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Mobile laboratories for implementing anti-epidemic measures in the territories of natural plague foci perform an important function of ensuring sanitary-epidemiological well-being of the country. The aim of the study was to determine the range of areas and tactics for deploying mobile laboratories to ensure the current tasks of immunological monitoring over the health status of the population at risk in the territory of a natural plague focus. Retrospective analysis of the results of long-term immunological monitoring of the risk contingent vaccinated against plague was carried out. The possibilities and prospects for using a mobile laboratory for immunological monitoring within the framework of ensuring the tasks and functions of the Rospotrebnadzor have been determined. Mobile laboratories for immunological monitoring expand the capacities to solve operational issues related to the provision of specific prophylaxis of particularly dangerous infectious diseases, and their deployment as part of other mobile units of the Sanitary-Epidemiological Service will contribute to a more accurate delimitation of the territory, volume, scope and priority of any given preventive measure.

Introduction: Cytomegalovirus (CMV) infection is a major cause of transplantation‐related morbidity and mortality. This study assessed the utility of the QuantiFERON monitor (QFM; Qiagen) for the prediction of early CMV infection and viral burden. Methods: QuantiFERON‐CMV (QF‐CMV; Qiagen) and QFM were measured at the post‐allogeneic hematopoietic stem cell transplantation (HSCT) week 4. CMV DNA was measured at every visit until post‐HSCT week 24. The QFM cutoff specific to CMV infection was established. Result: At the post‐HSCT week 4, the QFM cutoff predicting CMV infection was 86.95 IU/mL. While QF‐CMV results at the post‐HSCT week 4 were associated with high‐level CMV infection (CMV DNA ≥ 5,000 IU/mL) but not with CMV infection (CMV DNA ≥ 500 IU/mL), QFM was associated with both CMV infection and high‐level CMV infection. Both indeterminate QF‐CMV and nonreactive QFM were associated with increased peak CMV DNA. Conclusion: Low QFM is a risk factor for CMV infection and increased CMV viral loads. QFM at post‐HSCT week 4 can be utilized as an assay to predict the risk and burden of early CMV infection in HSCT recipients, in conjunction with other risk factors. [ABSTRACT FROM AUTHOR]

Relevance. COVID-19 is accompanied by high production of cytokines - immune molecules, imbalanced regulation of which leads to disruption of the immune response. Therefore, the study of cytokine status in both vaccinated and re-vaccinated patients is necessary to develop an immunoprophylaxis strategy for COVID-19. Aim. To evaluate the cytokine status in COVID-19 survivors and vaccinated employees of a health care facility against this disease. Materials and Methods. Production levels of cytokines (IL-4, IL-10, IFN-γ and TNF-α) were determined by quantitative enzyme immunoassay in serum of 144 volunteers. Results and discussion. A significant 1.5-fold increase in IL-10 and 2.0-fold increase in TNF-α was found in the group of re-invaccinated overinfected volunteers, and a 2.0-fold decrease in IL-4 compared with the vaccinated group. An inverse correlation between IL-4 and IFN-γ levels and timing after the disease was detected. A significant decrease in IL-4 in survivors (Omicron variant) and an increase in IL-10 in the survivors at the beginning of the pandemic as compared to other re-vaccinees was established. In the vaccinated group, regardless of the vaccine used, IL-10 and TNF-α decreased significantly after revaccination. No statistically significant differences were found between the cytokine status parameters in the survivors before and after vaccination. The predominance of the cellular component of immunity was established in all the examined groups, and the highest proportion of persons with a humoral immune response was detected among the vaccinated volunteers, while the highest proportion of persons with a mixed immune response was detected among the overexperienced non-vaccinated patients. Conclusions. The results obtained supplement the available data on coronavirus infection and indicate the need for further research.

The vulnerability of healthcare and laboratory to potential infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has thus far been analyzed through the lens of the acute phase of the pandemic, including remote-based work, as well as emergency settings that are different from routine healthcare operations. However, as lockdowns ease and activities return to an identifiable pre-pandemic routine, the safety considerations also require to shift accordingly. As laboratory workers are likely to continue being exposed to unidentified SARS-CoV-2 positive samples through routine blood collection and processing operations, coronavirus disease 2019 (COVID-19) might have to be re-considered as an occupational disease within this context. Additionally, as per many such occupational diseases, a surveillance system is implemented for the medium- and long-term. This manuscript presents the views on the possible surveillance scenarios for laboratory staff, viewed from an immunological and biosafety perspective.

Abstract Background Immunosuppression after kidney transplantation is mainly guided via plasma tacrolimus trough level, which cannot sufficiently predict allograft rejection and infection. The plasma load of the non-pathogenic and highly prevalent torque teno virus (TTV) is associated with the immunosuppression of its host. Non-interventional studies suggest the use of TTV load to predict allograft rejection and infection. The primary objective of the current trial is to demonstrate the safety, tolerability and preliminary efficacy of TTV-guided immunosuppression. Methods For this purpose, a randomised, controlled, interventional, two-arm, non-inferiority, patient- and assessor-blinded, investigator-driven phase II trial was designed. A total of 260 stable, low-immunological-risk adult recipients of a kidney graft with tacrolimus-based immunosuppression and TTV infection after month 3 post-transplantation will be recruited in 13 academic centres in six European countries. Subjects will be randomised in a 1:1 ratio (allocation concealment) to receive tacrolimus either guided by TTV load or according to the local centre standard for 9 months. The primary composite endpoint includes the occurrence of infections, biopsy-proven allograft rejection, graft loss, or death. The main secondary endpoints include estimated glomerular filtration rate, graft rejection detected by protocol biopsy at month 12 post-transplantation (including molecular microscopy), development of de novo donor-specific antibodies, health-related quality of life, and drug adherence. In parallel, a comprehensive biobank will be established including plasma, serum, urine and whole blood. The date of the first enrolment was August 2022 and the planned end is April 2025. Discussion The assessment of individual kidney transplant recipient immune function might enable clinicians to personalise immunosuppression, thereby reducing infection and rejection. Moreover, the trial might act as a proof of principle for TTV-guided immunosuppression and thus pave the way for broader clinical applications, including as guidance for immune modulators or disease-modifying agents. Trial registration EU CT-Number: 2022-500024-30-00

