BK Polyomavirus-Specific 9mer CD8 T Cell Responses Correlate With Clearance of BK Viremia in Kidney Transplant Recipients: First Report From the Swiss Transplant Cohort Study.

BK polyomavirus (BKPyV) causes premature kidney transplant (KT) failure in 1-15% of patients. Because antivirals are lacking, most programs screen for BKPyV-viremia and, if positive, reduce immunosuppression. To evaluate the relationship of viremia and BKPyV-specific immunity, we examined prospectively cryopreserved plasma and peripheral blood mononuclear cells at the time of transplantation (T0) and at 6 mo (T6) and 12 mo (T12) after transplant from 28 viremic KT patients and 68 nonviremic controls matched for the transplantation period. BKPyV IgG seroprevalence was comparable between cases (89.3%) and controls (91.2%; p = 0.8635), but cases had lower antibody levels (p = 0.022) at T0. Antibody levels increased at T6 and T12 but were not correlated with viremia clearance. BKPyV-specific T cell responses to pools of overlapping 15mers (15mer peptide pool [15mP]) or immunodominant CD8 9mers (9mer peptide pool [9mP]) from the early viral gene region were not different between cases and controls at T0; however, clearance of viremia was associated with stronger 9mP responses at T6 (p = 0.042) and T12 (p = 0.048), whereas 15mP responses were not informative (T6 p = 0.359; T12 p = 0.856). BKPyV-specific T cells could be expanded in vitro from all patients after transplant, permitting identification of 78 immunodominant 9mer epitopes including 50 new ones across different HLA class I. Thus, 9mP-responses may be a novel marker of reconstituting CD8 T cell function that warrants further study as a complement of plasma BKPyV loads for guiding immunosuppression reduction.

The "ABC" of Virus-Specific T Cell Immunity in Solid Organ Transplantation.

Transplant patients are at increased risk of viral complications due to impaired control of viral replication, resulting from HLA mismatching between graft and host and the immunosuppression needed to avert alloimmune reactions. In the past decade, quantitative viral load measurements have become widely available to identify patients at risk and to inform treatment decisions with respect to immunosuppressive drugs and antiviral therapies. Because viral loads are viewed as the result of viral replication and virus-specific immune control, virus-specific T cell monitoring has been explored to optimize management of adenovirus, BK polyomavirus and cytomegalovirus ("ABC") in transplant patients. Although most studies are descriptive using different technologies, the overall results show that the quantity and quality of virus-specific T cells inversely correlate with viral replication, whereby strong cellular immune responses are associated with containment of viral replication. The key obstacles to the introduction of assays for virus-specific T cells into clinical practice is the definition of reliable cutoffs for clinical decision making, the poor negative predictive value of some assays, and the absence of interventional trials justifying changes of antiviral treatment or immunosuppression. More clinical research is needed using optimized assays and targets before standardization and commutability can be envisaged as achieved for viral load testing.

Characterization of Immunodominant BK Polyomavirus 9mer Epitope T Cell Responses.

Uncontrolled BK polyomavirus (BKPyV) replication in kidney transplant recipients (KTRs) causes polyomavirus-associated nephropathy and allograft loss. Reducing immunosuppression is associated with clearing viremia and nephropathy and increasing BKPyV-specific T cell responses in most patients; however, current immunoassays have limited sensitivity, target mostly CD4(+) T cells, and largely fail to predict onset and clearance of BKPyV replication. To characterize BKPyV-specific CD8(+) T cells, bioinformatics were used to predict 9mer epitopes in the early viral gene region (EVGR) presented by 14 common HLAs in Europe and North America. Thirty-nine EVGR epitopes were experimentally confirmed by interferon-γ enzyme-linked immunospot assays in at least 30% of BKPyV IgG-seropositive healthy participants. Most 9mers clustered in domains, and some were presented by more than one HLA class I, as typically seen for immunodominant epitopes. Specific T cell binding using MHC class I streptamers was demonstrated for 21 of 39 (54%) epitopes. In a prospective cohort of 118 pediatric KTRs, 19 patients protected or recovering from BKPyV viremia were experimentally tested, and 13 epitopes were validated. Single HLA mismatches were not associated with viremia, suggesting that failing immune control likely involves multiple factors including maintenance immunosuppression. Combining BKPyV load and T cell assays using immunodominant epitopes may help in evaluating risk and reducing immunosuppression and may lead to safe adoptive T cell transfer.

KIR3DL2 (CD158k) is a potential therapeutic target in primary cutaneous anaplastic large-cell lymphoma.

