Hand-assisted Laparoscopic Donor Nephrectomy and Cytokine Changes.

BACKGROUND: Laparoscopic living-donor nephrectomy (LDN) exerts systemic effects causing transaminitis and increased urinary neutrophil gelatinase-associated lipocalm (NGAL) excretion. Hand-assisted laparoscopic donor nephrectomy, which tends to be shorter with less pneumoperitoneum, may be hypothesized to produce less systemic stimulation than total laparoscopic LDN. METHODS: Serial urine and serum samples were collected from 15 patients undergoing HALDN. Samples were analyzed for NGAL and kidney injury molecule 1 (KIM-1) levels preoperatively and 24 hours post-surgery. Data relating to alanine aminotransferase, creatinine, and estimated glomerular filtration rate was also analyzed in 48 live donors preoperatively and at 24 hours and 48 hours post-surgery and compared to published data on LDN. RESULTS: Expected changes to creatinine and estimated glomerular filtration rates were observed in the donors. Compared to the preoperative levels, alanine aminotransferase levels showed a significant decrease at 24 hours (P = .004) and were not significantly different from baseline levels at 48 hours (P = .08). Serum KIM-1 and NGAL levels remained unchanged (P = .89 and P = .14, respectively) at 24 hours after donation. Similarly, urinary levels of KIM-1 and NGAL were not statistically significantly different after donation. Mean operating time for this cohort was 1 hour, 36 minutes. CONCLUSIONS: In contrast to other published data, our cohort did not exhibit changes to liver function tests or biomarker changes after donor nephrectomy. This could be because of the lower operative time (96 minutes vs. 216 minutes) or because of the intermittent release of the pneumoperitoneum in the hand-assisted method which may exert less of a systemic inflammatory response.

In treatment-naïve and antiretroviral-treated subjects with HIV, reduced plasma adiponectin is associated with a reduced fractional clearance rate of VLDL, IDL and LDL apolipoprotein B-100.

AIMS/HYPOTHESIS: We hypothesised that loss of peripheral fat in HIV patients would result in decreased plasma adipocytokines, in particular adiponectin, and that this decrease would be associated with changes in VLDL, IDL and LDL apolipoprotein B kinetics. METHODS: Plasma adiponectin, leptin and other cytokines were measured in uninfected control subjects (n=12) and three HIV-positive groups comprising treatment-naïve patients (n=15) and patients on triple antiretroviral therapy containing protease inhibitors (PI, n=15) or non-nucleoside reverse transcriptase inhibitors (NNRTI, n=25). VLDL, IDL and LDL apolipoprotein B kinetics were measured with an infusion of [1-(13)C] leucine. Regional body fat was measured with a dual energy X-ray absorptiometry scan. Insulin resistance was calculated using homeostasis model assessment (HOMA). RESULTS: Adiponectin (median [interquartile range]) was reduced in the treatment-naive (5.4 microg/ml [4.7-8.5]), PI (5.0 microg/ml [3.3-6.4]) and NNRTI (5.0 microg/ml [3.1-6.7]) groups compared with controls (9.7 microg/ml [6.9-13.3]) (p<0.05). In all subjects adiponectin correlated positively with HDL-cholesterol levels, the VLDL, IDL and LDL apolipoprotein B fractional clearance rates, and with the limb fat:lean body mass ratio (all p<0.01). Adiponectin correlated negatively with plasma triglyceride levels and HOMA (p<0.001). In a linear regression model that included HOMA, adiponectin was an independent predictor of VLDL and HDL-cholesterol levels and the IDL fractional clearance rate. TNF was higher in treatment-naive and PI subjects, and soluble TNF receptor superfamily, members 1A and 1B (previously known as TNF receptors 1 and 2) was higher in PI patients than in control subjects (p<0.05). CONCLUSIONS/INTERPRETATION: Adiponectin levels are significantly reduced in treated and untreated HIV patients and are predictive of VLDL and IDL apolipoprotein B fractional clearance rates. Adiponectin may have a direct effect on lipoprotein metabolism, which may be independent of insulin.

Effects of resistance training on myosin function studied by the in vitro motility assay in young and older men.

It is generally believed that the maximum shortening velocity (V(o)) of a skeletal muscle fiber type does not vary unless a change in myosin heavy chain (MHC) isoform composition occurs. However, recent findings have shown that V(o) of a given fiber type can change after training, suggesting the hypothesis that the function of myosin can vary without a change in isoform. The present study addressed the latter hypothesis by studying the function of isolated myosin isoforms by the use of the in vitro motility assay (IVMA) technique. Four young (age 23-29 yr, YO) and four elderly men (age 68-82 yr, EL) underwent a 12-wk progressive resistance training program of the knee extensor muscles and to one pre- and one posttraining biopsy of the vastus lateralis muscle. The significant increase in one-repetition maximum posttraining in both YO and EL indicated that training was effective. After training, MHC isoform composition showed a shift from MHC(2X) toward MHC(2A) in YO and no shift in EL. The velocity of sliding (V(f)) of actin filaments on pure myosin isoforms extracted from single fibers was studied in IVMA. One hundred sixty IVMA samples were prepared from 480 single fibers, and at least 50 filaments were analyzed in each experiment. Whereas no training-induced change was observed in V(f) of myosin isoform 1 either in YO or in EL, a significant increase in V(f) of myosin isoform 2A after training was observed in both YO (18%) and EL (19%). The results indicate that resistance training can change the velocity of the myosin molecule.

Power output of the lower limb during variable inertial loading: a comparison between methods using single and repeated contractions.