Background: Despite recent advances and refinements in perioperative management of kidney transplantation (KT), early renal graft injury (eRGI) remains a critical problem with serious impairment of graft function as well as short- and long-term outcome. Serial monitoring of peripheral blood innate immune cells might be a useful tool in predicting post-transplant eRGI and graft outcome after KT. Methods: In this prospective study, medical data of 50 consecutive patients undergoing KT at the University Hospital of Leipzig were analyzed starting at the day of KT until day 10 after the transplantation. The main outcome parameter was the occurrence of eRGI and other outcome parameters associated with graft function/outcome. eRGI was defined as graft-related complications and clinical signs of renal IRI (ischemia reperfusion injury), such as acute tubular necrosis (ATN), delayed graft function (DGF), initial nonfunction (INF) and graft rejection within 3 months following KT. Typical innate immune cells including neutrophils, natural killer (NK) cells, monocytes, basophils and dendritic cells (myeloid, plasmacytoid) were measured in all patients in peripheral blood at day 0, 1, 3, 7 and 10 after the transplantation. Receiver operating characteristics (ROC) curves were performed to assess their predictive value for eRGI. Cutoff levels were calculated with the Youden index. Significant diagnostic immunological cutoffs and other prognostic clinical factors were tested in a multivariate logistic regression model. Results: Of the 50 included patients, 23 patients developed eRGI. Mean levels of neutrophils and monocytes were significantly higher on most days in the eRGI group compared to the non-eRGI group after transplantation, whereas a significant decrease in NK cell count, basophil levels and DC counts could be found between baseline and postoperative course. ROC analysis indicated that monocytes levels on POD 7 (AUC: 0.91) and NK cell levels on POD 7 (AUC: 0.92) were highly predictive for eRGI after KT. Multivariable analysis identified recipient age (OR 1.53 (95% CI: 1.003–2.350), p = 0.040), recipient body mass index > 25 kg/m2 (OR 5.6 (95% CI: 1.36–23.9), p = 0.015), recipient cardiovascular disease (OR 8.17 (95% CI: 1.28–52.16), p = 0.026), donor age (OR 1.068 (95% CI: 1.011–1.128), p = 0.027), <0.010), deceased-donor transplantation (OR 2.18 (95% CI: 1.091–4.112), p = 0.027) and cold ischemia time (CIT) of the renal graft (OR 1.005 (95% CI: 1.001–1.01), p = 0.019) as clinically relevant prognostic factors associated with increased eRGI following KT. Further, neutrophils > 9.4 × 103/μL on POD 7 (OR 16.1 (95% CI: 1.31–195.6), p = 0.031), monocytes > 1150 cells/ul on POD 7 (OR 7.81 (95% CI: 1.97–63.18), p = 0.048), NK cells < 125 cells/μL on POD 3 (OR 6.97 (95% CI: 3.81–12.7), p < 0.01), basophils < 18.1 cells/μL on POD 10 (OR 3.45 (95% CI: 1.37–12.3), p = 0.02) and mDC < 4.7 cells/μL on POD 7 (OR 11.68 (95% CI: 1.85–73.4), p < 0.01) were revealed as independent biochemical predictive variables for eRGI after KT. Conclusions: We show that the combined measurement of immunological innate variables (NK cells and monocytes on POD 7) and specific clinical factors such as prolonged CIT, increased donor and recipient age and morbidity together with deceased-donor transplantation were significant and specific predictors of eRGI following KT. We suggest that intensified monitoring of these parameters might be a helpful clinical tool in identifying patients at a higher risk of postoperative complication after KT and may therefore help to detect and—by diligent clinical management—even prevent deteriorated outcome due to IRI and eRGI after KT. [ABSTRACT FROM AUTHOR]

Pig kidney xenotransplantation has the potential to alleviate the current shortage of deceased and living human organs and provide patients with end‐stage renal disease with a greater opportunity for long‐term survival and a better quality of life. In recent decades, advances in the genetic engineering of pigs and in immunosuppressive therapy have permitted the resolution of many historical obstacles to the success of pig kidney transplantation in nonhuman primates. Pig kidney xenotransplantation may soon be translated to the clinic. Given the potential risks of kidney xenotransplantation, particularly of immunologic rejection of the graft, potential patients must be carefully screened for inclusion in the initial clinical trials and immunologically monitored diligently post‐transplantation. We provide an overview of the immunological methods we believe should be used to (i) screen potential patients for the first clinical trials to exclude those with a higher risk of rejection, and (ii) monitor patients with a pig kidney graft to determine their immunological response to the graft. [ABSTRACT FROM AUTHOR]

The article covers the issues related to the scientific substantiation and methodological support of immunological monitoring of persons vaccinated against plague upon epidemic indications. The problematic issues of the methodology for the assessment of immunological efficiency (efficacy) of plague live vaccine (PLV) are noted. The current tasks and possible prospects for the introduction of immunological monitoring of persons vaccinated against plague upon epidemic indications have been defined. The algorithm of efficacy estimation of plague live vaccine in vaccinated (revaccinated) persons has been tested under real conditions. Analysis of the results of efficacy evaluation of plague live vaccine among vaccinated (revaccinated) people against plague living in the territories of natural foci of this infection has been performed. Demonstrated is the possibility of using immunological monitoring results in creating an objective basis for improving the strategy of specific plague prevention in natural foci of this infection. The priority areas for further optimization of the specific plague prevention in the territories of natural foci of the infection, including those related to the formation of individual regimen revaccination tactics, taking into account the possibilities of creating modern and effective vaccines, are outlined.

Relevance. In 2005 International Health Regulations, the plague, is on the list of dangerous infectious diseases that can cause emergency situations of interstate importance. Even single cases of human plague are considered as the basis for carrying out preventive measures. The paper presents the results of immunological monitoring conducted on the territory of the Republic of Kalmykia in order to assess the immunological efficacy and safety of the plague live vaccine The paper presents the results of immunological monitoring in the territory of the Kalmyk Republic over the individuals vaccinated against plague due to epidemiological reasons. Materials and methods. Studies of immunological efficacy of live plague vaccine were conducted alongside stepwise assessment of cellular and humoral components of innate and adaptive immunity in persons revaccinated against plague, using a complex of advanced informative tests. Results and conclusions. It is established that before the second revaccination all the surveyed persons retained expressed immune response by the mixed or cellular type, characterized by high level of spontaneous and induced production of Th1-associated cytokines. Activation of Th1 immune reaction was registered one month after the scheduled revaccination; immune response change-over from Th1 to Th2 type – after 6 months of observation, and retention of adaptive immunity by mixed type at the moderate level – in a year. Specific humoral immunity developed in 85% of the surveyed persons, but throughout the whole investigation the dynamics of antibody titers to plague microbe F1 individualized and did not coincide with cellular immunity indicators. Performed complex study has confirmed the relative safety of the live plague vaccine.

