Estimated aortic stiffness is independently associated with cardiac baroreflex sensitivity in humans: role of ageing and habitual endurance exercise.

We hypothesised that differences in cardiac baroreflex sensitivity (BRS) would be independently associated with aortic stiffness and augmentation index (AI), clinical biomarkers of cardiovascular disease risk, among young sedentary and middle-aged/older sedentary and endurance-trained adults. A total of 36 healthy middle-aged/older (age 55-76 years, n=22 sedentary and n=14 endurance-trained) and 5 young sedentary (age 18-31 years) adults were included in a cross-sectional study. A subset of the middle-aged/older sedentary adults (n=12) completed an 8-week-aerobic exercise intervention. Invasive brachial artery blood pressure waveforms were used to compute spontaneous cardiac BRS (via sequence technique), estimated aortic pulse wave velocity (PWV) and AI (AI, via brachial-aortic transfer function and wave separation analysis). In the cross-sectional study, cardiac BRS was 71% lower in older compared with young sedentary adults (P<0.05), but only 40% lower in older adults who performed habitual endurance exercise (P=0.03). In a regression model that included age, sex, resting heart rate, mean arterial pressure (MAP), body mass index and maximal exercise oxygen uptake, estimated aortic PWV (ß±s.e.=-5.76±2.01, P=0.01) was the strongest predictor of BRS (model R(2)=0.59, P<0.001). The 8-week-exercise intervention improved BRS by 38% (P=0.04) and this change in BRS was associated with improved aortic PWV (r=-0.65, P=0.044, adjusted for changes in MAP). Age- and endurance-exercise-related differences in cardiac BRS are independently associated with corresponding alterations in aortic PWV among healthy adults, consistent with a mechanistic link between variations in the sensitivity of the baroreflex and aortic stiffness with age and exercise.

In vitro generation of mature, naive antigen-specific CD8(+) T cells with a single T-cell receptor by agonist selection.

Peripheral blood T cells transduced with a tumor-specific T-cell receptor (TCR) face problems of auto-reactivity and lack of efficacy caused by cross-pairing of exogenous and endogenous TCR chains, as well as short term in vivo survival due to activation and growth factor-induced differentiation. We here studied an alternative strategy for the efficient generation of naive CD8(+) T cells with a single TCR. TCR-transduced human postnatal thymus-derived and adult mobilized blood-derived hematopoietic progenitor cells (HPCs) were differentiated to CD4(+)CD8(+) double-positive T cells using OP9-Delta-like 1 (OP9-DL1) cultures. Addition of the agonist peptide induced double positive cells to cross-present the peptide, leading, in the absence of co-stimulation, to cell cycle arrest and differentiation into mature CD8(+) T cells. Comprehensive phenotypic, molecular and functional analysis revealed the generation of naive and resting CD8(+) T cells through a process similar to thymic positive selection. These mature T cells show a near complete inhibition of endogenous TCRA and TCRB rearrangements and express high levels of the introduced multimer-reactive TCR. Upon activation, specific cytokine production and efficient killing of tumor cells were induced. Using this strategy, large numbers of high-avidity tumor-specific naive T cells can be generated from readily available HPCs without TCR chain cross-pairing.

Immunotherapy with gene-modified T cells: limiting side effects provides new challenges.

Genetic tools have been developed to efficiently engineer T-cell specificity and enhance T-cell function. Chimeric antigen receptors (CAR) use the antibody variable segments to direct specificity against cell surface molecules. T-cell receptors (TCR) can redirect T cells to intracellular target proteins, fragments of which are presented in the peptide-binding groove of HLA molecules. A recent clinical trial with CAR-modified T cells redirected against the B-cell lineage antigen CD19 showed dramatic clinical benefit in chronic lymphocytic leukaemia patients. Similarly, impressive clinical responses were seen in melanoma and synovial cell carcinoma with TCR-modified T cells redirected against the melanocyte lineage antigen MART-1 and the testis-cancer antigen NY-ESO-1. However, on and off-target toxicity was associated with most of these clinical responses, and fatal complications have been observed in some patients treated with gene modified T cells. This review will discuss factors that might contribute to toxic side effects of therapy with gene modified T cells, and outline potential strategies to retain anticancer activity while reducing unwanted side effects.