BACKGROUND: KIR3DL2, an inhibitory receptor expressed by natural killer cells and a subset of normal CD8(+) T cells, is aberrantly expressed in neoplastic cells in transformed mycosis fungoides and Sézary syndrome. Anti-KIR3DL2 targeted antibody therapy has shown potent activity in preclinical models for these diseases. OBJECTIVES: To examine the expression of KIR3DL2 and its potential use as a therapeutic target in patients with primary cutaneous anaplastic large-cell lymphoma (pcALCL), the most aggressive cutaneous CD30(+) lymphoproliferative disease. METHODS: Samples from 11 patients with pcALCL and three CD30(+) lymphoproliferative disease cell lines - Mac1, Mac2a and Mac2b - were used in KIR3DL2 expression studies using immunohistochemistry, flow cytometry and reverse-transcriptase quantitative polymerase chain reaction. The effect of IPH4102, a monoclonal humanized IgG1 targeting KIR3DL2, was assessed by in vitro cytotoxicity assays against Mac1, Mac2a and Mac2b using allogeneic peripheral blood mononuclear cells as effectors. RESULTS: KIR3DL2 mRNA and protein were found in all human samples of pcALCL, and in the Mac2a and Mac2b cell lines. KIR3DL2 protein expression was present on 85·8 ± 14·0% of CD30(+) skin-infiltrating tumour cells. In vitro functional studies showed that KIR3DL2(+) Mac2a and Mac2b pcALCL lines are sensitive to antibody-derived cytotoxicity mediated by IPH4102, through activation of natural killer cells, in a concentration-dependent manner. CONCLUSIONS: pcALCL tumour cells express KIR3DL2, and we provide preclinical proof of concept for the use of IPH4102, a humanized anti-KIR3DL2 antibody, to treat patients with primary cutaneous CD30(+) ALCL.

Prevention of hepatitis C virus infection by adoptive allogeneic immunotherapy using suicide gene-modified lymphocytes: an in vitro proof-of-concept.

Hepatitis C virus (HCV)-induced, end-stage liver disease is a major indication for liver transplantation, but systematic graft reinfection accelerates liver disease recurrence. Transplantation recipients may be ineligible for direct-acting antivirals, owing to toxicity, resistance or advanced liver disease. Adoptive immunotherapy with liver graft-derived, ex vivo-activated lymphocytes was previously shown to prevent HCV-induced graft reinfections. Alternatively, the applicability and therapeutic efficacy of adoptive immunotherapy may be enhanced by 'ready for use' suicide gene-modified lymphocytes from healthy blood donors; moreover, conditional, prodrug-induced cell suicide may prevent potential side effects. Here, we demonstrate that allogeneic suicide gene-modified lymphocytes (SGMLs) could potently, dose- and time-dependently, inhibit viral replication. The effect occurs at effector:target cell ratios that exhibits no concomitant cytotoxicity toward virus-infected target cells. The effect, mediated mostly by CD56+ lymphocytes, is interleukin-2-dependent, IFN-γ-mediated and, importantly, resistant to calcineurin inhibitors. Thus, post-transplant immunosuppression may not interfere with this adoptive cell immunotherapy approach. Furthermore, these cells are indeed amenable to conditional cell suicide; in particular, the inducible caspase 9 suicide gene is superior to the herpes simplex virus thymidine kinase suicide gene. Our data provide in vitro proof-of-concept that allogeneic, third-party, SGMLs may prevent HCV-induced liver graft reinfection.

Human de novo papillary renal-cell carcinomas in a kidney graft: evidence of recipient origin with adenoma-carcinoma sequence.

Papillary renal-cell carcinoma (pRCC) is unusual for its occurrence in kidneys with chronic dysfunction, for its frequent multifocality and for its common association with papillary adenoma, a benign renal lesion morphologically indistinguishable from pRCC. Concomitant development of papillary adenoma and pRCC in five transplanted kidneys, where donor and recipient characteristics are well established, provided a unique opportunity for molecular studies of de novo pRCC carcinogenesis. We aimed to study this tumor type to determine whether or not the different papillary tumors have the same origin, and whether or not papillary adenomas are precursor lesions of pRCC. We performed XY-FISH in sex-mismatched kidney transplants, and polymorphic microsatellite DNA and high-resolution melting of mitochondrial DNA analyzes in all five patients on laser-microdissected tumor cells, then compared these molecular profiles to donor and recipient profiles. This study (i) identified the recipient origin of de novo papillary adenomas and pRCCs in a kidney transplant, (ii) demonstrated an identical origin for precursor cells of papillary adenomas and pRCCs and (iii) showed additional genetic alterations in pRCCs compared to papillary adenomas. This molecular approach of papillary tumors developed in transplanted kidney identified successive steps in carcinogenesis of human de novo papillary renal-cell carcinoma.