Abstract Background One of today’s greatest hurdles for cancer immunotherapy is the absence of information regarding which tumor antigens are already recognized by patients receiving immunotherapies, and whether those therapies then boost or generate an immune response against tumor proteins. For CD8+ T cells in particular, patient-specific immune recognition and responses at the level of individual tumor antigens are rarely characterized. Because of this, some immunologists have turned to serum antibodies as an alternative measure of antigen-specific anti-tumor immunity. In this work, we sought to simultaneously interrogate serum IgG and CD8+ T cell recognition of individual tumor antigens to determine whether antigen-specific serum IgG antibodies provide a window into the behavior of antigen-specific CD8+ T cell responses. Using antibody-based assays to evaluate immune response repertoires and focus T cell antigen exploration could afford substantial advantages for discovering and monitoring the anti-cancer immune responses of patients enrolled on clinical trials. Methods We vaccinated female BALB/c mice with a novel combination of an autophagosome-enriched vaccine derived from 4T1 mammary carcinoma along with poly-I:C adjuvant, then screened serum for IgG binding to arrays of 15mer peptides containing known mutation sites in 4T1. Simultaneously, we primed CD8+ T cell cultures from these same animals with 8-11mer peptides derived from these antigens. These primed T cells were then stimulated to measure recognition of the peptides or live 4T1 cells by IFNγ release. Results Vaccinated animals demonstrate increases in antigen-specific CD8+ T cell recognition of 4T1 tumor cells and peptides. For proteins confirmed in 4T1 cells and vaccine by mass spectrometry, there is a correlation between this increased CD8+ T cell IFNγ release and serum IgG binding to individual peptide antigens. Conclusions These results suggest it is possible to observe some features of a patient’s antigen-specific T cell repertoire via an antibody surrogate, which has implications for tumor antigen discovery and clinical monitoring of antigen-specific anti-tumor immunity.

Immunotherapy has become an important approach for treating different tumours which has shown significant efficacy in numerous clinical trials, especially those using new checkpoint inhibitors and adoptive cell therapy, which have rapidly become widespread after being approved. However, analysis of peripheral immune biomarkers before and after immunotherapy and their relationship to clinical responses and disease prognosis have rarely been performed in clinical trials. In this review, we examine dynamic changes in the immune system before and after therapy by analyzing recent clinical trials of immunotherapy in patients with cancer that focused on checkpoint inhibitors and adoptive cell therapy. Our aim was to identify circulating biomarkers which can specifically predict clinical response and prognosis, as well as toxicities of immunotherapy. Through this approach, we hope to advance our understanding of the mechanisms of immunotherapy with the goal of developing individualized treatment for cancer patients. [ABSTRACT FROM AUTHOR]

Abstract. We have performed clinical and immunological investigation in the patients with trauma of face bones before and after stable mandibular ostheosynthesis. Blood samples for analysis were taken upon admission of the patient to clinics, and following treatment (3, 10, and 1-2 months). The patients with initially retarded bone consolidation exhibited low levels of monocytes and lactoferrine before surgical treatment. It was shown that the consecutive stages of bone regeneration (inflammation, osteoblastic proliferation, collagenogenesis, and ossification) are accompanied by certain changes in immune parameters. In particular, we observed increased levels of IgМ, TNF, and activation of leucopoiesis after treatment. The results obtained allow us of discriminating some natural reactions of immune system in cases of normal and retarded bone consolidation. For each of these criteria, diagnostic sensitivity and informativity of tests are calculated, thus providing an opportunity to predict retarded consolidation in surgical treatment of the face bones. (Med. Immunol., 2007, vol. 10, N 4-5, pp 371-388).

Summary: Despite its impact on quality of life and potential for complications, specific risk and protective factors for herpes zoster (HZ) after kidney transplantation (KT) remain to be clarified. We included 444 patients undergoing KT between November 2008 and March 2013. Peripheral blood lymphocyte subpopulations were measured at baseline and months 1 and 6. The risk factors for early (first post‐transplant year) and late HZ (years 1–5) were separately assessed. We observed 35 episodes of post‐transplant HZ after a median follow‐up of 48.3 months (incidence rate: 0.057 per 1000 transplant‐days). Median interval from transplantation was 18.3 months. Six patients (17.1%) developed disseminated infection. Postherpetic neuralgia occurred in 10 cases (28.6%). The receipt of anti‐cytomegalovirus (CMV) prophylaxis with (val)ganciclovir decreased the risk of early HZ [adjusted hazard ratio (aHR): 0.08; 95% CI: 0.01–1.13; P‐value = 0.062], whereas the natural killer (NK) cell at month 6 was protective for the occurrence of late HZ [aHR (per 10‐cells/μl increase): 0.94; 95% CI: 0.88–1.00; P‐value = 0.054]. In conclusion, two easily ascertainable factors (whether the patient is receiving anti‐CMV prophylaxis and the NK cell count at month 6) might be potentially useful to tailor preventive strategies according to individual susceptibility to post‐transplant HZ. [ABSTRACT FROM AUTHOR]

Background The transmembrane glycoprotein CD30 contributes to regulate the balance between Th1 and Th2 responses. Previous studies have reported conflicting results on the utility of its soluble form ( sCD30) to predict post-transplant infection. Methods Serum sCD30 was measured by a commercial ELISA assay at baseline and post-transplant months 1, 3, and 6 in 100 kidney transplant ( KT) recipients (279 monitoring points). The impact of sCD30 levels on the incidence of overall, bacterial and opportunistic infection during the first 12 months after transplantation was assessed by Cox regression. Results There were no differences in serum sCD30 according to the occurrence of overall or opportunistic infection. However, sCD30 levels were higher in patients with bacterial infection compared to those without at baseline ( P=.038) and months 1 ( P<.0001), 3 ( P=.043), and 6 after transplantation ( P=.006). Patients with baseline sCD30 levels ≥13.5 ng/mL had lower 12-month bacterial infection-free survival (35.0% vs 80.0%; P<.0001). After adjusting for potential confounders, baseline sCD30 levels ≥13.5 ng/mL remained as an independent risk factor for bacterial infection (adjusted hazard ratio [ aHR]: 4.65; 95% confidence interval [ CI]: 2.05-10.53; <.001). Analogously, sCD30 levels ≥6.0 ng/mL at month 1 acted as a risk factor for subsequent bacterial infection ( aHR: 5.29; 95% CI: 1.11-25.14; P=.036). Conclusion Higher serum sCD30 levels were associated with an increased risk of bacterial infection after KT. We hypothesize that this biomarker reflects a Th2-polarized T-cell response, which exerts a detrimental effect on the immunity against bacterial pathogens. [ABSTRACT FROM AUTHOR]