Effects of sleep on the cardiovascular and thermoregulatory systems: a possible role for hypocretins.

Sleep influences the cardiovascular, endocrine, and thermoregulatory systems. Each of these systems may be affected by the activity of hypocretin (orexin)-producing neurons, which are involved in the etiology of narcolepsy. We examined sleep in male rats, either hypocretin neuron-ablated orexin/ataxin-3 transgenic (narcoleptic) rats or their wild-type littermates. We simultaneously monitored electroencephalographic and electromyographic activity, core body temperature, tail temperature, blood pressure, electrocardiographic activity, and locomotion. We analyzed the daily patterns of these variables, parsing sleep and circadian components and changes between states of sleep. We also analyzed the baroreceptor reflex. Our results show that while core temperature and heart rate are affected by both sleep and time of day, blood pressure is mostly affected by sleep. As expected, we found that both blood pressure and heart rate were acutely affected by sleep state transitions in both genotypes. Interestingly, hypocretin neuron-ablated rats have significantly lower systolic and diastolic blood pressure during all sleep stages (non-rapid eye movement, rapid eye movement) and while awake (quiet, active). Thus, while hypocretins are critical for the normal temporal structure of sleep and wakefulness, they also appear to be important in regulating baseline blood pressure and possibly in modulating the effects of sleep on blood pressure.

Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells.

Advances in tumour immunotherapy.

The clinical goal of tumour immunotherapy is to provide either active or passive immunity against malignancies by harnessing the immune system to target tumours. Although vaccination is an effective strategy to prevent infectious disease, it is less effective in the therapeutic setting for cancer treatment, which might be related to the low immunogenicity of tumour antigens and the reduced immunocompetence of cancer patients. Recent advances in technology have led to the development of passive immunotherapy approaches that utilize the unique specificity of antibodies and T cell receptors to target selected antigens on tumour cells. These approaches are likely to benefit patients and alter the way that clinicians treat malignant disease. In this article we review recent advances in the immunotherapy of cancer, focusing on new strategies to enhance the efficacy of passive immunotherapy with monoclonal antibodies and antigen-specific T cells.

Retroviral transfer of a dominant TCR prevents surface expression of a large proportion of the endogenous TCR repertoire in human T cells.

The latent membrane protein-2 (LMP2) of Epstein-Barr virus is a potential target for T-cell receptor (TCR) gene therapy of Hodgkin lymphoma and nasopharyngeal carcinoma. Here, we modified a human leukocyte antigen-A2-restricted, LMP2-specific TCR to achieve efficient expression following retroviral TCR gene transfer. The unmodified TCR was poorly expressed in primary human T cells, suggesting that it competed inefficiently with endogenous TCR chains for cell surface expression. In order to improve this TCR, we replaced the human constant region with murine sequences, linked the two TCR genes using a self-cleaving 2A sequence and finally, codon optimized the TCR-alpha-2A-beta cassette for efficient translation in human cells. Retroviral transfer of the modified TCR resulted in efficient surface expression and HLA-A2/LMP2 pentamer binding. The transduced cells showed peptide-specific interferon-gamma and interleukin-2 production and killed target cells displaying the LMP2 peptide. Importantly, the introduced LMP2-TCR suppressed the cell surface expression of a large proportion of endogenous TCR combinations present in primary human T cells. The design of dominant TCR is likely to improve TCR gene therapy by reducing the risk of potential autoreactivity of endogenous and mispaired TCR combinations.

Antigen-specific cellular immunotherapy of leukemia.

