Cell-mediated cytotoxicity: a predictor of chronic rejection in pediatric HLA haploidentical renal transplants

BACKGROUND: Recipient antidonor cytotoxic T-cell activity has been associated with graft loss and acute rejection in renal allograft recipients. The role of immunologic mechanisms in the development of chronic graft rejection is controversial. We analyzed all living related renal transplants performed at Children's Hospital (Boston, MA) from 1983 to 1995 to assess whether cell-mediated cytotoxicity, determined in vitro and measured before transplantation, was predictive of chronic rejection. METHODS: Eighty-three patients were studied retrospectively. Fifty-seven patients with one haplotype-matched renal transplants from living related donors were studied to determine the association between cell-mediated lympholysis (CML) level, acute rejection, chronic rejection, and graft failure. Acute rejection was defined by the decision to treat. Chronic rejection was defined by histology and/or the absolute serum creatinine value using an increasing serum creatinine level >1.0 mg/dl for children less than 3, a creatinine level >1.5 mg/dl for children between 3 and 10 years of age, and a creatinine level >2.0 mg/dl for children above 10 years of age. Return to dialysis or retransplantation was considered graft failure. RESULTS: Of the 57 haploidentical patients, there were 33 males and 24 females. The mean age at transplant was 11.1 years (SD=6.7). Twelve patients developed chronic rejection, 24 patients developed acute rejection, and 7 patients had graft failure. Pretransplant cytotoxic T lymphocyte activity was associated with chronic rejection (P=0.001) and graft failure (P=0.013) but only marginally with acute rejection (P=0.058). Controlling for age and sex, Cox's proportional hazards model revealed that CML level was predictive of time to chronic rejection (P<0.01) but not acute rejection (P=0.11). It was estimated that every 1-unit increase in CML level raises the monthly risk of chronic rejection by 7%. Ten children received HLA-identical kidneys from their siblings. There were no episodes of chronic rejection after 5 years. Two patients with high CML levels had episodes of acute rejection; both patients responded to treatment. CONCLUSION: Our data demonstrate an association between pretransplant cell-mediated cytotoxicity and the occurrence of chronic rejection in living related one-haploidentical renal transplants in pediatric patients.     

In vitro diagnosis of axillary lymph node metastases in breast cancer by spectrum analysis of radio frequency echo signals

Axillary lymph node status is of particular importance for staging and managing breast cancer. Currently, axillary lymph node dissection is performed routinely in cases of invasive breast cancer because of the lack of accurate noninvasive methods for diagnosing lymph node metastasis. We investigated the diagnostic ability of ultrasonic tissue characterization based on spectrum analysis of backscattered echo signals to detect axillary lymph node metastasis in breast cancer in vitro compared with in vitro B-mode imaging. Immediately after surgery, individual lymph nodes were isolated from axillary tissue. Each lymph node was scanned in a water bath using a 10-MHz instrument, and radio frequency data and B-mode images were acquired. Spectral parameter values were calculated, and discriminant analysis was performed to classify metastatic and nonmetastatic lymph nodes. Forty histologically characterized axillary lymph nodes were enrolled in this study, including 25 nonmetastatic and 15 metastatic lymph nodes. A significant difference existed in the spectral parameter values (slope and intercept) for metastatic and nonmetastatic lymph nodes. Spectral parameter-based discriminant function classification of metastatic vs. nonmetastatic lymph nodes provided a sensitivity of 93.3%, specificity of 92.0%, and overall accuracy of 92.5%. In comparison, B-mode ultrasound images of in vitro lymph nodes provided a sensitivity of 73.3%, specificity of 84.0%, and overall accuracy of 80.0%. Receiver operating characteristic (ROC) analysis comparing the efficacy of both methods gave an ROC curve area of 0.9888 for spectral methods, which was greater than the area of 0.8980 for B-mode ultrasound. Hence, this in vitro study suggests that the diagnostic ability of spectrum analysis may prove to be markedly superior to that of B-mode ultrasound in detecting axillary lymph node metastasis in breast cancer. Because of these encouraging results, we intend to conduct an investigation of the ability of spectral methods to classify metastatic axillary lymph nodes in vivo.     

Intravascular ultrasound predictors of restenosis after balloon angioplasty of the femoropopliteal artery

The skepticism surrounding the potential benefits of resistance exercise training prevalent just decades ago has evolved over the years to an understanding of the integral nature muscular overload plays in the training programs for athletes. The science of training elite athletes is progressing rapidly, as insights into the physiological adaptations resulting from varying program configurations become available. Resistance training impacts several body systems, including muscular, endocrine, skeletal, metabolic, immune, neural, and respiratory. An understanding and appreciation of basic scientific principles related to resistance training is necessary in order to optimize training responses. Careful selection of the acute program variables in a workout to simulate sports-specific movements is required for optimal transfer of gains made in training to competition. Thus, whether athletes require predominantly eccentric, isometric, slow-velocity, or high-velocity strength or power in their athletic event will dictate the time commitment to each component and form the basis for designing individual workouts. Program variation over a training period is essential to maximize gains and prevent overtraining.     

