Antibody-epitope conjugates deliver immunogenic T-cell epitopes more efficiently when close to cell surfaces.

Antibody-mediated delivery of immunogenic viral CD8+ T-cell epitopes to redirect virus-specific T cells toward cancer cells is a promising new therapeutic avenue to increase the immunogenicity of tumors. Multiple strategies for viral epitope delivery have been shown to be effective. So far, most of these have relied on a free C-terminus of the immunogenic epitope for extracellular delivery. Here, we demonstrate that antibody-epitope conjugates (AECs) with genetically fused epitopes to the N-terminus of the antibody can also sensitize tumors for attack by virus-specific CD8+ T cells. AECs carrying epitopes genetically fused at the N-terminus of the light chains of cetuximab and trastuzumab demonstrate an even more efficient delivery of the T-cell epitopes compared to AECs with the epitope fused to the C-terminus of the heavy chain. We demonstrate that this increased efficiency is not caused by the shift in location of the cleavage site from the N- to the C-terminus, but by its increased proximity to the cell surface. We hypothesize that this facilitates more efficient epitope delivery. These findings not only provide additional insights into the mechanism of action of AECs but also broaden the possibilities for genetically fused AECs as an avenue for the redirection of multiple virus-specific T cells toward tumors.

Immunoglobulin G4: an odd antibody.

Despite its well-known association with IgE-mediated allergy, IgG4 antibodies still have several poorly understood characteristics. IgG4 is a very dynamic antibody: the antibody is involved in a continuous process of half-molecules (i.e. a heavy and attached light-chain) exchange. This process, also referred to as 'Fab-arm exchange', results usually in asymmetric antibodies with two different antigen-combining sites. While these antibodies are hetero- bivalent, they will behave as monovalent antibodies in most situations. Another aspect of IgG4, still poorly understood, is its tendency to mimic IgG rheumatoid factor (RF) activity by interacting with IgG on a solid support. In contrast to conventional RF, which binds via its variable domains, the activity of IgG4 is located in its constant domains. This is potentially a source of false positives in IgG4 antibody assay results. Because regulation of IgG4 production is dependent on help by T-helper type 2 (Th2) cells, the IgG4 response is largely restricted to non-microbial antigens. This Th2-dependency associates the IgG4 and IgE responses. Another typical feature in the immune regulation of IgG4 is its tendency to appear only after prolonged immunization. In the context of IgE-mediated allergy, the appearance of IgG4 antibodies is usually associated with a decrease in symptoms. This is likely to be due, at least in part, to an allergen-blocking effect at the mast cell level and/or at the level of the antigen-presenting cell (preventing IgE-facilitated activation of T cells). In addition, the favourable association reflects the enhanced production of IL-10 and other anti-inflammatory cytokines, which drive the production of IgG4. While in general, IgG4 is being associated with non-activating characteristics, in some situations IgG4 antibodies have an association with pathology. Two striking examples are pemphigoid diseases and sclerosing diseases such as autoimmune pancreatitis. The mechanistic basis for the association of IgG4 with these diseases is still enigmatic. However, the association with sclerosing diseases may reflect an excessive production of anti-inflammatory cytokines triggering an overwhelming expansion of IgG4-producing plasma cells. The bottom line for allergy diagnosis: IgG4 by itself is unlikely to be a cause of allergic symptoms. In general, the presence of allergen-specific IgG4 indicates that anti-inflammatory, tolerance-inducing mechanisms have been activated. The existence of the IgG4 subclass, its up-regulation by anti-inflammatory factors and its own anti-inflammatory characteristics may help the immune system to dampen inappropriate inflammatory reactions.

Anatomical branches of the superior rectal artery in the distal rectum.

OBJECTIVE: The aim of this experimental study was to study the arterial supply of the corpus cavernosum recti in the inner wall of the distal rectum in relation to haemorrhoidal ligation therapy. METHOD: In 10 nonfixed human cadavers, the arterial vasculature of the rectum was studied using the Araldite casting method. Subsequently, the specimens were treated with methylbenzoate in order to obtain semitransparent specimens in which the corpus cavernosum recti could be studied. RESULTS: Specimens were obtained permitting study of the arterial vasculature of the rectum and corpus cavernosum recti at all levels. The superior rectal artery was found to supply the corpus cavernosum recti which consisted of a variable number of equally spaced twisting arteries. CONCLUSION: The distal rectum is supplied by the superior rectal artery. The supplying arteries of the corpus cavernosum recti are not confined to the strict locations described in the literature. This finding is of importance in surgical treatment of haemorrhoidal disease.