Advances in cellular and molecular immunology have led to the characterization of leukemia-specific T-cell antigens and to the development of strategies for effective augmentation of T-cell immunity in leukemia patients. While several leukemia-related antigens have been identified, this review focuses on the Wilms' tumor 1 (WT1) antigen and the proteinase 3 (Pr3) antigen that are overexpressed in leukemic cells and are already being used in the clinical setting. Moreover, WT1 is also overexpressed in a vast number of nonhematological solid tumors, thereby expanding its use as a promising target for cancer vaccines. Examples of spontaneous immune responses against WT1 and Pr3 in leukemia patients are presented and the potential of WT1 and Pr3 for adoptive T-cell immunotherapy of leukemia is discussed. We also elaborate on the use of professional antigen-presenting cells loaded with mRNA encoding WT1 exploiting the advantage of broad HLA coverage for therapeutic vaccination purposes. Finally, the summarized data underscore the potential of WT1 for the manipulation of T-cell immunity in leukemia and in cancer in general, that will likely pave the way for the development of more effective and generic cancer vaccines.

Wilms' tumour gene 1 (WT1) in human neoplasia.

The transcription factor Wilms' tumour gene 1 (WT1) is important as a prognostic marker as well as in the detection and monitoring of minimal residual disease in leukaemia and myelodysplastic syndromes. Evidence has accumulated over the past decade to show that WT1 is a key molecule for tumour proliferation in a large number of human neoplasms most prominent in acute leukaemias, making it a suitable target for therapeutic strategies. Based on animal results, showing safety and efficacy of immunization with WT1 peptides and protein, early clinical trials in leukaemia have recently been initiated. The First International Conference on WT1 in Human Neoplasia was held in Berlin, March 11--12, 2004. This report reviews the current knowledge on the role of WT1 in tumour promotion and as a diagnostic and therapeutic target, and summarizes the data presented and discussed in this meeting.

Exploiting T cell receptor genes for cancer immunotherapy.

Adoptive antigen-specific immunotherapy is an attractive concept for the treatment of cancer because it does not require immunocompetence of patients, and the specificity of transferred lymphocytes can be targeted against tumour-associated antigens that are poorly immunogenic and thus fail to effectively trigger autologous T cell responses. As the isolation and in vitro expansion of antigen-specific lymphocytes is difficult, 'conventional' adoptive T cell therapy can only be carried out in specialized centres in small numbers of patients. However, T cell receptor (TCR) genes isolated from antigen-specific T cells can be exploited as generic therapeutic molecules for 'unconventional' antigen-specific immunotherapy. Retroviral TCR gene transfer into patient T cells can readily produce populations of antigen-specific lymphocytes after a single round of polyclonal T cell stimulation. TCR gene modified lymphocytes are functionally competent in vitro, and can have therapeutic efficacy in murine models in vivo. TCR gene expression is stable and modified lymphocytes can develop into memory T cells. Introduction of TCR genes into CD8(+) and CD4(+) lymphocytes provides an opportunity to use the same TCR specificity to produce antigen-specific killer and helper T lymphocytes. Thus, TCR gene therapy provides an attractive strategy to develop antigen-specific immunotherapy with autologous lymphocytes as a generic treatment option.

WT1-targeted immunotherapy of leukaemia.

Since malignant cells are derived from normal cells, many tumour-associated antigens are also expressed in normal tissues. For examples, WT1 is expressed at elevated levels in most leukaemias, but it is also expressed at reduced levels in normal CD34+ haematopoietic stem cells and in progenitor cells of other tissues. Antigen expression in normal tissues is likely to trigger immunological tolerance and thus blunt T cell responses. This could explain the observation that WT1 vaccination in mice frequently fails to stimulate high avidity cytotoxic T cell responses. In order to circumvent tolerance, we have isolated from HLA-A2-negative donors high avidity CTL specific for HLA-A2-presented peptide epitopes of WT1. These allorestricted CTL efficiently kill HLA-A2-positive leukaemia cells but not normal CD34+ haematopoietic stem cells. However, adoptive cellular therapy with allorestricted CTL could only be performed in leukaemia patients rendered tolerant to the infused CTL by prior allogeneic stem cell transplantation. In order to circumvent this limitation, we propose to exploit the TCR of allorestricted CTL as therapeutic tool. TCR gene transfer can be used to take advantage of the specificity of allorestricted CTL and transfer it to patient CTL, while avoiding the transfer of immunogenic alloantigens from the donor CTL to the patient.

