Quantifying selection in immune receptor repertoires

The efficient recognition of pathogens by the adaptive immune system relies on the diversity of receptors displayed at the surface of immune cells. T-cell receptor diversity results from an initial random DNA editing process, called VDJ recombination, followed by functional selection of cells according to the interaction of their surface receptors with self and foreign antigenic peptides. Using high-throughput sequence data from the β-chain of human T-cell receptors, we infer factors that quantify the overall effect of selection on the elements of receptor sequence composition: the V and J gene choice and the length and amino acid composition of the variable region. We find a significant correlation between biases induced by VDJ recombination and our inferred selection factors together with a reduction of diversity during selection. Both effects suggest that natural selection acting on the recombination process has anticipated the selection pressures experienced during somatic evolution. The inferred selection factors differ little between donors or between naive and memory repertoires. The number of sequences shared between donors is well-predicted by our model, indicating a stochastic origin of such public sequences. Our approach is based on a probabilistic maximum likelihood method, which is necessary to disentangle the effects of selection from biases inherent in the recombination process.The T-cell response of the adaptive immune system begins when receptor proteins on the surface of these cells recognize a pathogen peptide displayed by an antigen-presenting cell. The immune cell repertoire of a given individual is comprised of many clones, each with a distinct surface receptor. This diversity, which is central to the ability of the immune system to defeat pathogens, is initially created by a stochastic process of germline DNA editing (called VDJ recombination) that gives each new immune cell a unique surface receptor gene. This initial repertoire is subsequently modified by selective forces, including nonpathogen-related thymic selection against excessive (or insufficient) recognition of self proteins, which are also stochastic in nature. Because of this stochasticity and the large T-cell diversity, these repertoires are best described by probability distributions. In this paper, we apply a probabilistic approach to sequence data to obtain quantitative measures of the overall (not necessarily pathogenic) selection pressures that shape T-cell receptor repertoires.New receptor genes are formed by randomly choosing alleles from a set of genomic templates for the subregions (V, D, and J) of the complete gene. Insertion and deletion of nucleotides in the junctional regions between the V and D and D and J genes greatly enhance diversity beyond pure VDJ combinatorics (1). The most variable region of the gene is between the last amino acids of the V segment and the beginning of the J segment; it codes for the Complementarity Determining Region 3 (CDR3) loop of the receptor protein, a region known to be functionally important in recognition (2). Previous studies have shown that immune cell receptors are not uniform in terms of VDJ gene segment use (3–6) or probability of generation (1) and that certain receptors are more likely than others to be shared by different individuals (4, 7). The statistical properties of the immune repertoire are, thus, rather complex, and their accurate determination requires sophisticated methods.Recent advances in sequencing technology have made it possible to sample the T-cell receptor diversity of individual subjects in great depth (8). The availability of such data has, in turn, led to the development of sequence statistics-based approaches to the study of immune cell diversity (9, 10). In particular, we recently quantitatively characterized the preselection diversity of the human T-cell repertoire by learning the probabilistic rules of VDJ recombination from out-of-frame DNA sequences that cannot be subject to functional selection and whose statistics therefore reflect only the recombination process (1). After generation, T cells undergo a somatic selection process in the thymus (11) and later in the periphery (12). Cells that pass thymic selection enter the peripheral repertoire as naive T cells, and the subset of naive cells that eventually engage in an immune response will survive as a long-lived memory pool. Although we now understand the statistical properties of the initial repertoire of immune receptors (1) and despite some theoretical studies of thymic selection at the molecular level (13, 14), a quantitative understanding of how selection modifies those statistics to produce the naive and memory repertoires is lacking.In this paper, we build on our understanding of the preselection distribution of T-cell receptors to derive a statistical method for identifying and quantifying selection pressures in the adaptive immune system. We apply this method to naive and memory DNA sequences of human T-cell β-chains obtained from peripheral blood samples of nine healthy individuals. Our goal is to characterize the likelihood that any given sequence, after it is generated, will survive selection for the ensemble of properties needed to pass into the peripheral repertoire(s). Our analysis reveals strong and reproducible signatures of selection on specific amino acids in the CDR3 sequence and on the usage of V and J genes. Most strikingly, we find significant correlation between the generation probability of a sequence and the probability that it will pass selection. This correlation suggests that natural selection, which acts on very long timescales to shape the generation mechanism itself, may have tuned it to anticipate somatic selection, which acts on single cells throughout the lifetime of an individual. The quantitative features of selection inferred from our model vary very little between donors, indicating that these features are universal. In addition, our measures of selection pressure on the memory and naive repertoires are statistically indistinguishable, consistent with the hypothesis that the memory pool is a random subsample of the naive pool.     

