Readings for Week 4 – Leonore A. Herzenberg Summary

Apologies for the (very) late posting - I was convinced that I was scheduled to post next week's summary instead of this one. It wasn't until I checked GoogleDocs that I realized my mistake. My deepest regrets.

Antigen-specific B-1a antibodies induced by Francisella tularensis LPS provide long-term protection against F. tularensis, LVS challenge

Francisella tularensis (Ft) the etiologic agent of tularemia, is a gram-negative bacterium with an extremely low infectious dose. Respiratory forms of human tularemia may have a mortality rate exceeding 30%. Most research into Ft pathogenesis therefore focuses on understanding immune responses to a “Live Vaccine Strain” (LVS) of Ft. Herzenberg and her colleagues demonstrate that injecting wild-type mice with as little as 0.1 ng of Ft LVS lipopolysaccharide (Ft-LPS) is sufficient to generate a rapid antibody response. All wild-type mice pretreated with 100 ng (Ft-LPS) two days prior to challenge with 1000 CFU of the live virus survived the infection, while all wild-type mice pretreated with saline died (Fig 1a). These results showed that (Ft-LPS) confers protection against lethal Ft LVS infection in wild-type mice, effectively immunizing them against the pathogen. Subsequent tests showed that the glycolipod compound lipid A, purified from Ft-LPS, is not sufficient to induce the same protective response (Fig 1b). Herzenberg deduces that the core carbohydrate component of Ft-LPS, missing from lipid A, is essential for recognition of the antigen and induction of a protective immune response. Finally, Herzenberg demonstrates that uMT mutant mice, which lack mature B cells, are unable to mount a protective immune response against Francisella tularensis , even when pretreated with Ft-LPS (Fig 1c). She concludes that B cells are necessary for Ft-LPS immunization and protection against the live virus.
Taking this research a step further, Herzenberg shows that Ft-LPS immunization is sufficient to stimulate the production of antigen-specific B-1a cells in the spleen and Per C as soon as three days after the initial injection. The presence of the IgK light chain bound to each cell makes them easily detectable by Hi-D FACS analysis (Fig 2A). These cells rapidly proliferate and bind flurochrome-labeled Ft-LPS, as evidenced by concurrently increasing levels of Anti Ft-LPS in serum.
Herzenberg and her colleagues found that the magnitude of this antibody response was equivalent in wild-type, T-deficient, and Toll-like receptor (TLR4)-deficient mice. They conclude that Ft-LPS immunization is therefore both T-Cell independent and TLR-independent, and that the response is governed exclusively by B-1a lymphocytes. T-deficient mice do eventually succumb to the virus, however, suggesting that T-cells are nonetheless important for long-term survival of Ft infection.
Summary: Herzenberg concludes that antigen-specific B-1a antibody responses are required for the immune system to mount a protective response against a live vaccine strain of Francisella tularensis. The causative antigen is Ft-LPS, not Ft-lipid A. The protective antibody response is T-Cell and TLR-4 independent, but cannot persist when B cell development is disrupted.

I also found some cool (albeit somewhat dated) articles on other research applications for B1a cells, including their role in autoimmunity and the mechanisms of their development. Check them out if you like.

"Role of B1a cells in autoimmunity"
http://www.ncbi.nlm.nih.gov/pubmed/16890894

"CD11b expression distinguishes sequential stages of peritoneal B-1 development."
http://www.ncbi.nlm.nih.gov/pubmed/18375763

Medullary Dendritic Cells are Responsible for the Humoral Response to Influenza Virus

It has long been known that sinus-lining macrophages play an important role in limiting the spread of viruses to the body by the usual phagocytic mechanisms. However, it remained unknown whether or not these macrophages were also responsible for the virus-specific humoral response following infection. In other words, do the macrophages present viral antigens to B cells in the lymph nodes and thus induce the generation of viral-specific antibodies?

Researchers from the Program in Cellular and Molecular Medicine and the Immune Disease Institute at Children's Hospital Boston seeking to answer this question have discovered an interesting and unexpected result. Using a wide variety of approaches, their experiments suggest that while macrophages are important for limiting the systemic spread of viruses, they are not involved in generation of the humoral response after all. The cells that are responsible for this immunity are the medullary dendritic cells of the lymph nodes. They utilize the lectin receptor SIGN-R1 to capture lymph-borne influenza virus and promote humoral immunity.

As of yet, not very much is understood about these medullary DCs. However, studies such as this one are vital for the advancement of vaccine technology. Knowing which cells are responsible for which aspects of the immune response opens up the opportunity to target those cells in such a way as to create more effective and efficient vaccines.