Infections continue to be one of the leading causes of morbidity and mortality in liver transplant recipients. We retrospectively reviewed the symptomatic infectious episodes that occurred during the first year post-transplant to determine time of onset, causative pathogens and cell-mediated immunity response patterns. Ninety-eight of the 202 (48.5%) recipients enrolled developed at least one infectious episode. The total number of infectious episodes was 135: 77 (57.1%) bacterial, 45 (33.3%) viral and 13 (9.6%) fungal. The most frequently isolated bacteria were Escherichia coli (21 isolates) and Klebsiella pneumoniae (19 isolates). Overall, extended-spectrum beta lactamase-producing and methicillin-resistant organisms were responsible for 29 (29/77; 37.7%) infectious episodes. Members of the herpes virus group, in particular cytomegalovirus (34/45 viral infections, 75.5%), were detected. Candida species (9 isolates) followed by Aspergillus species (4 isolates) were isolated. The majority of infections (63%) occurred during the early post-transplant phase (<1 month), whereas only 8/135 episodes (5.9%) were detected after the sixth month (late phase). Significantly lower median ImmuKnow intracellular ATP values in patients who developed bacterial and fungal infections compared to infection-free patients were observed ( P < 0.0001 and P = 0.0016, respectively), whereas patients who developed a viral infection had a median intracellular ATP level not statistically different compared to uninfected patients ( P = 0.4). Our findings confirm that bacteria are responsible for the majority of symptomatic infections and occur more frequently during the first month post-transplant. The ImmuKnow measurements can be a useful tool for identifying patients at high risk of developing infection, particularly of fungal and bacterial etiology. [ABSTRACT FROM AUTHOR]

Background: Optimal HIV treatment monitoring remains a big challenge in resource limited settings. Guidelines recommend the use of clinical and immunological criteria in resource limited settings due to unavailability of viral load monitoring; however their utility is questionable. This study aimed at assessing the accuracy of immunological criteria in detecting treatment failure among HIV infected Tanzanian adults receiving first line ART. Methods: A clinic based cross sectional study was conducted between February and July 2011 at Bugando Medical centre (BMC) HIV care and treatment clinic (CTC) involving HIV infected patients aged 18 years and above, receiving first line ART; followed up for at least 1 year. Viral load was tested for every enrolled patient. Standard WHO criteria were used to define immunological failure. Virological failure was defined as one viral load measurement of >5000 copies/ml and was used as a gold standard. A 2 × 2 table was used to assess the accuracy of immunological criteria in detecting treatment failure. Results: A total of 274 HIV-infected adults were enrolled into the study. Out of these, 65.7% were females, the median age was 39 years (IQR 33–45), the median BMI 21.9 kg/m2 (IQR 19.7–24.0). Out of the 274 study participants 156 (56.9%) had immunological failure. Only 60 of the study participants (21.9%) had viral load >5000. Only 42 patients (70%) were found to have both immunological failure and virological failure. The sensitivity of immunological criteria in detecting treatment failure was 70%, specificity 46.7%, positive predictive and negative predictive values of 26.9 and 84.7% respectively. Conclusion: WHO immunological criteria have low sensitivity and positive predictive value for detecting treatment failure. Relying on CD4 counts for treatment monitoring would therefore lead to misclassifications of treatment failure that could result into unnecessary or delayed switch to second line ART. Access to viral load monitoring is important to avoid these misclassifications. [ABSTRACT FROM AUTHOR]

Résumé But de l’étude La qualité du suivi immunologique est primordiale car elle conditionne le succès de la greffe rénale. L’objectif de ce travail est de décrire l’expérience de l’unité d’immunologie du CHU Ibn Sina de Rabat relative au suivi immunologique des patients transplantés entre 2001 et 2014. Patients et méthodes Les prélèvements des patients proviennent des services de néphrologie de différents hôpitaux publics et privés du Maroc. Les examens réalisés dans le cadre du suivi immunologique sont le groupage ABO, le typage HLA-A, B, DR, DQ, la recherche et l’identification des anticorps anti-HLA et le cross-match . Résultats Cent quatorze patients ont bénéficié d’un suivi immunologique pré- et post-greffe dans notre laboratoire. Le pourcentage de receveurs ayant entre 2 et 5 sérums stockés est de 60,5 avant la greffe et 56,1 après la greffe. Les patients immunisés représentent 22,8 % avant la greffe et 17,6 % après la greffe. Quatre-vingt dix-sept patients ont toujours un greffon fonctionnel alors que 4 d’entre eux avaient des DSA de faible intensité avant la greffe. Cinq rejets immunologiques ont été déclarés alors que les cross-match étaient négatifs et qu’aucun DSA n’a été identifié avant la greffe. La survie des patients et des greffons à un an est de 98,2 % et 92,7 % respectivement. Conclusion La mise en place d’un suivi immunologique régulier est parfois difficile à instaurer dans notre contexte, cependant, les résultats obtenus sont satisfaisants en termes de survie du greffon et des patients. Purpose of study The quality of the immunological monitoring is crucial because it determines the success of the kidney transplantation. The scope of this work is to describe the experience of the department of immunological unity of the Ibn Sina university hospital in Rabat regarding the immunological monitoring of patients transplanted between 2001 and 2014. Patients and methods Patient samples were collected from nephrology services of different public and private hospitals of Morocco. The tests conducted in the context of immunological monitoring are ABO typing, HLA-A, B, DR, DQ typing, anti-HLA antibodies detection and identification and cross-match. Results One hundred and fourteen benefited from a pre- and post-transplant immunological monitoring in our laboratory. The percentage of recipients having between 2 and 5 stored sera is 60.5 before transplantation and 56.1 after transplantation. Immunized patients account for 22.8% before the transplant and 17.6% after transplantation. Ninety-seven patients still have a functional graft, while 4 of them had DSA of low intensity before transplantation. Five immunological rejections were reported while the cross-match were negative and no DSA was identified before transplantation. Patient survival and graft at 1 year was 98.2% and 92.7% respectively. Conclusion Conducting regular immunological monitoring is sometimes difficult in our context, however, the results are satisfactory in terms of graft and patients survival. [ABSTRACT FROM AUTHOR]

The article presents a study which examines the reduction of newly produced T and B lymphocytes and T-cell receptor repertoire (TCR) diversity in the peripheral blood of multiple sclerosis (MS) patients treated with fingolimod. The study is investigated by real-time polymerase chain reaction (PCR), immune phenotyping, and spectratyping. Results show that newly produced T and B lymphocytes were reduced while T-cell repertoire increased after 12 months of treatment.

Activation of cellular immunity, mainly cytotoxic T cells, plays an important role in the · mechanisms of cyclic acute exacerbation of hepatocellular damage (AE) in hepatitis B virus (HBV) carriers. Monitoring of the dynamics of immunoreactivity is clinically important to manage the patients and recognize the mechanism of liver damage. Serum neopterin has been widely- recognized as a marker of actiyation of cell-mediated immunity. We observed the clinical course of five hepatitis Be antigen I HBeAg ) positive HBV carriers with recurrent AE for mean eighteen months by monitoring of serum neopterin using a RIA kit. Alanine amino transferase (ALT) and HBeAg were also measured serially as markers for hepatocne necrosis and viral replication, respectively During follow-up period. each patient had several episodes of AE alternating with normal or nearly normal liver function. The levels of serum neopterin changed dynamically related to those of HBeAg and ALT Temporal analysis of these makers before, during and after the episodes of AE showed a close involvement of the viral replication prior to the hepatocellular injury as well as the activation of cellular immunity following the viral replication. Immunological monitoring of serum neopterin could provide useful information about the dynamic interaction between virus replication, cellular immunoreactivity and hepatocellular damage. It was thought that serum neopterin can be used as a complementary marker to follow and manage chronic hepatitis B patients.