Illness perceptions of stroke survivors: Predictors and changes over time - A 1 year follow-up study.

OBJECTIVE: To describe the illness perceptions (IP) of stroke patients in the first year post stroke; to identify patient clusters with comparable IP trajectories and determine their associations with health. METHODS: This prospective study included consecutive stroke patients after medical rehabilitation. Three and 12 months post stroke they completed the Brief Illness Perception Questionnaire (B-IPQ) and questionnaires on physical and mental health. All eight IP and their changes over time were described. Clusters of patients with comparable IP trajectories were constructed by k-means clustering, with subsequent comparison of patient characteristics. Multivariable logistic regression analyses were conducted to determine the association between IP clusters and 12-month mental health. RESULTS: Hundred-and-eighty-four patients were included (men n = 107 [58.2%]; mean age 61.1 [SD 12.7] years). At 3 months, the scores of the IP coherence (mean 3.0, SD 2.3) and treatment control (mean 3.2, SD 2.5) were lowest (best), and consequences (mean 6.1, SD 2.8) and anticipated timeline (mean 6.0, SD 2.7) were highest (worst). At 12 months, the timeline and treatment control scores had significantly worsened. Three clusters of the trajectories of IP were identified, and designated as 'favourable', 'average', and 'unfavourable'. The unfavourable cluster was significantly associated with worse physical and mental health at 3 months (unadjusted) and depressive symptoms at 12 months. CONCLUSION: Stroke patients' IP partly changed between 3 and 12 months post stroke. Patients with an unfavourable IP trajectory had a higher chance of depressive symptoms at 12 months. Illness perceptions could be considered as an additional target of treatment.

In situ depletion of CD4+ T cells in human skin by Zanolimumab.

CD4(+) T cells, in activated or malignant form, are involved in a number of diseases including inflammatory skin diseases such as psoriasis, and T cell lymphomas such as the majority of cutaneous T cell lymphomas (CTCL). Targeting CD4 with an antibody that inhibits and/or eliminates disease-driving T cells in situ may therefore be a useful approach in the treatment of inflammatory and malignant skin diseases. Depletion of CD4(+) T cells in intact inflamed human skin tissue by Zanolimumab, a fully human therapeutic monoclonal antibody (IgG1, kappa) against CD4, was studied in a human psoriasis xenograft mouse model. Zanolimumab treatment was shown to induce a significant reduction in the numbers of inflammatory mononuclear cells in upper dermis. This reduction in inflammatory mononuclear cells in situ was primarily due to a significant reduction in the numbers of skin-infiltrating CD4(+), but not CD8(+) CD3(+) T cells. The capacity of Zanolimumab to deplete the CD4(+) T cells in the skin may be of importance in diseases where CD4(+) T cells play a central role. Indeed, in a phase II clinical trial Zanolimumab has shown a dose-dependent clinical response in patients with CTCL and the antibody is currently in a phase III clinical trial for CTCL, a disease for which there is no safe and effective treatment available today.

Can near infrared spectroscopy measure the effect of pressure on oxygenation of sacral soft tissue?

OBJECTIVE: To test whether near infrared spectroscopy (NIRS) is applicable for the examination of the influence of external pressure on oxygenation of the soft tissues in the sacral area. METHOD: Tissue oxygenation was measured in 33 healthy volunteers in the prone position. A NIRS probe was positioned over the sacrum and external pressure was applied in 10 mmHg increments, from 20 mmHg to 200 mmHg and then decreased. At each level, tissue oxygen saturation (StO2) was measured. To test reproducibility, the protocol was repeated in six volunteers, in whom the thickness of the soft-tissue envelope at different levels of external pressure was assessed using ultrasound. RESULTS: There was wide variability in StO2 courses between the 33 subjects, with a non-linear relationship between pressure and StO2. The only consistent finding was that the StO2 was significantly higher after decreasing pressure than at the initial pressure of 20 mmHg, which is indicative of reactive hyperaemia. Despite the application of high external pressures, reasonable tissue oxygenation was maintained in 19 of 33 subjects. Reproducibility of the measurements was poor. Comparison of soft-tissue thickness with corresponding StO2 values showed that, with increasing pressure, the percentage decrease in tissue thickness was higher than the decrease in tissue oxygenation. CONCLUSION: This study confirms that NIRS is not useful for assessing tissue oxygenation in pressure ulcer research due to unacceptable inter-individual variability and poor reproducibility of measurements.

