Armin A. Weiser

Expression levels of the putative zinc transporter LIV-1 are associated with a better outcome of breast cancer patients.

We investigated the expression pattern of the breast cancer associated gene LIV‐1 on mRNA and protein level in 111 human breast cancer patients by in situ hybridization as well as immunohistochemistry and focused on the unknown potential of LIV‐1 expression levels as a prognostic marker. To our knowledge, this is the first study on endogenous LIV‐1 protein expression. Results of our study indicate that LIV‐1 mRNA and protein expression levels are only weakly correlated, suggesting posttranscriptional regulatory mechanisms. Furthermore, LIV‐1 mRNA quantity in combination with a positive ER status seem to represent a better marker than the progesterone receptor status according to the prognostic significance for relapse free survival (RFS). A negative correlation of LIV‐1 protein levels with tumor size, grade and stage reflects an association of LIV‐1 protein expression with less aggressive tumors. High LIV‐1 protein expression seems to be associated with a longer relapse free and overall survival in breast cancer patients with invasive ductal carcinoma. This association, however, seems to be dependent from other prognostic markers. Our data suggest that LIV‐1 is a promising candidate for a novel marker for breast cancer patients with better outcome. Furthermore, our study presents a revised cDNA sequence of LIV‐1 and demonstrates the localization of endogenous LIV‐1 in the endoplasmic reticulum. (Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience. wiley.com/jpages/0020‐7136/suppmat/index.html).

Recirculation of germinal center B cells: a multilevel selection strategy for antibody maturation.

Summary:  Optimization of antibody affinity is a hallmark of the humoral immune response. It takes place in hundreds of transient microstructures called germinal centers (GCs). Their function and time‐dependent behavior are subjects of active investigation. According to a generally accepted notion, their individual kinetics follows the average kinetics of all GCs present in the observed lymphatic tissue. In this review, we challenge this view and point out, with the help of mathematical simulations, that inferring the kinetics of individual GCs from cross‐sectional evaluation of GC kinetics is virtually impossible. Thus, the time course of individual GCs is open to conjecture. For instance, one possible interpretation is that GCs exist for a time span considerably shorter than that of the observed average kinetics. We explore the implications of different temporal organizations of GCs in the light of the hypothesis that GC B‐cell emigrants recolonize GC niches. This assumption leads to a view where GCs work in parallel but are linked by recirculation of B‐cell emigrants. In this view, interleaved global and local competition provide for an implementation of multiple levels of B‐cell selection in affinity maturation. The concepts of iteration, all‐or‐none behavior, and phasic mutation schedule are discussed in the light of this hypothesis.

Is There a Typical Germinal Center? A Large-Scale Immunohistological Study on the Cellular Composition of Germinal Centers during the Hapten-Carrier–Driven Primary Immune Response in Mice

Germinal centers (GCs) are complex, multicell-type, transient structures that form in secondary lymphatic tissues in response to T cell-dependent stimulation. This process is crucial to the adaptive immune response because it is the source of affinity maturation and long-lived B cell memory. Our previous studies showed that the growth of murine splenic GCs is nonsynchronized, involving broad-volume distributions of individual GCs at any time. This raises the question whether such a thing as a typical GC exists. To address this matter, we acquired large-scale confocal data on GCs throughout the course of the 2-phenyl-5-oxazolone chicken serum albumin-driven primary immune response in BALB/c mice. Semiautomated image analysis of 3457 GC sections revealed that, although there is no typical GC in terms of size, GCs have a typical cellular composition in that the cell ratios of resident T cells, macrophages, proliferating cells, and apoptotic nuclei are maintained during the established phase of the response. Moreover, our data provide evidence that the dark zone (DZ) and light zone (LZ) compartments of GCs are about the same size and led us to estimate that the minimal cell loss rate in GCs is 3% per hour. Furthermore, we found that the population of GC macrophages is larger and more heterogeneous than previously thought, and that despite enrichment of T cells in the LZ, the DZ of murine splenic GCs is not poor in T cells. DZ and LZ differ in the T cell-to-macrophage ratio rather than in the density of T cells.

Broad Volume Distributions Indicate Nonsynchronized Growth and Suggest Sudden Collapses of Germinal Center B Cell Populations

Immunization with a T cell-dependent Ag leads to the formation of several hundred germinal centers (GCs) within secondary lymphoid organs, a key process in the maturation of the immune response. Although prevailing perceptions about affinity maturation intuitively assume simultaneous seeding, growth, and decay of GCs, our previous mathematical simulations led us to hypothesize that their growth might be nonsynchronized. To investigate this, we performed computer-aided three-dimensional reconstructions of splenic GCs to measure size distributions at consecutive time points following immunization of BALB/c mice with a conjugate of 2-phenyl-oxazolone and chicken serum albumin. Our analysis reveals a broad volume distribution of GCs, indicating that individual GCs certainly do not obey the average time course of the GC volumes and that their growth is nonsynchronized. To address the cause and implications of this behavior, we compared our empirical data with simulations of a stochastic mathematical model that allows for frequent and sudden collapses of GCs. Strikingly, this model succeeds in reproducing the empirical average kinetics of GC volumes as well as the underlying broad size distributions. Possible causes of GC B cell population collapses are discussed in the context of the affinity-maturation process.

Affinity maturation of B cells involves not only a few but a whole spectrum of relevant mutations

Affinity maturation of B lymphocytes within germinal centers involves both diversification of their B-cell receptors (BCRs) by somatic hypermutation (SHM) and a crucial receptor-mediated selection step. However, in contrast to recent advances in revealing the molecular mechanism of SHM, the fundamentals of the selection process are still poorly understood, i.e. it is often not clear how and how many mutations contribute to improving a BCR during the response against a given antigen. A general drawback in assessing the mutations relevant to the selection process is the difficult task of rating the relative contributions of selection and intrinsic biases to the experimentally observed mutation patterns of BCRs. The approach proposed here is premised on statistical comparison of the frequency distributions of nucleotide substitutions as observed in datasets of hypermutated BCRs against their frequency distribution expected under the null hypothesis of no selection. Thereby, we show that the spectrum of mutations relevant to maturation of canonical anti-(4-hydroxy-3-nitrophenyl)acetyl BCRs is much broader than previously acknowledged, going beyond the scope of single key mutations. Moreover, our results suggest that maturation not only involves selection by means of affinity but likewise expression and stabilization of BCRs.

Mapping discontinuous protein-binding sites via structure-based peptide libraries: combining in silico and in vitro approaches

To perform their various functions, protein surfaces often have to interact with each other in a specific way. Usually, only parts of a protein are accessible and can act as binding sites. Because proteins consist of polypeptide chains that fold into complex three‐dimensional shapes, binding sites can be divided into two different types: linear sites that follow the primary amino acid sequence and discontinuous binding sites, which are made up of short peptide fragments that are adjacent in spatial proximity. Such discontinuous binding sites dominate protein–protein interactions, but are difficult to identify.