Recent successes with adeno-associated virus (AAV)-based gene therapies fuel the hope for new treatments for hereditary diseases. Pre-existing as well as therapy-induced immune responses against both AAV and the encoded transgenes have been described and may impact on safety and efficacy of gene-therapy approaches. Consequently, monitoring of vector- and transgene-specific immunity is mandated and may rationally guide clinical development. Next to the humoral immune response, the cellular response is central in our understanding of the host reaction in gene therapy. But in contrast to the monitoring of antibodies, which has matured over many decades, sensitive and robust monitoring of T cells is a relatively new development. To make cellular immune assessments fit for purpose, investigators need to know, control and report the critical assay variables that influence the results. In addition, the quality of immune assays needs to be continuously adjusted to allow for exploratory hypothesis generation in early stages and confirmatory hypothesis validation in later stages of clinical development. The concept of immune assay harmonization which includes use of field-wide benchmarks, harmonization guidelines, and external quality control can support the context-specific evolution of immune assays. Multi-center studies pose particular challenges to sample logistics and quality control of sample specimens. Cooperative groups need to define if immune assessments should be performed in one central facility, in peripheral labs or including a combination of both. Finally, engineered reference samples that contain a defined number of antigen-specific T cells may become broadly applicable tools to control assay performance over time or across institutions.

Background Autophagy regulates innate and adaptive immune responses to pathogens and tumors. We have reported that autophagosomes derived from tumor cells after proteasome inhibition, DRibbles (Defective ribosomal products in blebs), were excellent sources of antigens for efficient cross priming of tumor-specific CD8+ T cells, which mediated regression of established tumors in mice. But the activity of DRibbles in human has not been reported. Methods DRibbles or cell lysates derived from HEK293T or UbiLT3 cell lines expressing cytomegalovirus (CMV) pp65 protein or transfected with a plasmid encoding dominant HLA-A2 restricted CMV, Epstein-Barr virus (EBV), and Influenza (Flu) epitopes (CEF) were loaded onto human monocytes or PBMCs and the response of human CMV pp65 or CEF antigen-specific CD4+ and CD8+ memory T cells was detected by intracellular staining. The effect of cytokines (GM-CSF, IL-4, IL-12, TNF-α, IFN-α and IFN-γ) TLR agonists (Lipopolysaccharide, Polyinosinic-polycytidylic acid (poly(I:C), M52-CpG, R848, TLR2 ligand) and CD40 ligand on the cross-presentation of antigens contained in DRibbles or cell lysates was explored . Results In this study we showed that purified monocytes, or human PBMCs, loaded with DRibbles isolated from cells expressing CMV or CEF epitopes, could activate CMV- or CEF-specific memory T cells. DRibbles were significantly more efficient at stimulating CD8+ memory T cells compared to cell lysates expressing the same antigenic epitopes. We optimized the conditions for T-cell activation and IFN-γ production following direct loading of DRibbles onto PBMCs. We found that the addition of Poly(I:C), CD40 ligand, and GM-CSF to the PBMCs together with DRibbles significantly increased the level of CD8+ T cell responses. Conclusions DRibbles containing specific viral antigens are an efficient ex vivo activator of human antigen-specific memory T cells specific for those antigens. This function could be enhanced by combining with Poly(I:C), CD40 ligand, and GM-CSF. This study provides proof-ofconcept for applying this strategy to activate memory T cells against other antigens, including tumor-specific T cells ex vivo for immunological monitoring and adoptive immunotherapy, and in vivo as vaccines for patients with cancer. [ABSTRACT FROM AUTHOR]

Recent successes with adeno-associated virus (AAV)-based gene therapies fuel the hope for new treatments for hereditary diseases. Pre-existing as well as therapy-induced immune responses against both AAV and the encoded transgenes have been described and may impact on safety and efficacy of gene therapy approaches. Consequently, monitoring of vector- and transgene-specific immunity is mandated and may rationally guide clinical development. Next to the humoral immune response, the cellular response is central in our understanding of the host reaction in gene therapy. But in contrast to the monitoring of antibodies, which has matured over many decades, sensitive and robust monitoring ofT cells is a relatively newdevelopment.To make cellular immune assessments fit for purpose, investigators need to know, control and report the critical assay variables that influence the results. In addition, the quality of immune assays needs to be continuously adjusted to allow for exploratory hypothesis generation in early stages and confirmatory hypothesis validation in later stages of clinical development.The concept of immune assay harmonization which includes use of field-wide benchmarks, harmonization guidelines, and external quality control can support the context-specific evolution of immune assays. Multi-center studies pose particular challenges to sample logistics and quality control of sample specimens. Cooperative groups need to define if immune assessments should be performed in one central facility, in peripheral labs or including a combination of both. Finally, engineered reference samples that contain a defined number of antigen-specific T cells may become broadly applicable tools to control assay performance over time or across institutions. [ABSTRACT FROM AUTHOR]

Ionizing radiation is one of the major risk factors for cancer diseases. The question on the rate of malignant neoplasms (MNs) and their early detection in Chernobyl nuclear accident (ChNA) liquidators is still open. In this work, the results of a long-term immunological monitoring of the ChNA liquidators who live in the northwestern region of Russia have been analyzed with an attempt to detect the predictors of cancer in this population cohort. The rate of newly detected MN cases in the cohort of followup ChNA liquidators monitored in 1990-2009 was 8.856% (89 cases per 1005 persons), which is somewhat higher as compared with the average rate for the total population. As for the rate of individual MN types, lung, gastric, and prostate cancers were prevalent, which matches the worldwide trend in MN abundance. It was shown that 1-3 years before the MN diagnosis, liquidators displayed detectable changes in their immune status, including a decrease in the percentages of CD3 and CD4 T lymphocytes, and, to a lesser degree, B lymphocytes; a decrease in the CD4/CD8 ratio; increase in the relative and absolute contents of CD16 lymphocytes; increase in the absolute CD8 T lymphocyte counts; prevalence of CD316/56 (NK-T) cells over CD3-16/56 (NK) cells; and increase in the activity of phagocytes. The patients who displayed one or several of the listed characteristics should be ascribed to the MN risk group to be assessed for cancer markers, be more comprehensively examined, and be the subjects of dynamic observation. [ABSTRACT FROM AUTHOR]