BetaAPP and furin mRNA concentrates in immature senile plaques in the brain of Alzheimer patients

This study examined the possibility that in Alzheimer disease (AD) beta-amyloid precursor protein (betaAPP) mRNA is delivered to senile plaques (SPs) via dendritic processes. BetaAPP mRNA was detected in SPs by in situ hybridization, using a 1.4-kb cRNA in which both [35S]-UTP and [35S]-CTP were incorporated together. The betaAPP mRNA was compared with that of furin, a proteolytic enzyme putatively involved in betaAPP processing, and its orthologue proprotein convertase PCI served as a control. Human presenile AD cases with mostly immature SPs and AD cases generally with mature SPs were analyzed. To decrypt SPs after hybridization, brain sections were stained with thioflavin S. To establish relationships between the density of dystrophic fibers, the degree of plaque maturation, and the concentration of mRNA in SPs, the plaque maturity markers Abeta(1-42) and Abeta(1-40) peptides were co-localized with neurofilament protein 200 and compared with microtubule-associated protein 2 (MAP 2). The results suggest that immature, Abeta(1-42)- and dystrophic dendrite-containing SPs (but not mature SPs containing Abeta(1-40) and missing dystrophic dendrites) are capable of concentrating specific mRNAs. Dystrophic dendrites may thus serve as a route for the transport of specific mRNAs from the cell bodies to SPs.     

Comparison of three models of neuropathic pain in mice using a new method to assess cold allodynia: The double plate technique

The recent identification of receptors sensitive to cold stimuli increased the significance of using mice to study cold allodynia, one of the important features of neuropathic pain. However, commonly used techniques (simple cold plate and acetone technique) may be inappropriate to study cold allodynia in mice because of problems of interpretation. We have developed a new method for assessing aversion to a cold non-noxious stimulus. It consists of calculating the time that mice spend on a non-noxious cold plate during their explorative behavior versus a thermoneutral one. We used three different models of neuropathic pain: chronic constriction injury of the sciatic nerve (CCI), partial sciatic nerve ligation (PSL) and chronic constriction of the saphenous nerve (CCS) with their respective sham groups and naive animals to assess the double plate in comparison to the acetone drop technique. All operated mice displayed cold allodynia with both methods. The response to acetone and the time spent on the cold plate were correlated (r = −0.93) and we also showed that the CCI mice were more sensitive to cold. Pharmacological validation of this technique showed that CCI induced cold allodynia was alleviated by gabapentin. In conclusion, the double plate technique provides a new, relevant method for assessing cold allodynia in mice. The advantages and drawbacks with the other techniques are discussed.     

Advanced cancer patients’ and caregivers’ use of a Question Prompt List

Objective

The objective of this study was to provide insight into how advanced cancer patients and their caregivers use a Question Prompt List (QPL) during a consultation and for preparation for future consultations.

Methods

Audiotaped consultations and follow-up phone calls of 28 advanced cancer patients were coded and content analyzed. Questions asked and concerns expressed in consultations were coded for initiator, content, inclusion in the QPL and exact wording. Patients’ reported and future use of the QPL were coded from the phone calls.

Results

The majority of patients reported that they used the QPL. Questions asked by patients and caregivers predominately coincided with questions from the prognosis section of the QPL. Questions were rarely asked literally from the QPL, instead questions were tailored to patients’ own circumstances.

Conclusion

QPLs are useful to stimulate discussion on prognosis among advanced cancer patients and caregivers. Patients tailored questions from the QPL to their own circumstances which may suggest high involvement and engagement. The development of more specific tailored communication interventions for advanced cancer patients is warranted.

Practice implications

Implementation of QPLs in the advanced cancer setting may be beneficial for patients, caregivers and healthcare providers to facilitate discussion of topics such as prognosis.     