Endothelial cell apoptosis in severe drug-induced bullous eruptions.

BACKGROUND: Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are characterized by extensive keratinocyte apoptosis mediated by cytotoxic proteins. Similar features have been found in another severe dysimmune syndrome, allogeneic acute graft-versus-host disease, where endothelial cell apoptosis has been recently characterized. OBJECTIVES: To determine whether endothelial cell apoptosis occurs in dermal vessels of TEN and SJS, and whether it is linked to expression of cytotoxic proteins. METHODS: Skin biopsies of eight patients with severe drug-induced bullous eruptions (four TEN, four SJS), eight with drug-induced urticaria and eight healthy controls were compared. Blood vessel damage was studied by electron microscopy and quantified by CD31 immunostaining. Apoptotic cells, characterized by electron microscopy, were quantified on terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling assay. Immunohistochemistry was also used to characterize and quantify inflammatory cells and granzyme B, tumour necrosis factor (TNF)-alpha and Fas ligand (FasL) expression. RESULTS: Endothelial cell apoptosis was observed in all TEN and SJS cases: it occurred in 85% of the vessel sections. It occurred in one case of drug-induced urticaria, in 5% of vessel sections, but not in healthy controls. Numbers of CD68+ macrophages and CD8+ T lymphocytes were significantly higher in TEN and SJS compared with both other groups; granzyme B and TNF-alpha but not FasL were expressed. CONCLUSIONS: Characterization of endothelial cell apoptosis in TEN and SJS is important to assess a factor worsening skin damage, with possible extension to other organs. It may also be useful for the development of novel therapeutic strategies.

Cloning, sequencing and characterization of the ccpA gene from Enterococcus faecalis.

Enzymes involved in the metabolism of complex carbon and energy sources are unnecessary under conditions of abundant, readily metabolisable nutrients such as glucose or fructose. The repression of these enzymes by glucose has been termed carbon catabolite repression. Mechanisms involved in the carbon catabolite repression in gram-positive bacteria are known to differ from those of gram-negative bacteria such as Escherichia coli. It appears to be mediated by transcriptional repression, requiring trans-acting CcpA, a member of the LacI-GalR family of bacterial regulatory proteins and a cis-acting consensus sequence, designated cre. Here, we report the cloning and characterisation of the chromosomal ccpA gene from Enterococcus faecalis JH2-2. This gene is predicted to encode a 333 amino acids protein with nearly 75% identity to CcpA of Lactobacillus casei.

Histochemical and immunohistochemical protocols for routine biopsies embedded in Lowicryl resin.

Tissue processing and analysis require good preservation of both the shape and content of cells. Lowicryl resin is one of the few embedding media that allow good preservation of both tissue architecture and cellular contents. Therefore, different histochemical and immunohistochemical reactions can be applied to semithin sister sections from one biopsy. Further examination of a zone of interest can be carried out under the electron microscope. The hydrophilic property of Lowicryl resins makes possible different histochemical reactions; however, the technique used for paraffin sections must be adapted for each reaction. Antigenic preservation of cells by low temperature embedding allows immunolabeling on either semithin sections or in the zone of interest on ultrathin sections. We have shown the application and adaptation of different histochemical and immunohistochemical reactions on semithin and ultrathin sections from hepatic biopsies that were large, but thin. The variety of techniques that can be used on sister Lowicryl sections of a single biopsy makes this medium useful for extensive pathological studies of precious needle biopsies.

MicroRNA187 overexpression is related to tumor progression and determines sensitivity to bortezomib in peripheral T-cell lymphoma.

MicroRNAs (miRs) are involved in tumorigenesis by regulating tumor suppressor genes and/or oncogenes. MiR187 was overexpressed in peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) and associated with high Ki67 expression, elevated lactate dehydrogenase, advanced International Prognostic Index and poor prognosis of patients. In vitro, ectopic expression of miR187 in T-lymphoma cell lines accelerated tumor cell proliferation, whereas treatment with miR187 inhibitor reduced cell growth. MiR187 downregulated tumor suppressor gene disabled homolog-2 (Dab2), decreased the interaction of Dab2 with adapter protein Grb2, resulting in Ras activation, phosphorylation/activation of extracellular signal-regulated kinase (ERK) and AKT, and subsequent stabilization of MYC oncoprotein. MiR187-overexpressing cells were resistant to chemotherapeutic agents like doxorubicin, cyclophosphamide, cisplatin and gemcitabine, but sensitive to the proteasome inhibitor bortezomib. Bortezomib inhibited T-lymphoma cell proliferation by downregulating miR187, dephosphorylating ERK and AKT and degrading MYC. In a murine xenograft model established with subcutaneous injection of Jurkat cells, bortezomib particularly retarded the growth of miR187-overexpressing tumors, consistent with the downregulation of miR187, Ki67 and MYC expression. Collectively, these findings indicated that miR187 was related to tumor progression in PTCL-NOS through modulating Ras-mediated ERK/AKT/MYC axis. Although potentially oncogenic, miR187 indicated the sensitivity of T-lymphoma cells to bortezomib. Cooperatively targeting ERK and AKT could be a promising clinical strategy in treating MYC-driven lymphoid malignancies.