Molecular cloning of CYP1A from the estuarine fish Fundulus heteroclitus and phylogenetic analysis of CYP1 genes: update with new sequences

Since we published a phylogenetic analysis of the CYP1A subfamily in 1995, several additional full-length sequences have been reported, including three members of an entirely new subfamily, CYP1B. Two avian sequences were recently published, so that CYP1A sequence data are now available from three of the five major vertebrate lineages. The two new branches that have been added to the CYP1 family tree significantly add to our understanding of P450 evolution. The inclusion of the CYP1Bs to the phylogenetic analysis allows us to root inferred trees. Addition of the avian CYP1As indicates that the CYP1A1/CYP1A2 duplication present in the mammalian lineage may have occurred after the divergence of birds and mammals. The number of fish species from which full-length coding regions of CYP1A genes have been sequenced has increased from four (trout, plaice, toadfish, and scup) to nine. These include CYP1A sequences from tomcod, butterflyfish, sea bream, sea bass, and the full-length sequence of CYP1A from the killifish Fundulus heteroclitus that is reported here. Phylogenetic analyses incorporating the new fish CYP1A sequences support our original conclusion that the fish CYP1As are monophyletic and indicate that the genes are evolving at very different rates in different species.     

Cerebral blood flow velocity and vasomotor reactivity before and after shunting surgery in patients with normal pressure hydrocephalus

The purpose of this study was to evaluate pre- and post-shunting haemodynamic changes and their correlation with the clinical results in normal pressure hydrocephalus (NPH). Accordingly, eleven demented patients with clinical signs suggestive of NPH received examinations of cerebral blood flow velocity (BFV) and vasomotor reactivity (VMR) by transcranial Doppler sonography with carbogen testing before and after shunt treatment. Computerized tomography (CT), clinical assessment and neuropsychological grading were performed prior to and at 3 months following surgery. A control group consisting of 10 patients was included to establish baseline data. The pre-operative CBF studies in the anterior cerebral artery (ACA) and the middle cerebral artery (MCA) revealed the NPH patients did not have significant decreases of BFVs, but had significant decreases of carbogen VMR (P < 0.05). After shunting, there were no significant changes of the BFVs as compared with the pre-shunting data. The post-shunting VMR of the ACA was significantly higher than the pre-shunting one (p < 0.05), but there was no variation in that of the MCA. Both the values of post-shunting VMR in ACA and the post-shunting increase in VMR in MCA of the 7 shunt-responsive patients who improved mentally and in other symptoms were significantly higher than those of patients without improvement (p < 0.05). In addition, the five patients with gait improvement showed significantly higher values of post-shunting VMR of ACA and the post-shunting increase of VMR for both ACA and MCA when compared with those patients without gait improvement (p < 0.05, respectively). Our study supports the view that patients with NPH had various degrees of impaired VMR in both the ACA and the MCA, but showed insignificant reduction in BFVs, indicating a compensatory mechanism of CBF over time to accommodate the subnormal state of cerebral perfusion pressure. Shunt placement would improve the VMR in responsive patients. Postoperatively, an increase of VMR tends to accompany improvement of the functional state: that in the MCA alone is associated with symptomatic improvement in mental function and that increase in VMR in both the ACA and the MCA with improvement in gait, respectively.     