It was demonstrated that malignant neoplasms (MNs) most frequently develop in the liquidators involved in the cleanup activities after the Chernobyl nuclear accident (ChNA) in 1986 (43.75%) and the liquidators involved in long-term activities, one-quarter of whom also participated in the cleanup in 1986. The specific features of the immune status (IS) depending on the period and year of the cleanup activities in the ChNA area were determined. In the cohort with a confirmed MN diagnosis, the observed deviations in the people who participated in these activities only in 1986 and on a permanent basis, including 1986, coincided in their pattern but differed in the magnitude of changes in immunological characteristics. Long-term participation during the extreme period and, correspondingly, higher exposure caused an increase in the contents of CD8 T cells, CD16 lymphocytes, and activated T lymphocytes, as well as decreases in the total immunoregulatory index, percentage of CD3-16/56 (NK), and total IgE and a more pronounced deficiency in B lymphocytes. Differences between the groups of liquidators who participated in the cleanup activities in 1986 and 1987 were detected. The liquidators-1987 display the most pronounced deviations in the IS characteristics. Permanent presence in the ChNA area in 1986 and 1987 caused a similar effect but the magnitude of changes in the content of CD4 T lymphocytes (↓), content of CD8 T lymphocytes (↑), and immunoregulatory index (↓) was considerably higher in liquidators-1987. The permanent cleanup activities in Chernobyl in 1987 caused a deficiency in CD4 T lymphocytes; more pronounced increase in the content of CD8 T lymphocytes; decrease in CD4/CD8 index; change in the ratio of NK-T to NK cells; elevation in the counts of CD95, HLA-DR, and activated T lymphocytes; and decrease in the level of total IgE. Any increase in the expression of cell activation markers was undetectable in the liquidators-1986 involved in the cleanup activities on a long-term basis. The specific features of the IS changes depending on the dose of external γ-irradiation were determined. It was shown that the MN rate in liquidators increases with age relative to the number of examined people in each age cohort. The differences in the IS age-related dynamics in liquidators with and without MNs were detectable in stable contents of CD3, CD4, and CD8 T lymphocytes; immunoregulatory index; and contents of CD95 cells and serum IgA at ages of 40 to 70 years old with a subsequent decrease in these parameters and elevation in the content of CD8 T lymphocytes with age in the absence of MNs; continuous increase in CD3-16/56 (NK) cells in the presence of MNs; and decreases in these values after 70 years in the absence of MNs. In both groups of liquidators older than 70 years (with and without MNs), a deficiency in the T-cell component (CD3 and CD4 T lymphocytes and CD4/CD8 ratio) and an increase in the counts of CD8 T lymphocytes are characteristic of the IS. A growing deficiency in CD4 T lymphocytes during the monitoring on the background of increasing content of CD8 T lymphocytes, which additionally contributes to the weakening of immune regulation due to progressing abnormalities in the ratio of regulatory T-lymphocyte subpopulations, can be regarded as one of the characteristics that suggest an adverse life expectancy prognosis for people with MNs. [ABSTRACT FROM AUTHOR]

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Background: Pretranspiant screening in living donor kidney transplantation includes human leukocyte antigen matching, and panel reactive antibody analysis, whereas T cell mediated anti-donor reactivity is not assessed routinely. We investigated I cell reactivity after living related kidney transplantation between two monocygotic twins and in consequence correlated the withdrawal of individual immunosuppressive medication with immunological findings. Methods: lmmunosuppression consisted of mycophenolate mofetil, glucocorticoid single shot, and induction therapy with antithymocyte immunoglobulin. Results: FACS analysis of recipient peripheral blood cells revealed a normal haemogram after transplantation, showing non-activated CD4 and CD8 cells. Mixed lymphocyte reaction did not reveal donor-specific T cell activity. IFN-γ and IL-10 ELISA of supernatants of recipient cells cocultivated with donor cells support the lack of Th1 and Th2 cell differentiation. Conclusion: Based on immunological findings on days 5 and 20 MMF-therapy was reduced and stopped. Immunological monitoring on day 90 confirmed the absence of immune reactions directed against donor tissue. [ABSTRACT FROM AUTHOR]

Over the last half century, kidney and liver transplantation have been recognized as the treatment of choice for adult and children with end-stage renal or liver failure. Infants present a relative naïve immune system, but they are capable of mounting both cellular and humoral immune responses to the foreign antigens presented by the allograft. Immune monitoring is a way of measuring functional and molecular correlates of immune reactivity which may provide clinically useful information for identifying patients who have an increase risk of acute rejection prior to clinical symptoms or develop transplant tolerance. However, although numerous assays have been shown to predict rejection, to date no assays have been demonstrated to detect or predict transplantation tolerance. This is a summary of the published literature on promising antigen-specific and non-antigen-specific assays used for immunological monitoring in solid organ transplantation. This work also attempts to review their applicability to pediatric transplantation, specifically, pediatric kidney and liver recipients. [ABSTRACT FROM AUTHOR]

Abstract: A series of cancer vaccines have been evaluated in clinical trials with encouraging results, but the demonstration of clinical benefit in confirmatory studies has so far proven to be difficult. The development of cancer vaccines is hampered by a range of issues particular to this field of research. On 12th March 2008, the Biotherapy Development Association convened a workshop to discuss issues faced by scientists and clinicians involved in the development of cancer vaccines. This paper is a review of the field, based on discussions held at the BDA workshop, and describes biological barriers encountered in generating effective immune responses to tumours, methodological obstacles encountered in the improvement of immunological monitoring which aims to improve inter-laboratory and inter-trial comparisons, challenges in clinical trial design and problems posed by the lack of specific regulation for cancer vaccines and the impact on their development. Ultimately, a number of general solutions are posed: (1) better patient selection, (2) use of multi-modal treatments that affect several aspects of the immune system at once, (3) a requirement for the development of good biomarkers to stratify patients for selection prior to trial and as surrogates for clinical response and (4) harmonisation of SOPs for immunological monitoring of clinical trials. [Copyright &y& Elsevier]

We are investigating the use of Alpha Fetoprotein (AFP) as a tumor rejection antigen for hepatocellular carcinoma (HCC). We recently completed vaccination of 10 AFP+/HLA-A2.1+ HCC subjects with AFP peptide-pulsed autologous dendritic cells (DC). There were increased frequencies of circulating AFP-specific T cells and of IFNγ-producing AFP-specific T cells after vaccination. In order to better understand the lack of association between immune response and clinical response, we have examined additional aspects of the AFP immune response in patients. Here, we have characterized the cell surface phenotype of circulating AFP tetramer-positive CD8 T cells and assessed AFP-specific CD4 function. Before vaccination, HCC subjects had increased frequencies of circulating AFP-specific CD8 T cells with a range of naïve, effector, central and effector memory phenotypes. Several patients had up-regulated activation markers. A subset of patients was assessed for phenotypic changes pre- and post-vaccination, and evidence for complete differentiation to effector or memory phenotype was lacking. CD8 phenotypic and cytokine responses did not correlate with level of patient serum AFP antigen (between 74 and 463,040 ng/ml). Assessment of CD4+ T cell responses by ELISPOT and multi-cytokine assay did not identify any spontaneous CD4 T cell responses to this secreted protein. These data indicate that there is an expanded pool of partially differentiated AFP-specific CD8 T cells in many of these HCC subjects, but that these cells are largely non-functional, and that a detectable CD4 T cell response to this secreted oncofetal antigen is lacking. [ABSTRACT FROM AUTHOR]