Use of the allogeneic TCR repertoire to enhance anti-tumor immunity.

It is well established that antigen-specific T lymphocytes can inhibit tumor growth in humans and in mice, leading to complete tumor elimination in some cases. However, in many cases T cell immunity is unable to successfully control tumor progression. Since tumors are derived from normal tissues, most antigens are shared with normal tissues, although expression levels are usually elevated in malignant cells. Nevertheless, low-level expression in normal cells can be sufficient to render autologous T cells tolerant and thus unable to mount effective immune responses against tumors. Here, we review how allogeneic T cells can be used to isolate T cells that effectively recognise and kill tumor cells, but not normal cells with low level of antigen expression. The TCR of allogeneic T cells can be introduced into patient T cells to equip them with anti-tumor specificity that may not be present in the autologous T cell repertoire.

Prospects for immunotherapy of malignant disease.

The majority of T cell-recognized tumour antigens in humans are encoded by genes that are also present in normal tissues. Low levels of gene expression in normal cells can lead to the inactivation of high-avidity T cells by immunological tolerance mechanisms. As a consequence, low-avidity T cell responses in patients are often inadequate in providing tumour protection. Recently, several technologies have been developed to overcome tolerance, allowing the isolation of high-affinity, HLA-restricted receptors specific for tumour-associated peptide epitopes. Furthermore, transfer of HLA-restricted antigen receptors provides an opportunity to empower patient T cells with new tumour-reactive specificities that cannot be retrieved from the autologous T cell repertoire.

Cardiovascular responses to angiotensin II in atherosclerosis.

Atherosclerosis is associated with increased angiotensin II AT1-receptor expression and vascular hyperresponsiveness to angiotensin II. Nevertheless, atherosclerosis is often not accompanied by hypertension. We studied if the hypertensive effect of angiotensin II is more pronounced in atherosclerosis. Rabbits were fed a high-cholesterol diet (n = 10) for 12 weeks, followed by a standard diet for another 6 weeks. Control animals received the standard diet (n = 8) for 18 weeks. After 18 weeks, haemodynamic measurements were performed during a baseline recording and during an intravenous infusion of angiotensin II (0.4 microg kg-1 min-1). Atherosclerosis in the high-cholesterol group was verified by histological and lipidchemical tissue examinations. During angiotensin II infusion, total peripheral resistance (TPR) increased more in the high-cholesterol group than in controls (+81.6 +/- 12.4 vs. +40.6 +/- 9.7 mmHg min L-1, P < 0.05). While cardiac output and stroke volume (SV) decreased more in the high-cholesterol group (P < 0.05), reflex bradycardia was stronger in the control group (P < 0.05), indicating a reduced baroreceptor reflex sensitivity in atherosclerosis. Despite the larger increase in TPR and the reduced baroreceptor reflex sensitivity in the high-cholesterol group, maximum blood pressure response to angiotensin II was similar in both groups. The lack of a greater blood pressure response to angiotensin II in the high-cholesterol group could be the result of the early stages of heart failure. Under resting conditions, heart failure seems to be fully compensated, as baseline haemodynamic parameters were similar in the high-cholesterol group and in controls. However, during angiotensin II infusion, the compensatory mechanisms do not prevent a stronger fall in cardiac output and SV. Therefore, the blood pressure response to angiotensin II is not exaggerated in atherosclerotic animals, as vascular hyperresponsiveness to angiotensin II is opposed by the stronger fall in cardiac output and SV.

The CD68 protein as a potential target for leukaemia-reactive CTL.