The inter-heavy chain disulfide bonds of IgG4 are in equilibrium with intra-chain disulfide bonds.

Unlike other immunoglobulin G (IgG) subclasses, IgG4 antibodies in plasma have been reported to be functionally monovalent. In a previous paper, we showed that the apparent monovalency of circulating IgG4 antibodies is caused by asymmetry of plasma IgG4-a large fraction has two antigen-binding sites resulting in bispecificity. We postulated that the generation of bispecific antibodies was caused by a post-secretion mechanism, involving the exchange of IgG4 half-molecules (i.e. one heavy and one light chain). This hypothesis was based on the observed instability of the inter-heavy chain disulfide bonds of IgG4. To investigate this instability, we constructed IgG4 mutants and analyzed the covalent interaction between the heavy chains by sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. The mutation to serine of one of the hinge cysteines involved in the inter-heavy chain bond formation, Cys226, resulted in a more stable rather than a more labile inter-heavy chain linkage. Moreover, we confirmed that mutating the IgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequence Cys-Pro-Pro-Cys also markedly stabilizes the covalent interaction between the heavy-chains. These two observations suggested an explanation for the observed instability of the inter-heavy chain disulfide bonds: the formation of an alternative, intra-chain cystine. Obviously, this intra-chain cystine cannot be formed in the mutant where Cys226 is replaced by Ser, and cannot easily be formed in the mutant with the IgG1 hinge sequence (Cys-Pro-Pro-Cys) due to the restricted torsional freedom of prolines. We, therefore, postulate that the lack of a covalent heavy-chain interaction in a subpopulation of IgG4 reflects an equilibrium between inter- and intra-chain cystines. Based upon the published structure of the IgG4-related hinge-deleted IgG1 myeloma protein Mcg, we propose a model for the two forms of IgG4 and for the half-molecule exchange reaction, which might result in the formation of bispecific IgG4 antibodies.

Comparison and uncertainty evaluation of different calibration protocols and ionization chambers for low-energy surface brachytherapy dosimetry.

PURPOSE: A surface electronic brachytherapy (EBT) device is in fact an x-ray source collimated with specific applicators. Low-energy (<100 kVp) x-ray beam dosimetry faces several challenges that need to be addressed. A number of calibration protocols have been published for x-ray beam dosimetry. The media in which measurements are performed are the fundamental difference between them. The aim of this study was to evaluate the surface dose rate of a low-energy x-ray source with small field applicators using different calibration standards and different small-volume ionization chambers, comparing the values and uncertainties of each methodology. METHODS: The surface dose rate of the EBT unit Esteya (Elekta Brachytherapy, The Netherlands), a 69.5 kVp x-ray source with applicators of 10, 15, 20, 25, and 30 mm diameter, was evaluated using the AAPM TG-61 (based on air kerma) and International Atomic Energy Agency (IAEA) TRS-398 (based on absorbed dose to water) dosimetry protocols for low-energy photon beams. A plane parallel T34013 ionization chamber (PTW Freiburg, Germany) calibrated in terms of both absorbed dose to water and air kerma was used to compare the two dosimetry protocols. Another PTW chamber of the same model was used to evaluate the reproducibility between these chambers. Measurements were also performed with two different Exradin A20 (Standard Imaging, Inc., Middleton, WI) chambers calibrated in terms of air kerma. RESULTS: Differences between surface dose rates measured in air and in water using the T34013 chamber range from 1.6% to 3.3%. No field size dependence has been observed. Differences are below 3.7% when measurements with the A20 and the T34013 chambers calibrated in air are compared. Estimated uncertainty (with coverage factor k = 1) for the T34013 chamber calibrated in water is 2.2%-2.4%, whereas it increases to 2.5% and 2.7% for the A20 and T34013 chambers calibrated in air, respectively. The output factors, measured with the PTW chambers, differ by less than 1.1% for any applicator size when compared to the output factors that were measured with the A20 chamber. CONCLUSIONS: Measurements using both dosimetric protocols are consistent, once the overall uncertainties are considered. There is also consistency between measurements performed with both chambers calibrated in air. Both the T34013 and A20 chambers have negligible stem effect. Any x-ray surface brachytherapy system, including Esteya, can be characterized using either one of these calibration protocols and ionization chambers. Having less correction factors, lower uncertainty, and based on measurements, performed in closer to clinical conditions, the TRS-398 protocol seems to be the preferred option.