Late occurring CMV disease is an important problem after allogeneic SCT and has been associated with poor CMV-specific immunity. We conducted a prospective study of 58 patients studied at 3–6 months after allo-SCT, to base the antiviral therapy on monitoring of CMV-specific immunity. Reactivation of CMV was measured by quantitative PCR, and intracellular IFN-γ production was analysed by FACS and enzyme-linked immunospot. Antiviral therapy was deferred in patients with documented CMV-specific immunity without symptoms of CMV disease or severe GVHD. Nineteen episodes of CMV reactivation were assessable. The strategy was correctly applied in 16/19 episodes. Therapy was deferred in 5/19 (none of these patients developed CMV disease) and was given according to the strategy in 11/19 episodes. Two patients received antiviral therapy despite having T cell-specific immunity. There was a tendency that patients with late CMV reactivation had weak CD8 T cell immunity at 3 months (P=0.06). The donors' serostatus influenced the strength of both CD4 and CD8 immunity at 3 months after SCT (P<0.01). There was no effect as regards the type of conditioning, donor type, stem cell source or acute GVHD. Monitoring the immunity of SCT patients may allow more targeted use of antiviral therapy.Bone Marrow Transplantation (2007) 40, 865–869; doi:10.1038/sj.bmt.1705825; published online 27 August 2007 [ABSTRACT FROM AUTHOR]

Since the early 1990s, great efforts have been made in the field of human cytomegalovirus (HCMV) diagnosis. Besides HCMV diagnosis in immunosuppressed patients (solid organ transplant recipients, hematopoietic stem cell transplant patients and AIDS patients), diagnosis in connection with congenital HCMV infection is of great interest. This review focuses on the development and clinical utility of serological assays, as well as on virological tests (molecular and nonmolecular assays). Interpretation of these tests is strongly dependent on the patient group (solid organ transplant recipients and hematopoietic stem cell transplant patients) and whether the tests are used for screening, risk stratification or diagnosis. Furthermore, a better understanding of HCMV infection has led to new approaches in HCMV diagnosis and monitoring. Thus, assays for viral resistance testing and assays for monitoring the HCMV-specific cellular immune response are increasingly important for the guidance of antiviral therapy. [ABSTRACT FROM AUTHOR]

Abstract: Cell mediated immunity (CMI) was assessed by the ImmuKnow assay in 12 patients after kidney transplantation, who presented with viral infection. Treatment included lowering of immunosuppression in all cases and antiviral treatment if indicated. The assay was repeated during the follow up. The ImmuKnow assay at time of presentation of viral infections was 56.8±58.2 (range 3–178; median 22) ATP ng/ml. With the clearance of viral infection and lowering of immunosuppression, the assay showed an increase in the level of CMI at 194.5±118.9 (range 53–409; median 150) ATP ng/ml. There was viral clearance or stabilization in all cases and there was no incidence of allograft rejection. The ImmuKnow assay of CMI can be used to titrate initial immunosuppression reduction and its subsequent increase, in patients with viral infection after transplantation. [Copyright &y& Elsevier]

Introduction: The use of monoclonal antibodies against the α-chain of the membrane-bound interleukin-2 receptor (IL-2Rα) as immune suppressants causes characteristic changes in the levels of soluble interleukin-2 receptor (sIL-2R) in serum and urine. Methods: 38 kidney transplant patients were included in this study. 28 of them received an induction therapy with the IL-2R antibody basiliximax (Simulect®) in addition to standard immunosuppression, 10 patients constituted the control group. Results: Time courses of sIL-2R levels of Simulect patients with and without complications after transplantation have been compared. It turned out that of a total of 18 cases with complications 15 cases could be identified by their elevated sIL-2R levels, which corresponds to a sensitivity of 83%. Conclusion: Acute rejection, CMV infection, extrarenal bacterial infection and pyelonephritis in the transplant all cause a significant increase of the sIL-2R level even after application of Simulect. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Background: Monitoring recipient''s alloreactivity has shown to be critical for limiting overimmunosuppression besides allowing preemptive treatment of acute rejection (AR). Methods: Flow cytometry and real time RT-PCR were performed in urine of kidney transplant recipients with AR (n =13) and compared with pyelonephritis (n =10), chronic allograft nephropathy (n =13), acute tubular necrosis (n =13) and stable graft function (n =11). Expression of CD3, CD4, CD8, HLA-DR, Fas-L, ICAM-1 and CD25 were assessed using flow cytometry. mRNA of perforin, granzyme B and Fas-L were quantified by real time RT-PCR. Results: Frequencies of CD3+, HLA-DR+, Fas-L+, ICAM-1+ and CD25+ cells were significantly higher in AR group (p <0.05). ROC curves showed sensitivity from 70% to 91% and specificity from 30% to 100%, whereas the highest sensitivity and specificity was 91% and 100% respectively, for Fas-L+ cells. Levels of mRNA of perforin, granzyme B and Fas-L were significantly augmented in AR, while the sensitivity and specificity ranged from 85% to 88% and from 55% to 100%, respectively. Conclusions: Analyses of immune activation markers by flow cytometry and real time RT-PCR are equally useful for noninvasive monitoring kidney allografts. [Copyright &y& Elsevier]

Immunological monitoring of transplant recipients is an attractive concept. Cytokines provide an obvious focus for research, as they are central to the human immune response. This study aimed to identify cytokines whose sequential gene expression differentiated rejectors from non-rejectors immediately following renal transplantation. Forty-five renal transplant recipients (15 rejectors) and 13 living donors were recruited. Total RNA was extracted from the peripheral blood mononuclear cells and reverse transcribed. Cytokine gene expression levels of IL-4, IL-10, TNF-α and TGF-β1 were measured using TaqMan. IL-10 expression increased significantly following donor surgery. IL-4 and TNF-α patterns clearly differentiated between rejectors and non-rejectors. In the rejectors significant increases occurred more than 48 h before clinical graft dysfunction. Negative predictive values were 76% and 80% for IL-4 and TNF-α, respectively. This study has identified two cytokines (IL-4 and TNF-α) whose gene expression patterns differentiate between rejecting and non-rejecting renal transplant recipients making immunological monitoring possible. [ABSTRACT FROM AUTHOR]