[Optical biometry of eyes corrected by phakic intraocular lenses]. / Biométrie optique des yeux avec implants phaques.

PURPOSE: The aim of this study was to determine whether eye length measurements obtained with the IOL Master (Zeiss Humphrey) before and after phakic IOL implantation would show any changes. METHODS: In a prospective study, we used the IOL Master to measure optical biometry in 25 myopic eyes of 15 patients before and after phakic IOL implantation (PRL, ICL, Artisan). The differences between both axial length measurements were calculated and compared using a nonparametric Wilcoxon test. RESULTS: The difference between the preoperative and postoperative measurements ranged from -0.16 mm to 0.06 mm and averaged -0.016 mm, which was not statistically significant (p=0.20). Both measurements correlated in a highly positive manner (r=0.999; p<0.0001). The reproducibility of the preoperative and postoperative axial length measurements was very high (coefficient of variation=0.09% and 0.07%, respectively). The precision was 26 microm for preoperative measurements and 19 microm for postoperative measurements. CONCLUSION: Our results showed that postoperative measurements of axial length are highly comparable to preoperative measurements and that optical biometry can achieve highly precise and reliable axial length measurements in eyes with phakic IOLs. This application becomes clinically relevant in evaluating eyes with phakic IOLs that might require cataract surgery. Hence, accurate axial length measurements in eyes with phakic IOLs will be extremely important when cataract occurs in these eyes and when the preoperative measurements are no longer available.

[Ultrasound biomicroscopy of posterior chamber phakic intraocular lenses: a comparative study between ICL and PRL models]. / Biomicroscopie ultrasonore des implants phaques de chambre postérieure: étude comparative des modèles ICL et PRL.

PURPOSE: To evaluate the anatomic relationships of the implantable contact lens (ICL) and the phakic refractive lens (PRL) posterior chamber phakic intraocular lenses (PCP IOL) using ultrasound biomicroscopy (UBM). MATERIAL AND METHODS: Seventeen phakic myopic eyes corrected with ICL, and 14 phakic myopic eyes that had had PRL implantation, were examined retrospectively using UBM. The main parameters measured and compared were anterior chamber depth, central and peripheral distance between PCP IOL and the crystalline lens, and exact lens haptic position. RESULTS: The mean distance between the PCP IOL and the central endothelium was 2398+/-203 microm and 2640+/-230 microm in the ICL and PRL groups, respectively. The central vault between the implant and the crystalline lens was greater in eyes with ICL (ICL, 402+/-194 microm; PRL, 256+/-187 microm, p<0.05). However, the incidence of lens contact on the peripheral level was higher in the ICL group (41%) than in the PRL group (29%), and the difference between the two implants in the peripheral crystalline lens-PCP IOL distance was significant (p<0.05). Both IOL haptics appeared to be correctly positioned in the sulcus in 13 (76%) eyes of the ICL group, and on the zonule in eight eyes (57%) of the PRL group. CONCLUSIONS: PCP IOL implantation is a safe procedure for the correction of high myopia with regard to refractive results. UBM provides a unique tool to noninvasively evaluate the relations of these implants within the posterior chamber, and helps to analyze the mechanisms of crystalline lens and iris complications.

The reliability of specific sacro-occipital technique diagnostic tests.

Four interexaminer and one intraexaminer agreement studies were performed on specific diagnostic tests commonly employed within sacro-occipital technique (SOT). Ten of the tests were evaluated in more than one interexaminer study. Of these, only one test (bilateral supine leg raise with cervical compaction) had at least fair reliability more than once. Six of these 10 tests obtained poor agreement in more than one study. One examiner out of two had a number of excellent and fair intraexaminer values, whereas the other examiner generally had poor results. There may have been some treatment effect as a comparison of the combined intraexaminer diagnosis for two observers after no treatment and after treatment showed that the repeatability diminished from Kappa of 0.36 in untreated cases (which were expected to have high agreement of before and after treatment findings) to a Kappa of 0.27 for those subjects having received treatment (which were expected to have low agreement of before and after treatment findings). It appears unlikely that SOT tests can be reproduced to a sufficiently high degree to constitute useful clinical procedures.

