Cellular responses to cytomegalovirus in immunosuppressed patients: circulating CD8+ T cells recognizing CMVpp65 are present but display functional impairment

The availability of tetrameric complexes of HLA class I molecules folded with immunodominant peptides makes it possible to utilize flow cytometry for rapid and highly specific visualization of virus specific CD8+ T cells. An alternate technique is to incubate whole blood with specific antigens and to subsequently detect and characterize responding T cells (e.g. by performing intracellular staining of interferon-gamma). By using an HLA-A2 tetramer construct folded with the same immunodominant CMV-peptide as that used for peptide pulsing, we monitored both the presence and functional capacity of CMV-specific CD8+ T cells. In addition T cell activation was assayed by determination of CD38 and CD69 expression. Twelve organ transplant patients and 31 healthy blood donors with latent CMV infection were investigated using CMV pp65 tetramer staining and intracellular staining of interferon-gamma after CMV pp65 peptide pulsing or CMV lysate pulsing. CMV-specific T cells were detected in similar absolute numbers as well as frequencies of T cells in the two groups investigated. However, the CMV-specific CD8+ T cells in immunosuppressed individuals showed a decreased functional response to the CMV-peptide, as evidenced by reduced interferon-gamma production when compared to healthy blood donors (19%; 42%, P < 0.005). In addition, CD38 expression was markedly higher in immunosuppressed patients compared to healthy blood donors (24%; 6%, P < 0.005). In a case report we demonstrate that reactivation of CMV can occur in an immunosuppressed patient with high number of CMV-specific T cells, but without functional capacity. Hence, these findings reflect impaired activation of cytotoxic T cells controlling latent CMV infection in immunosuppressed patients.     

Cruciate ligament integrity in osteoarthritis of the knee

OBJECTIVE: To evaluate, using magnetic resonance imaging (MRI), the prevalence of anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) rupture in knees with symptomatic osteoarthritis (OA) compared with those without OA, and the relationship to pain and recalled injury. METHODS: MRI and plain radiography of the knee were performed in a group of 360 subjects with painful knee OA (cases; 66.7% male, mean age 67.1 years) and 73 without knee pain (controls; 57.5% male, mean age 66.1 years). MRIs were read for the presence or absence of complete or partial ACL or PCL tear. Subjects with knee pain were asked to quantify severity of pain on a visual analog scale and to report whether they could recall a significant knee injury (requiring use of a cane or crutches). We compared the prevalence of ACL and PCL rupture in those with and those without knee pain and also evaluated whether, in cases, there was any association with recalled knee injury. RESULTS: The proportion of cases who had complete ACL rupture was 22.8%, compared with 2.7% of controls (P = 0.0004). PCL rupture was rare both in cases (0.6%) and in controls (0%). Cases with ACL rupture had more severe radiologic OA (P < 0.0001) and were more likely to have medial joint space narrowing (P < 0.0001) than cases with intact ACLs, but did not have higher pain scores. Among cases, only 47.9% of those with complete ACL tears reported a previous knee injury, compared with 25.9% of those without complete ACL tears (P = 0.003). CONCLUSION: ACL rupture is more common among those with symptomatic knee OA compared with those without knee OA. Fewer than half of subjects with ACL rupture recall a knee injury, suggesting that this risk factor for knee OA is underrecognized.     

Simultaneous generation of cytomegalovirus-specific CD8+ and CD4+ T lymphocytes by use of dendritic cells comodified with pp65 mRNA and pp65 protein

Cytomegalovirus (CMV) disease remains a severe complication in patients who have undergone transplantation. Viremia can be prevented and treated by the adoptive transfer of donor-derived CMV-directed T cells. To ensure long-term protection against CMV disease, it is important to transfer CMV antigen-specific T cells that represent both the CD8+ and the CD4+ subsets. In the present study, we used as stimulators dendritic cells (DCs) that were electroporated with in vitro-transcribed 5'-capped polyadenylated messenger RNA (mRNA) that encoded the CMV pp65 protein (i.e., pp65 mRNA). These DCs could efficiently activate CMV-directed CD8+ T cells, as assayed by tetramer staining, interferon- gamma production, and cytolytic activity. We also used DCs that were pulsed with a recombinant pp65 protein to activate CMV-directed CD4+ T cells. When DCs were comodified with pp65 mRNA and pp65 protein, large numbers of CMV-directed CD8+ and CD4+ T cells were generated simultaneously. The approach outlined in the present study can be adapted for a clinical protocol that circumvents potential virus-related biohazards and is available to all patients independently of their human leukocyte antigen haplotype.     

Cell cycle progression of B-chronic lymphocytic leukemia cells induced to differentiate by TPA

The cell cycle transition and differentiation-associated surface antigen expression was studied in a clone of B cell chronic lymphocytic leukemia (B-CLL) with phenotypic properties similar to those of resting B lymphocytes. Differentiation was induced with TPA (12-O-tetradecanoyl- phorbol-13-acetate) and defined and quantitated by morphological and functional markers. Changes in the cell cycle position were determined by flow cytometry of acridine orange-stained cells. The uninduced B-CLL cells represented a homogeneous population with the same cell cycle position (GO) as resting normal peripheral blood lymphocytes. After five days of TPA stimulation, 56% of the B-CLL cells were found in G1A, 9% in G1B, and 3% in the S + G2/M phase, of which 2% was accounted to proliferating T cells. The cell cycle transition of the differentiating B-CLL cells was also examined using cell cycle-associated surface antigens as markers. HLA-DR and CD23 antigens were present already on noninduced cells. The former had a high constant expression, while the amount of CD23 increased upon induction. The 4F2 antigen was absent on noninduced cells but present on 86% of the induced cells. HH1 (CD37) was expressed by the majority of the cells before TPA treatment and decreased to almost undetectable levels within 24 hours. Two antigens related to late stages of the cell cycle, the interleukin 2 (IL 2; CD25) and the transferrin receptor, were present on about 20% of the induced cells. Experiments with enriched T cells showed that T but not B cells incorporated 3H-thymidine. Taken together these results and previous work on the induction of the protooncogene c-myc and c-fos suggest that this B-CLL clone represents GO cells that undergo differentiation without concomitant proliferation when exposed to TPA.     