The period of immune deficiency following stem cell transplantation (SCT) results in significant morbidity and mortality. Whilst supportive therapies have partially improved the outcome of infective episodes, disease relapse remains a considerable obstacle to improvement in overall outcomes. An increased understanding of the importance of the immune system in preventing relapse has derived from studies in the allogeneic setting. Increasing awareness of autologous anti-tumor responses has also focused interest on enhancing such activity. Successful application of some of these newer therapies, such as tumor vaccination approaches, may depend critically on reconstitution of functional immune reactivity. Whilst early recovery of innate immunity (myeloid series and natural killer (NK) cells) results in reconstitution of protective immunity against many bacterial pathogens, both the absolute levels and function of T and B lymphocytes remain abnormal for many months or years. Incorporation of T-cell depletion, choice of graft type (both donor and source), development of graft-vs.-host disease and level of residual thymic activity can all influence aspects of the reconstitution process. Advances in immunological monitoring are providing new insights, particularly into the recovery of specific T-cell subsets. This review focuses mainly on recent advances in the understanding of immune reconstitution in the allogeneic setting. [ABSTRACT FROM AUTHOR]

Zheng X-F, Pei G-X, Qiu Y-R, Zhu L-J, Gu L-Q. Serial monitoring of immunological parameters following human hand transplant. Clin Transplant 2004: 18: 119–123. © Blackwell Munksgaard, 2004 Although early successes have been achieved in human hand transplant, the changes of immunological parameters in the recipients and their relations to clinical events were not yet known. In two patients undergoing hand transplantation, we prospectively determined lymphocyte subsets using flow cytometry as well as the serum levels of interleukin (IL)-2, IL-10, tumor necrosis factor (TNF)- α, and interferon (IFN)- γ using enzyme linked immunosorbent assays during the first 6 months after transplantation. The decreases in CD , CD , CD T cell, the activated T cell (CD /CD , CD /HLA-DR+) as well as IL-2, IFN- γ and corresponding significant peak in IL-10 in human hand transplant during the first post-transplant week were observed. Then these parameters recovered to the pre-transplant level except for an even higher level of CD T cell. The low CD /CD ratio was been maintained constantly. After 7 wk, IL-2, IFN- γ, and IL-10 decreased to be maintained at a low or undetectable level except for slight increase in IL-10 at post-transplant month 5. There are no significant variation in TNF- α early after transplant. After 3 months, IL-10 was not detected again. The immunosuppressive agents had significantly effects on the immunological status in human hand transplant recipients. These profiles of immunological parameters would be useful data for the future immunomonitoring in human hand transplant recipients. [ABSTRACT FROM AUTHOR]

Inexpensive antiretroviral therapy (ART) might soon be available to treat human immunodeficiency virus (HIV) infections in resource-restricted areas of the globe. The number of CD4+ T-cells in the blood is the single most important laboratory parameter to select patients for therapy at the right time and to monitor the effect of ART. The question is asked whether flow cytometry is adaptable to change from an expensive and complicated scientific analytical instrumentation to become a practical diagnostic tool that can be widely operated. Recent studies indicate that a branch of clinical/practical flow cytometry is gaining a new identity by a confident simplification and improvement of clinical protocols. These new observations can be marshaled into three new areas of development. First, the changes in the choices of reagents and staining protocols have been initiated by the preferred use of primary immunological gating during the flow-cytometric analysis, exploiting the exquisite discriminating power of antibodies. The recent National Institutes of Health (NIH) and Centers for Disease Control and Prevention (CDC) guidelines introduced CD45-assisted protocols that are also at the heart of the PanLeucogating protocol that revitalized the testing of absolute CD4 counts on double-platform. With CD45 staining the leucocyte populations can be reliably studied in aging samples. It is now also documented that minimal technology, that is primary gating with CD4 (or CD8) antibodies, provides excellent CD4 (or CD8) T-cell counts. Consequently the optimal affordable protocol uses CD45 and CD4 antibodies in combination. The second area of development is the absolute counting facility on flow cytometers. Most frequently, microbeads are added to blood to define the sample volume in instruments that are not equipped with a volumetric microsyringe. The beads are, however, expensive, and the utilization of the stable flow rate of the excellent instruments might soon provide an answer to replace dear microbeads. With stable flow, the time span of analysis tells the sample volume and the absolute counts can be calculated. Finally, stabilized blood cell standards and blood stabilizing fluid, referred to as Transfix, have recently been introduced for quality assurance. These products are important to prove the precision and accuracy of the new affordable protocols and also to check, and guide if necessary, the performance of the laboratories even in remote areas. These recent developments provide evidence about the renaissance of affordable flow cytometry, a precise, costeffective and quantitative technology that is capable of providing CD4+ T-lymphocyte counts in as high volume as >400 patient assessment per day at the fraction of the cost of the current Western prices. These recent achievements need to be taken into consideration when alternative, nonflow CD4-counting methods are assessed. [Copyright &y& Elsevier]

In vivo-activated, committed donor-specific cytotoxic lymphocytes (cCTL) can be propagated and expanded from endomyocardial biopsies (EMB) in IL-2-enriched medium especially during an acute rejection episode. We report here our efforts to detect these cCTL by the same technique in peripheral blood at the moment of rejection and when no rejection was diagnosed. During or just before rejection, significantly less frequent (P < 0.01) donor reactive cCTL were found in PBL samples (two out of 20) than in the simultaneously taken EMB samples (13 out of 19). Donor B-LCL and/or third party B-LCL were lysed by 15 PBL samples. Inhibition studies revealed that this lysis was due to LAK-like cytotoxicity. The results show that peripheral blood does not reflect intra-graft events, which is probably the reason for the irreproducible results of diagnosis of rejection by monitoring immunological parameters in the peripheral blood. [ABSTRACT FROM AUTHOR]

In attempts to assess the possibility of allograft rejection, serial assays of spontaneous blastogenesis (SB) by uptake of tritium thymidine in 50 μl whole blood were performed in 72 consecutive renal allograft recipients. Of 79 clinically diagnosed rejection episodes, 65 (82 per cent) had an associated elevation of SB values, and 14 (18 per cent) showed false negative results. In 38 (59 per cent) of 65 episodes in which the elevations of SB values were observed, the elevations preceded the clinical diagnosis by 1-7 days. Although large doses of steroids did not affect the values of SB, false positive elevations which were mainly caused by systemic infections occurred in 12 cases. There was no significant association between severity of rejection crisis and the maximum value of SB in the course. The SB assay requiring only a small amount of blood can be accomplished within 4-5 hours and provides a simple means of assessing and predicting host's immune responsiveness to the histocompatibility antigens. [ABSTRACT FROM AUTHOR]