Determination of serum tumor markers TPS and CA 15-3 during monitoring of treatment in metastatic breast cancer patients.

Serum levels of tissue polypeptide specific antigen (TPS), a cytokeratin 18 marker, and CA 15-3 were determined in 42 patients with metastatic breast cancer during routine treatment follow-up. At the time of proved metastatic disease, 86% of the values for TPS were above the upper reference value as compared to 93% for CA 15-3. The combined use of TPS and CA 15-3 increased the overall sensitivity. The levels of the tumor markers followed the course of disease during a follow-up period of 6-10 months, even though the dynamics of the changes of tumor markers levels differed in some patients. An increase in the tumor marker level of 25% or more was seen in 60% (TPS) and 52% (CA 15-3), respectively of the studied patients. TPS appeared to indicate changes faster than CA 15-3. The overall results of this study suggest the combined use of TPS and CA 15-3 in the monitoring of breast cancer patients is preferable.

Psychological side-effects of the mass screening on cervical cancer.

The results are reported of a study on the psychological side-effects of mass screening for cervical cancer in Rotterdam. The reactions of participants towards a positive smear and towards the treatment which followed the positive smear were analysed. Women who had problems with a positive smear and with a serious disorder which necessitated an operation and hospitalization had problems with the treatment. However, most women who underwent the operation did not have lasting problems of either a psychological or somatic nature. Women who had been treated by their G.P.s or by a gynaecologist in most cases did not have problems with their treatment. The final conclusion was that mass screening for cervical cancer in Rotterdam caused psychological side-effects to occur among women with a positive smear. However, for the majority of women with a positive smear they were not of a lasting or serious nature.

Randomised trial of prostate cancer screening in The Netherlands: assessment of acceptance and motives for attendance.

OBJECTIVES: To assess motives for attending a randomised population based prostate cancer screening trial, and to assess acceptance of screening and invitation procedures. METHODS: First pilot of the European Randomised Study of Screening for Prostate Cancer (ERSPC; 1992/1993). Men aged 55-75 years, randomly selected from the population register of four city districts of Rotterdam, were invited by a single invitation for screening. Screening consisted of prostate specific antigen prescreening followed by either (1) digital rectal examination, transrectal ultrasound, and, on indication, biopsy, or (2) no additional screening. After screening, or in the case of non-attendance, a questionnaire was sent to a random sample of 600 attenders and 400 non-attenders, with a reminder after three weeks. OUTCOME MEASURES: In both attenders and non-attenders: knowledge of prostate cancer, attitudes towards screening, motives for attending, procedural aspects and sociodemographic characteristics. In attenders, acceptance of screening procedures. RESULTS: The response rate for the questionnaire was 76%: 94% in attenders and 42% in non-attenders. The main reasons for attending were expected personal benefit (76%) and scientific value (39%), and those for not attending were absence of urological complaints (41%) and anticipated pain or discomfort (24%). Uptake of screening was 32%, which increased to a sustained 42% in following years. Attenders, compared with non-attenders, were significantly younger, more often married, better educated, and had higher perceived health status, more knowledge about prostate cancer, and a more positive attitude towards screening. Information materials and invitation procedures were well accepted (high report marks and satisfaction, and 95% would attend for rescreening). A single prostate specific antigen determination was liked less than a combination of all three screening modalities. CONCLUSIONS: (1) The main reasons for attending are personal benefit and science, and those for not attending were absence of urological complaints and anticipated pain or discomfort; (2) knowledge, attitudes, and motives for attending are comparable with other screening programmes; hence, for population based prostate cancer screening, known health promotional aspects should be carefully considered; (3) prostate specific antigen, digital rectal examination and transrectal ultrasound are acceptable to attenders.