The power-inertial load relationship of the lower limb muscles was studied during a single leg thrust using the Modified Nottingham Power Rig (mNPR) and during cycling exercise in nine young male subjects. The relationship between peak power and inertial load showed a parabolic-like relationship for mNPR exertions, with a peak [937 (SD 246) W] at 0.158 kg m(2), this being significantly (P <0.05) different from the power generated at both the lowest [723 (162) W] and highest [756 (206) W] inertial loads. In contrast, for cycling exercise power output did not differ significantly between inertial loads, except at the lowest inertia where power output was significantly ( P<0.05) less compared with all other inertial loads. Maximum peak power output during cycling was 1,620 (336) W, which was significantly (P <0.05) greater than that recorded on the mNPR. However, a close association was observed between the mean power generated by each method (r=0.84, P<0.05). The results suggest that during a single contraction a range of inertial loads is required to allow peak power to be expressed. Above a certain critical value, this is unnecessary during cycling movements where the load can be repeatedly accelerated.

The cellular immunotherapy of viral infection.

Viral infections remain a major problem for the patient who has undergone a stem cell transplant and the introduction of increasingly more intensive SCT regimens suggests that this will remain a significant concern for the immediate future. Advances in basic immunology have led to an improved understanding of how immune responses control individual viral infections and this information is now being directly applied to patient care with encouraging results. The application of cellular immunotherapy to the immunocompromised patient is one of the most exciting areas of clinical medicine and offers the prospect of significant clinical impact.

Expression of IGF-I splice variants in young and old human skeletal muscle after high resistance exercise.

The mRNA expression of two splice variants of the insulin-like growth factor-I (IGF-I) gene, IGF-IEa and mechano growth factor (MGF), were studied in human skeletal muscle. Subjects (eight young, aged 25-36 years, and seven elderly, aged 70-82 years) completed 10 sets of six repetitions of single legged knee extensor exercise at 80 % of their one repetition maximum. Muscle biopsy samples were obtained from the quadriceps muscle of both the control and exercised legs 2.5 h after completion of the exercise bout. Expression levels of the IGF-I mRNA transcripts were determined using real-time quantitative RT-PCR with specific primers. The resting levels of MGF were significantly (approximately 100-fold) lower than those of the IGF-IEa isoform. No difference was observed between the resting levels of the two isoforms between the two subject groups. High resistance exercise resulted in a significant increase in MGF mRNA in the young, but not in the elderly subjects. No changes in IGF-IEa mRNA levels were observed as a result of exercise in either group. The mRNA levels of the transcription factor MyoD were greater at rest in the older subjects (P < 0.05), but there was no significant effect of the exercise bout. Electrophoretic separation of myosin heavy chain (MHC) isoforms showed the older subjects to have a lower (P < 0.05) percentage of MHC-II isoforms than the young subjects. However, no association was observed between the composition of the muscle and changes in the IGF-I isoforms with exercise. The data from this study show an attenuated MGF response to high resistance exercise in the older subjects, indicative of age-related desensitivity to mechanical loading. The data in young subjects indicate that the MGF and IGF-IEa isoforms are differentially regulated in human skeletal muscle.

Purification of cytomegalovirus-specific CD8 T cells from peripheral blood using HLA-peptide tetramers.

Cytomegalovirus (CMV) reactivation and disease remains an important clinical problem for patients after allogeneic stem cell transplantation. Impaired cellular immune control of viral replication is responsible for viral reactivation, and transfer of CMV-specific T cells from transplant donors can be effective in providing protection. Recent reports have indicated that the frequency of CMV-specific CD8(+) T cells in the peripheral blood of healthy donors is surprisingly high. Here we demonstrate that by using a combination of human leucocyte antigen (HLA) Class I-peptide tetramers and magnetic selection it is possible to select CMV-specific T cells from CMV antibody-positive individuals to high purity. Reliable purification of CMV-specific T cells up to 99.8% of CD8(+) cells was possible within hours, even when starting with a precursor frequency of < 0.1% of peripheral blood CD8(+) T cells. CMV-specific T cells remained functional after the selection process. This novel form of antigen-specific T-cell selection should facilitate the selection of T cells for cellular immunotherapy to treat or prevent CMV disease after transplantation. In addition, this technique could potentially be applied to many antigens including against other infective agents and tumour-specific antigens.

Direct visualization of cytomegalovirus-specific T-cell reconstitution after allogeneic stem cell transplantation.

Cytomegalovirus (CMV) remains an important cause of morbidity and mortality after allogeneic stem cell transplantation (SCT), but cytotoxic T lymphocytes (CTL) may play a critical role in controlling CMV reactivation. Fluorescent HLA-peptide tetramers containing immunodominant peptides from CMV were used to prospectively monitor the recovery of CMV CTL in recipients of allogeneic transplants from siblings (n = 13) or unrelated donors (n = 11). In patients given allografts from a sibling when both the patient and donor were seropositive for CMV before SCT, recovery of CMV-specific CTL was rapid and reached up to 21% of all CD8(+) T cells. Early reconstitution of CMV-specific immunity was not observed if either the donor or recipient was seronegative for CMV. In recipients of transplants from volunteer unrelated donors, recovery of CMV-specific CTL was delayed in comparison to that in recipients of transplants from siblings and no CTL were observed within the first 100 days after SCT. CTL numbers were increased after episodes of CMV reactivation but were suppressed by prednisolone therapy. Recovery of CMV-specific CTL to levels greater than 10 x 10(6)/L was associated with protection from CMV disease. It was concluded that use of HLA-peptide tetramers to quantify CMV CTL is valuable for studying T-cell responses after allogeneic SCT. It should allow prediction of CMV reactivation in individual patients and assist in the development of adoptive T-cell immunotherapy.