Computed tomography to assess pelvic lysis after total hip replacement

To assess the accuracy of a computer-assisted computed tomography image analysis program in determining the location and volume of periacetabular osteolysis, we designed an osteolysis model by implanting bilateral total hip replacements in human pelvic cadavers and creating osteolytic lesions of varying sizes. The volumes of 48 defects were measured physically, and axial computed tomography scans were obtained. The computed tomography images were processed with streak artifact reduction and segmentation algorithms. The location and volume of lesions were determined from these images. Eighty-one percent (39 lesions) were identified correctly from the computed tomography scans. Detection was location-dependent. More lesions were detected in the ilium (100%) and at the rim (89%) than in the ischium (78%) or the pubis (50%). Computed tomography overestimated lesion volume by a mean of 0.5 +/- 2.3 cm. The volumetric error was unrelated to lesion location but was dependent on lesion size. As lesion size increased above 10 cm, the mean percentage error decreased to 1.8%. Computed tomography image analysis can be used more accurately than plain radiographs to investigate the effectiveness of treatment and the natural history of pelvic osteolysis.     

Accuracy and reproducibility of manual and semiautomated quantification of MS lesions by MRI

PURPOSE: To evaluate the accuracy, reproducibility, and speed of two semiautomated methods for quantifying total white matter lesion burden in multiple sclerosis (MS) patients with respect to manual tracing and to other methods presented in recent literature. MATERIALS AND METHODS: Two methods involving the use of MRI for semiautomated quantification of total lesion burden in MS patients were examined. The first method, geometrically constrained region growth (GEORG), requires user specification of lesion location. The second technique, directed multispectral segmentation (DMSS), requires only the location of a single exemplar lesion. Test data sets included both clinical MS data and MS brain phantoms. RESULTS: The mean processing times were 60 minutes for manual tracing, 10 minutes for region growth, and 3 minutes for directed segmentation. Intra- and interoperator coefficients of variation (CVs) were 5.1% and 16.5% for manual tracing, 1.4% and 2.3% for region growth, and 1.5% and 5.2% for directed segmentation. The average deviations from manual tracing were 9% for region growth and 5.7% for directed segmentation. CONCLUSION: Both semiautomated methods were shown to have a significant advantage over manual tracing in terms of speed and precision. The accuracy of both methods was acceptable, given the high variability of the manual results.     

3D interleaved water and fat image acquisition with chemical-shift correction.

A new technique, 3D interleaved water and fat image acquisition with chemical-shift correction (3-DIWFAC), was developed to acquire 3D water and fat images in a single acquisition time and to combine the water and fat images to produce chemical-shift-free images. A 3D gradient-recalled-echo (GRE) sequence was implemented with a 1-3-3-1 binomial Shinnar-Le Roux spatial-spectral excitation, and with interleaved phase-encoding lines that alternate between water and fat excitations separated by half TR. Water-only and fat-only images were then realigned to remove chemical shift artifacts. Results from phantoms and human subjects demonstrated that the image contrast was the same as in the regular GRE sequence. With the chemical shift corrected, the shadow artifacts often seen at water and fat boundaries were removed. Since this sequence simultaneously provides water-only images showing cartilage and bone lesions, and water-fat images that depict soft tissue anatomy, it may be clinically useful in musculoskeletal imaging. Magn Reson Med 44:322-330, 2000.     

Diagnosis of femoropopliteal venous thrombosis with MR imaging: a comparison of four MR pulse sequences

In a prospective study, MR images were evaluated in seven patients with femoropopliteal venous thrombosis with symptoms of less than 5 days duration. T1-weighted (600/25 [TR/TE]), intermediate (2000/30), and T2-weighted (2000/100) spin-echo series and a gradient-recalled acquisition in the steady state (GRASS) series were compared. Using venography as the standard for diagnosis, we found GRASS to be the most sensitive of the MR techniques, showing thrombi in all patients. It provided good contrast between the low-intensity thrombus and high-intensity flowing blood and also between thrombus and intermediate- or high-intensity perivascular tissues. The T1-weighted series was the least sensitive technique. All thrombi showed heterogeneity in the transaxial image with differences in signal between the peripheral and central regions. A higher intensity signal in the center than in the periphery at some level of the thrombus was found in six of seven T2-weighted or GRASS images. Heterogeneity in the signal intensity was more frequent in distal portions of thrombi, whereas the most proximal extent was homogeneous in appearance in six of seven cases. The heterogeneous appearance may be related to the greater age of the distal thrombus, because deep venous thrombi are known to begin in the calf and extend proximally over time. We conclude, on the basis of our experience with a small number of patients, that the GRASS MR technique is more sensitive for detecting acute deep venous thrombosis than T1-weighted, intermediate, and T2-weighted MR images.