CD68, a haematopoietic differentiation marker of the monocyte-macrophage lineage, is expressed in various human malignancies including chronic and acute myeloid leukaemia (AML). While the majority of normal CD34(+) cells are negative for CD68 expression, CD34(+) cells from AML patients produce elevated amounts of this protein. The purpose of this study was to identify CTL epitopes in the human CD68 protein. Mouse CD68 was also analysed to search for epitopes that could be used in murine tumor model. Peptides binding to murine H2(b) class I molecules were identified and used to stimulate CTL responses from allogeneic donor mice to avoid immunological tolerance. High avidity CTL clones specific for three different peptide epitopes did not kill CD68-expressing murine target cells, indicating that endogenous antigen processing failed to produce sufficient amounts of these peptides. In contrast, allo-restricted human CTL specific for an HLA-A2-binding peptide of CD68 recognised not only picomolar concentrations of peptide, but also displayed low levels of killing against HLA-A2-positive K562 and THP-1 leukemia cell lines and blast cells from AML patients. These data suggest that human leukaemia cells express limited amounts of CD68-derived peptides, and that high avidity CTL capable of recognising sub-picomolar concentrations of peptides are required for efficient killing of leukaemia cells.

Sympathetic modulation of blood pressure variability.

Although sympathetic nervous activity (SNA) displays oscillations synchronous with the heart beat and respiration, and between 0.1-0.4 Hz, it is apparent that each of these frequencies does not have the same effect on the vasculature. Frequencies above 1 Hz do not produce oscillations in the vasculature but instead contribute to the mean level of vasoconstriction. Slower oscillations in SNA result in a cycle of vasoconstriction and vasodilation within the vasculature, the amplitude of which, generally decreases with increasing frequency. Some studies indicate that, within the same species, differences exist in the frequency responses between vascular beds, such as the skin and gut. This differential responsiveness is also found between the medullary and cortical vasculature regions of the rabbit kidney. Low-pass filter properties have been described in the iliac circulation of rats, and evidence has been provided that noradrenaline reuptake mechanisms are not the frequency limiting step of the vasculature response. Recent studies on isolated rat vascular smooth muscle cells suggest that sympathetic modulation of vascular tone is limited by the alpha-adrenoceptor signal transduction into the cells and not by an intrinsic inability of the cells to contract and relax at higher rates.

Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer.

We identified a tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the widely expressed human MDM2 oncoprotein and were able to bypass self-tolerance to this tumor antigen in HLA-A*0201 (A2.1) transgenic mice and by generating A2.1-negative, allo-A2.1-restricted human T lymphocytes. A broad range of malignant, as opposed to nontransformed cells, were killed by high-avidity transgenic mouse and allogeneic human CTLs specific for the A2.1-presented MDM2 epitope. Whereas the self-A2.1-restricted human T cell repertoire gave rise only to low-avidity CTLs unable to recognize the natural MDM2 peptide, human A2.1+ T lymphocytes were turned into efficient MDM2-specific CTLs upon expression of wild-type and partially humanized high-affinity T cell antigen receptor (TCR) genes derived from the transgenic mice. These results demonstrate that TCR gene transfer can be used to circumvent self-tolerance of autologous T lymphocytes to universal tumor antigens and thus provide the basis for a TCR gene transfer-based broad-spectrum immunotherapy of malignant disease.

Separation of antithrombin III variants by micellar electrokinetic chromatography.

The characterisation of proteins is still one of the most challenging analytical tasks in modern bioanalysis. Due to the complex structure of proteins, several analytical techniques are often required to get sufficient information. Antithrombin III (AT III), a high-molecular-mass plasma glycoprotein which is an important protease inhibitor and the main modulator of thrombin activity, circulates in plasma in two isoforms, the so-called AT III-alpha (90-95%) and -beta (5-10%). Micellar electrokinetic chromatography was used to analytically separate these AT III variants, which differ in their affinity to the polysaccharide heparin.