Low back pain.

Low back pain (LBP) affects a large proportion of the population and is an increasingly costly problem in the western world. This review highlights some of the recent theories relating to LBP and the conflicting evidence which has been brought to light. Theories relating to the causes of LBP have included single and multiple factors such as abnormal physical findings, mechanical, psychosocial and economic factors. The two lowest lumbar segments are most often afflicted in LBP sufferers, but the diagnosis of LBP is otherwise uncertain in most cases due to insufficient knowledge relating to the validity of clinical tests, inconsistent terminology and unclear symptomatic patterns. Despite a lack of understanding of the exact anatomical cause of LBP, an abundance of therapeutic models exist, most of which are purely empirical, and few methods have been shown to be clinically useful. Similarly, insufficient knowledge of the causes of LBP makes primary, secondary and tertiary prevention difficult.

The sensitivity and specificity of seven lumbo-pelvic orthopedic tests and the arm-fossa test.

Twenty-nine lumbosacral asymptomatic and 39 symptomatic patients who attended a chiropractic clinic were examined by a practitioner who was blinded to their symptoms. Seven lumbosacral orthopedic tests, along with the arm-fossa test were scrutinized for sensitivity, specificity and diagnostic competency values. Only the arm-fossa test and heel-buttock tests had a significantly higher percentage of positive findings in symptomatic than asymptomatic cases. These same tests were the only ones which could be considered to have an acceptable diagnostic value, when both the sensitivity and specificity were taken into consideration by Youden's index. The number of positive tests was unrelated to the presence of lumbosacral symptoms. Orthopedic tests which appeared to strain several adjacent anatomical structures were most commonly positive. No particular combination of tests could predict if the patient was symptomatic or asymptomatic. Only the heel-buttock test had some predictive value. It appears that these tests were of limited value in differentiating between the symptomatic and asymptomatic subjects who attended the study clinic.

A review of data reports published in the Journal of Manipulative and Physiological Therapeutics from 1986 to 1988.

Forty-six data reports which appeared in the Journal of Manipulative and Physiological Therapeutics from 1986 to 1988 were reviewed according to specific methodological criteria. There appears to be a need for improved study design in contemporary chiropractic research. Experimental and quasi-experimental studies were most commonly deficient in the areas of reliable methods of measurement, the use of blind assessors and blind, or at least naive, study subjects. Common flaws in surveys were: failure to test the survey instrument prior to the study, not stating the response rate, and omitting discussion of the question of confidentiality. The results confirmed findings obtained in a similar study of data reports of an Australian peer-reviewed chiropractic journal. The use of various types of checklists is recommended for authors and reviewers of chiropractic scientific material.

A review of the research papers published by the international College of Applied Kinesiology from 1981 to 1987.

Applied Kinesiology (AK) is a diagnostic and therapeutic approach used by a large number of chiropractors. AK seminars are conducted worldwide; during these seminars mention is frequently made of the presence of supportive research. A review was undertaken of the type and scientific quality of 50 papers which had been published between 1981 and 1987 by the International College of Applied Kinesiology, 20 of which were classified as research papers. These were subjected to further scrutiny relating to criteria considered crucial in research methodology, namely, a clear identification of sample size, inclusion criteria, blind and naive subjects and statistical analysis. Although some papers satisfied several of these criteria, none satisfied all seven of them. As none of the papers included adequate statistical analyses, no valid conclusions could be drawn concerning their report of findings.

Prevalence of spondylolisthesis, transitional anomalies and low intercrestal line in a chiropractic patient population.

Five hundred and thirty radiographs were screened for the presence of certain lumbosacral anomalies. The prevalence of spondylolisthesis was found to be 5.1%, lumbarization 6.0%, sacralization 5.5% and low intercrestal line 56.9%. There was no greater prevalence in patients suffering from low back pain when compared against those who did not. There was a propensity for a low intercrestal line among females. Contrary to previous claims that lumbarization is more common in men, we found a moderate predilection for this finding among women. No difference between the two sexes was found in the prevalence of sacralization, contradicting previous claims that is more common in females, nor was spondylolisthesis found more frequently in men, contrary to our expectations.

A survey of recently graduated chiropractors in Australia.

A survey was performed in Australia to study recently graduated chiropractors. Five general themes were investigated: a) personal and professional demographic profile, b) personal and professional attitudes, c) referral patterns, d) diagnostic procedures, and e) therapeutic procedures. Consideration is given to the possible development of the chiropractic profession in Australia.