Heat tolerance in Tuli-, Senepol-, and Brahman-sired F1 Angus heifers in Florida

We investigated heat tolerance and growth rate in two trials under ambient conditions in central Florida. Trial 1 (1994) involved 38 Brahman (B), 21 Senepol (S), 19 B x Angus (A), 20 S x A, and 20 Tuli (T) x A heifers. Trial 2 (1995) involved 13 A, 35 B, 30 S, 23 B x A, 17 S x A, and 28 T x A heifers. Measurements were made on three consecutive weeks during the hotter and cooler seasons of each year and included rectal temperature (RT, degrees C), respiration rate (RR, bpm), temperament score (TS; 1 = very docile, 5 = very aggressive), blood packed-cell volume (PCV), and plasma cortisol concentration (CORT). Data for RT were transformed (log10 [RT - 37]) before analysis. On the hottest date in Trial 1, log10 RT was not different between B (.39 +/- .011) and B x A (.37 +/- .016) or between T x A (.35 +/- .015) and B x A, but log10 RT was lower (P < .05) in S x A (.30 +/- .015) than in either S (.35 +/- .015) or T x A. On all dates in Trial 1, RR was lower (P < .05 to .001) and PCV was higher (P < .05 to .001) in B than in B x A. There were few differences in TS except on two dates when B scored higher (P < .01 to .001) than B x A, and these differences were associated with higher (P < .05) CORT in B than in B x A. Using initial BW as a covariate, adjusted ADG (kg) of T x A (.52 +/- .023) was not different from adjusted ADG of B x A (.57 +/- .024) or S x A (.54 +/- .023). On the hottest date in Trial 2, log10 RT and RR were higher (P < .001) in A (.59 +/- .017, 74 +/- 2.7) than in B (.47 +/- .010, 39 +/- 1.6), S (.42 +/- .011, 50 +/- 1.8), and crossbred heifers (.47 +/- .011, 60 +/- 1.8; .43 +/- .014, 55 +/- 2.4; and .50 +/- .012, 48 +/- 2.0 for T x A, S x A and B x A, respectively), and RR was higher (P < .001) in B x A than in B. On the coolest date in Trial 2, RR was slightly lower in B (32 +/- .5) than in A(34 +/- .7, P < .01) and B x A (36 +/- .6, P < .001) and was associated with higher PCV in B than in A. On both dates, TS and CORT were higher (P < .01) in B than in A. In Trial 2, adjusted ADG (kg) was higher (P < .01) in B (.43 +/- .017) than in A (.32 +/- .033), higher (P < .001) in S (.45 +/- .018) than in A, and higher (P < .001) in crossbreds (B x A [.53 +/- .023] + S x A [.44 +/- .025] + T x A [.46 +/- .019]) than in A. These data indicate that heat tolerance in F1 crosses of tropically adapted breeds (Tuli, Senepol, Brahman) with a temperate breed (Angus) is similar to heat tolerance displayed by purebred tropical breeds (Senepol, Brahman).     

Aggravated decrease in the activity of nucleus basalis neurons in Alzheimer's disease is apolipoprotein E-type dependent

As reported before, the metabolic activity of nucleus basalis neurons is reduced significantly in Alzheimer patients. Because the apolipoprotein E (ApoE) epsilon4 genotype is a major risk factor for Alzheimer's disease (AD), we determined whether the decrease in metabolic activity in nucleus basalis neurons in AD is ApoE-type dependent. The size of the Golgi apparatus (GA) was determined as a measure of neuronal metabolic activity in 30 controls and 41 AD patients with a known ApoE genotype by using an image analysis system in the nucleus basalis of Meynert. A polyclonal antibody directed against MG-160, a sialoglycoprotein of the GA, was used to visualize this organelle. There was a very strong reduction in the size of the GA in the nucleus basalis of AD patients. Furthermore, a strong and significant extra reduction in the size of the GA was found in the nucleus basalis neurons of AD patients with either one or two ApoE epsilon4 alleles compared with Alzheimer patients without ApoE epsilon4 alleles. Our data show that the decreased activity of nucleus basalis neurons in AD is ApoE epsilon4 dependent and suggest that ApoE epsilon4 participates in the pathogenesis of AD by decreasing neuronal metabolism.     

Molecular barriers to biomaterial thrombosis by modification of surface proteins with polyethylene glycol

For cardiovascular biomaterials, thrombosis, thromboembolism and vascular graft occlusion are believed to be precipitated by the adsorption of proteins containing adhesive ligands for platelets. Polyethylene-glycol-diisocyanate (PEG-diisocyanate, 3400 MW) may potentially react with protein amines to form molecular barriers on adsorbed proteins on biomaterials, thereby masking adhesive ligands and preventing acute surface thrombosis. To test this notion, PE, PTFE, and glass microconduits were pre-adsorbed with fibrinogen and treated with PEG-diisocyanate, non-reactive PEG-dihydroxyl, or remained untreated. Following perfusion of 111In-labeled platelets in whole human blood for 1 min (wall shear rate = 312 s(-1)), PEG-diisocyanate treated surfaces experienced 96% (PE), 97% (PTFE) and 94% (glass) less platelet deposition than untreated surfaces. Similar reductions were seen for PEG-diisocyanate versus PEG-dihydroxyl treatment. Low shear perfusions of plasma for 1 h prior to blood contact did not reduce the inhibitory effect of PEG-diisocyanate. Platelet adhesion onto collagen-coated glass coverslips and platelet deposition onto preclotted Dacron were also reduced by treatment with PEG-diisocyanate (93 and 91%, respectively). Protein-reactive PEG may thus have utility in forming molecular barriers on surface-associated proteins to inhibit acute thrombosis on cardiovascular biomaterials.     

Hepatic elimination of diazepam: interactions with albumin, desflurane and sevoflurane

We have studied the effect of sevoflurane and desflurane on the hepatic elimination of diazepam, by incubating slices of rat livers in a closed system. Protein free and protein containing (albumin 10 g litre-1) buffers were used to examine the effect of the anaesthetics on enzyme activity and diazepam binding to albumin. Both anaesthetics (in concentrations of 0.5, 1.5 and 3.0 mmol litre-1) reduced the elimination of diazepam slightly in the absence of albumin, while the presence of the protein increased elimination to a maximum of 30% at the greatest concentration of the anaesthetics. These data support previous observations that volatile anaesthetics may interact pharmacokinetically with both liver enzyme activity and drug binding to albumin.