we hope to achieve.

Multifunctional, high-level cytokine-producing Th1 cells in the lung, but not spleen, correlate with protection against Mycobacterium tuberculosis aerosol challenge in mice.

Boosting bacillus Calmette-Guérin (BCG)-primed mice with a recombinant adenovirus expressing Mycobacterium tuberculosis Ag 85A by different administration routes has very different effects on protection against aerosol challenge with M. tuberculosis. Mice boosted intradermally make very strong splenic CD4 and CD8 Th1 cytokine responses to Ag 85A, but show no change in lung mycobacterial burden over BCG primed animals. In contrast, intranasally boosted mice show greatly reduced mycobacterial burden and make a much weaker splenic response but a very strong lung CD4 and CD8 response to Ag 85A and an increased response to purified protein derivative. This effect is associated with the presence in the lung of multifunctional T cells, with high median fluorescence intensity and integrated median fluorescence intensity for IFN-gamma, IL-2, and TNF. In contrast, mice immunized with BCG alone have few Ag-specific cells in the lung and a low proportion of multifunctional cells, although individual cells have high median fluorescence intensity. Successful immunization regimes appear to induce Ag-specific cells with abundant intracellular cytokine staining.

Improving the adjustment of educationally disadvantaged students to medical school: the Summer Enrichment Program.

The Summer Enrichment Program (SEP) is a 6-week pre-matriculation program that targets students who may be at an educational disadvantage and/or may have difficulties adjusting to the rigors of medical school. The objective of the current study was to determine whether the SEP (a) eased the transition to the first year of medical school and (b) had an impact on academic performance during the first year of medical school. All students from groups underrepresented in medicine, who had been invited to participate in the SEP, and all Humanities and Medicine Program students who matriculated at Mount Sinai School of Medicine between 1999 and 2003 and were still matriculated during the 2003-2004 academic year were asked to respond to a survey distributed in the spring of 2004. In addition, student academic profiles were reviewed. Responses to the survey indicated that the SEP provided important emotional benefits for those students who chose to attend the program. Virtually all students who had attended had praise for the program and felt that it eased the transition to medical school, helped build confidence and facilitated social connections. In addition, those students from groups underrepresented in medicine who attended the SEP had less academic difficulty (fewer course failures) in their first year of medical school.