Chimeric immunoglobulin E reactive with tumor-associated antigen activates human Fc epsilon RI bearing cells.

Crosslinking of immunoglobulin E molecules that are bound to the Fc epsilon receptors expressed on mast cells or basophils triggers activation of these cells, resulting in the development of a type I hypersensitivity. Targeting this potent immune reaction towards tumors by using IgE that reacts with a tumor-associated antigen, may induce a local inflammation at the tumor site, and may therefore promote tumor regression. We have previously shown that murine IgE bound to tumor cells can activate murine mast cells to release TNF-alpha and histamine. To further investigate the therapeutic potential of IgE-mediated immunotherapy of carcinomas, we have developed human/murine chimeric versions, containing the murine variable regions and human constant regions, of both G250 and 323/A3 IgE. These chimeric IgEs are reactive respectively with the G250 renal cell carcinoma antigen and the Ep-CAM molecule, which is highly expressed by most carcinomas. Transfection of the respective chimeric heavy and light chain genes into recipient Sp2/0 myeloma cells yielded chimeric IgE-producing clones. Chimeric G250 and 323/A3 IgE reacted with tumor cells expressing the G250 antigen or Ep-CAM, respectively. To generate a cell line that expresses Fc receptors for human or chimeric IgE, the rat basophilic leukemia cell line RBL-7 was transfected with the human Fc epsilon RI alpha chain (RBL-7TZ) and subsequently tested for binding of chimeric IgE. Functional assays showed that both chimeric IgEs activated RBL-7TZ cells to release TNF-alpha when cultured with tumor cells that express the respective specific antigen. Furthermore, both chimeric IgEs were able to activate freshly isolated human basophils.

Quantification of the atrial contribution to diastolic filling during radionuclide angiography.

To define exactly the onset of late diastolic filling with respect to atrial contraction, the atrial contribution (AC) to left ventricular filling was quantified in 34 patients with a variety of diseases using radionuclide angiography. From the time-activity curve and its first derivative, a flow-volume loop was constructed. Using the flow-volume loop, the period between minimal flow and the moment of maximal end-diastolic counts was defined as the AC-interval and correlated with the PQ-interval on the electrocardiogram. The relative filling volumes within these time periods were closely related in all patients (r = 0.99, P < 0.0001). The correlation between the PQ-interval and AC-interval was also statistically significant (r = 0.82, P < 0.0001). In a subset of patients, the PQ-interval and AC-interval were not exactly the same. In these patients, the AC-interval was always longer than the PQ-interval, indicating the existence of passive diastasis flow before the onset of atrial contraction. This was more apparent in patients with low heart rates than in those with high heart rates. Despite the close correlation between the PQ-interval on the electrocardiogram and the AC-interval of the flow-volume loop, they may represent different entities. We conclude that the PQ-interval is better than the AC-interval for determining the moment of onset of atrial activity during radionuclide angiography.

[Internal and external haemorrhoids]. / Interne en externe hemorroïden.

In this article, we present 3 cases of patients with different types of haemorrhoidal disease. The first patient is a 27-year-old woman who had been experiencing incidental rectal blood loss without pain during defecation for 3 months. The second patient is a 76-year-old woman who had been bothered by varying degrees of pain from a swelling nearby the anus for 1 year. The third case involves a 31-year-old man who had had continuous severe pain in the anal area for 3 consecutive days. The first patient appeared to have internal hemorrhoids, whereas different forms of external hemorrhoids affected the patients in the other 2 cases. Internal haemorrhoids develop from the intraluminal corpus cavernosum recti; external haemorrhoids from the perianal marginal veins. Patients with internal haemorrhoids present with symptoms that include blood loss and prolaps feeling during defecation. In patients with external haemorrhoids pain is the prominent symptom. Internal haemorrhoids are treated either conservatively or surgically, depending upon their severity. Considering external haemorrhoidal disease surgical treatment provides the most rapid and persistent relief of symptoms.