Relevant scientific article:
Gonzalez, Santiago F., et. al. Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes. Nature Immunology 11, 427–434 (2010).
http://www.nature.com/ni/journal/v11/n5/abs/ni.1856.html#/

TB in Populations At Risk for HIV, Mexico

Image source: http://en.wikipedia.org/wiki/File:TB_in_sputum.png

Article: http://www.cdc.gov/eid/content/16/5/pdfs/757.pdf

In order to determine the rate of latent TB infection in Tijuana, Mexico (where there is both little routine TB screening and a rising epidemic of HIV infection) the University of California at San Diego School of Medicine and San Diego County Department of Health and Human Services alongside several universities and government agencies in Tijuana, Mexico conducted a study to examine latent TB in marginalized populations. They recruited from three at-risk populations for HIV-- injection drug users, female sex workers, and homeless persons-- and conducted risk assessment, rapid HIV testing, and TB screening.

The authors found that 57% of eligible recruits were positive for TB infection, 4.2% were HIV positive, and 2.2% were positive for both TB infection and HIV. 2 participants were diagnosed with active TB. From this data, the authors found that TB was exceptionally high in these marginalized populations (at risk for HIV infection), though actual HIV rates in these populations were lower than originally estimated. However, despite finding lower rates of HIV infection that expected, the rate of HIV infection in Tijuana is still higher than the rest of Baja California. From this data, the authors claim that if there is an increase in HIV infection in these populations that already show high rates of TB infection, the reactivation and spread of TB (due to compromised immunity) will increase more rapidly than TB control efforts will be able to contain.

This study shows that there is indeed a strong relationship between TB infection and risk factors for HIV infection. However, it seems as though these two epidemics are treated independently of one another in both prevention and treatment efforts. Both HIV and TB screening is insufficient in this region, though there is obvious room for overlap and coordination in conducting both screens simultaneously since they share common risk groups. Do you think there should be better coordination in screening and treatment across different diseases and infections, or should one singular disease that is deemed most critical (in this case, HIV) be the primary focus ?

Reading Summaries for Week 3 - Leonard Herzenberg

FACS innovation a view from Stanford

Leonard Herzenberg

The Fluorescence-Activated Cell-Sorter (FACS) sorts cell by putting a tiny electrical charge on a droplet carrying a desired cell, and then passes that through an electric field. Charged droplets move left or right, and fall into collection tubes down below. Uncharged droplets, which are empty or contain unwanted material, fall in to the waste collection vessel. Expansion on FACS capabilities have included the cloning of hybridoma cells, adding more fluorescence colors and developing the dual-laser machine, using ethidium monoazide (EMA) to identify dead cells, and improving the hardware and FACS software.

Genetics, FACS, Immunology, and Redox: A Tale of Two Lives Intertwined

Leonard Herzenberg, and Leonore Herzenberg

In this paper, Dr. Len and Lee Herzenberg detail their journey through academia, from their days at Caltech, to the Pasteur Institute in Paris, to the NIH, and finally to Stanford. They discuss some of the most influential researchers with whom they have cooperated. Caltech's environment fostered scientific exploration along the themes of genetics and somatic cells function. And at the Pasteur Institute in Paris, Len discovered that the galactoside-concentrating mechanism encoded by the permease gene in the LacZ operon is responsible for the autocatalytic increase in LacZ induction. These and other findings contributed to the discovery of the LacZ operon, which has laid the groundwork for modern molecular biology. At the NIH, Len discovered that adding pyruvate is sufficient for supporting clonal growth, and Lee worked on the characterization of enzymes in the histidine synthesis pathway in Salmonella. After moving to Stanford to join the burgeoning Genetics Department, Len and Lee studied mouse histocompatibility antigens to use as somatic cell markers in cultured cell lines, and Lee discovered a potential model for human Rh immunization during pregnancy. They then focused on the study of H-2 antigens and immunoglobulin allotypes, specifically a study of the genetics of the Ig heavy-chain (IgH) chromosome region. During this time, Len became increasingly aware of the need to characterize and isolate the different kinds of lymphocytes. Building on a machine constructed by a group at Los Alamos to sort cells by volume, Len added a fluorescence-detection system, and sought to measure the amount of fluorescence associated with individual cells and to sort cells according to this measure in addition to volume. He and his co-researchers titled the machine the Fluorescence-Activated Cell Sorter (FACS), perhaps the first ever biotech instrument, and used the machine to track the expression of genes encoding surface molecules that distinguish various kinds of lymphocytes and other cells. They made available to the scientific community a series of monoclonal reagents to mouse MHC and other cell-surface molecules. Since its first model, the FACS has been improved upon through the addition of cell surface markers and lasers, and through improvements in the software interface. One of the first applications of FACS in immunology further characterized the Ig isotype and allotype commitment of naive B cells and their memory (IgG+)-B-cell progeny. Len and Lee began to investigate B-cell subsets in the spleen, and continue to work on this today. They also study T-cell subsets in murine models and humans, and focus in particular on the loss of CD4 and CD8 in HIV, and the role of glutathione (GSH) in HIV.