An Exception to Tumour Neoangiogenesis in a Malignant Breast‐Lesion

Abstract: Magnetic resonance‐mammography is regarded as the most sensitive diagnostic modality in the detection of breast cancer. It uses the tumour neoangiogenesis to depict lesions after intravenous contrast agent injection. It is said, that for tumours exceeding a diameter of three millimetres contrast agent enhancement is mandatory. In our case report we describe a rare tumour growth condition. We observed a large invasive carcinoma (18 millimetres diameter) without contrast enhancement in breast MRI due to an almost missing tumour neoangiogenesis. The cancer had a low cellularity and a strong desmoplastic reaction.     

A two year observation of the process of applying recombinant IGF-1 to treat short stature in children with primary IGF-1 deficiency -- case reports of 3 patients

Growth deficiency is one of the most frequent causes of referral to Endocrinology Outpatient Clinic. IGF-1 (insulin-like growth factor 1) deficiency is one of the rarest causes of short stature. In 2009 in Poland a therapeutic programme was set up for children with severe primary IGF-1 deficiency. The authors present the data of three first polish patients qualified for the rhIGF-1 (recombinant human insulin-like growth factor 1) - mecasermin. The authors conclude that the treatment with rhIGF-1 significantly improves growth velocity in patients with IGF-1 deficiency. During two years of mecasermin treatment no serious side effects were noted.     

Combined assessment of 3q26 amplification and promoter methylation in patients with high grade cervical lesions show age specific differences

A considerable proportion of high grade cervical intraepithelial lesions (CIN2/3) are known to resolve on their own especially among young women. However, since reliable prognostic markers are still lacking, the diagnosis “CIN3” is still an indication for surgery which may result in overtreatment. It is conceivable that a combination of different, ideally independent molecular markers may provide more reliable results. In the present cross‐sectional study two established triage markers, 3q26 amplification and a methylation signature, were evaluated in an age‐dependent manner. The patient cohort comprised 60 patients with histologically confirmed CIN2/3 in two equally sized age groups (<30 years, ≥30 years). Cervical scrapes were analyzed by interphase fluorescence in situ hybridization for 3q26 amplification and methylation specific PCR (GynTect®) for six different genome regions. Both assays showed a significantly different pattern of test outcome independent of age (P = .001). Moreover, the combination of both assays differed significantly for double positive and double negative cases when comparing the two age groups: In patients <30 years there were clearly less cases with positive methylation signature and amplification of 3q26 as in women ≥30 years (23% vs 63%, Bonferroni adjusted P = .016). Of particular interest is the finding that double negative results were exclusive for the young age group (0% vs 27%, Bonferroni adjusted P = .020). Since regression of CIN2/3 characteristically occurs among young women it is tempting to speculate that a double negative test result could be prognostic for regression of CIN2/3. This will have to be investigated further in a prospective longitudinal intervention study.     

A phase II trial of temozolomide and IFN-alpha in patients with advanced renal cell carcinoma.

The combination of temozolomide (TEM) and interferon-alpha (IFN-alpha) previously demonstrated a 30% response rate in metastatic melanoma. A single institution, phase II trial evaluating the efficacy of TEM/IFN in patients with advanced renal cell carcinoma (RCC) was conducted. Safety and tumor response were the main outcomes. Eligible patients received 200 mg/m(2)/day TEM orally on days 1-5 every 28 days, with IFN 2.5 million U/m(2)/day subcutaneously (s.c.) three alternate days/week for days 1-15 first cycle, then 5 million U/m(2)/day s.c. 3 alternate days/week throughout each 28-day cycle. Efficacy was evaluated every 8 weeks, and dose-limiting toxicities (DLTs) were treated with dose reductions of the culprit drug. Sixteen patients (ages 37-67) were initially enrolled. Of the 14 evaluable patients, there was one minor response. Best response was stable disease, with 7 patients remaining on study for > or =6 months. Five were alive for more than 2 years, and 2 remain alive at 45 and 50 months after enrollment. DLTs included TEM-induced myelosuppression and IFN-induced fever/chills. Other toxicities were mild to moderate (grades 1-3). The combination of TEM/IFN proved quite tolerable. This regimen appears inactive in terms of response in this population with poor prognosis, but the patients with stable disease > or =6 months remain of interest.