Questions

Before EMA was introduced to indicate dead cells, how were dead cells identified? i.e. if dead cells caused significant false positives after fixation and permeabilization when using FACS, how did you previously compensate for that?

In what ways were you, as a scientist, affected by the McCarthyism that you so protested in the '50s?

By the way, so sorry about the delayed post!

Reading Summaries for Week 2 - Patricia P. Jones

The Invariant Chain of Murine Ia Antigens: Its Glycosylation, Abundance and Subcellular Localization
Ellen Sung and Patricia P. Jones

In this paper, Sung and Jones characterize the glycosylation, abundance, and subcellular localization of Ia antigen-associated invariant chain (Ii). By utilizing the antibiotic tunicamycin (inhibits the addition of N-linked carbohydrates to nascent polypeptides) and 2D polyacrylamide gel electrophoresis, they show that while there are various haplotypes of polymorphic chains, there appears to be only a single precursor molecule for Ii. They also show that Ii is likely to be glycosylated by two N-linked oligosaccharide units. In the second part of the study, they use various membrane extraction and immunoprecipitation procedures to show that Ii does not appear to be associated with any surface Ia antigens nor is it free in the cytosol, yet there does appear to be a pool of Ii chains within the cell that are not bound to Ia antigens. In their conclusions, they suggest the possibility that Ii is associated then only with intracellular polymorphic chains and raise the question: what exactly is the function of Ii?


Identification of an IFN-gamma Responsive Region in an Intron of the Invariant Chain Gene
Zhu A. Cao, Bethany B. Moore, David Quezada, Cheong-hee Chang, Patricia P. Jones

IFN-gamma is known to co-regulate the expression of MHC class II and invariant chain (Ii) in murine macrophages. In this paper, the researchers explore the mechanism by which IFN-gamma upregulates Ii mRNA. By using transient transfections of CAT reporter constructs, they were able to identify a 155-bp region (named Q) within the first intron of the Ii gene that contains an IFN-gamma responsive element. They further confirmed this by testing reporter constructs containing various mutations in gene promoter boxes--any mutation in the Q fragment eliminated IFN-gamma responsiveness. However, the Ii promoter region and upstream enhancer most likely work synergistically with fragment Q to mediate IFN-gamma induction of Ii. Sequence analysis of Q revealed homologous regions to motifs in MHC class II gene promoters and the Ii enhancer. It is likely then that expression of these various proteins is regulated by similar mechanisms.


Questions asked in class plus some:
If MHC haplotypes preferentially recognize and bind certain kinds of peptides that contain similar motifs, then how is it that all MHC haplotypes form a complex with the same invariant chain?

Why would nature put such stock in one single version of the invariant chain? What if there is a mutation? Have any mutations been reported/any research done involving mutated invariant chains?

How exactly does 2D-PAGE work?

How exactly does an intronic enhancer work?

What is the difference between N-linked and O-linked glycosylation (aside from the obvious, i.e. what physiological differences might occur)? Why choose to study N-linked instead of O-linked?

Feel free to add more in the comments!

Rise in oral cancer linked to HPV

http://www.bmj.com/cgi/content/extract/340/mar25_1/c1439
http://www.bmj.com/cgi/content/abstract/339/oct08_2/b3884
http://www.cancer.gov/cancertopics/hpv-oropharyngeal-cancer0507

The British Medical Journal indicated in a commentary that there has been a recent surge in oropharyngeal squamous cell carcinoma, which researchers believe may be due to the spread of human papillomavirus through oral sex. Apparently there has been a 22% increase in the number of cases in the U.S. between 1999 and 2006, a significant rise considering there was almost no increase between 1975 and 1999. Researchers are currently trying to determine just what treatment for oropharyngeal carcinoma is most effective and how this treatment might differ from treatments for other types of head and neck cancer.

As an added controversy, there is a vaccine available against HPV and yet the Harvard School of Public Health suggested in 2009 that vaccinating boys isn't cost effective, even though the link between oropharyngeal carcinoma and HPV has been know since at least 2007. This has left researchers wondering whether the sudden rise in this type of cancer "may alter the cost effectiveness of vaccinating boys before they become sexually active." Currently the CDC recommends that all 11 and 12 year old girls get vaccinated against HPV, so why not boys? Perhaps this study will compel the CDC to change their guidelines and include boys in their recommendations.