Unintegrated circular HIV-1 DNA in the peripheral mononuclear cells of HIV-1-infected subjects: association with high levels of plasma HIV-1 RNA, rapid decline in CD4 count, and clinical progression to AIDS

We observed 36 HIV-infected patients to evaluate whether the presence of tandem 2-long terminal repeat circular unintegrated HIV-1 DNA (2-LTR) in peripheral blood mononuclear cells (PBMC) at baseline was associated with acceleration of HIV disease. Detection of 2-LTR at baseline correlated with high plasma HIV-1 RNA levels (p < .01), recovery of culturable HIV-1 from plasma (p = .02), and progression to AIDS during follow-up (p = .01). More patients with 2-LTR (68%) than without 2-LTR (31%) had a decline in CD4 levels of >50 cells/mm3 over the first 18 months of follow-up (p = .04), and the average annual CD4 decline was 35% in patients with 2-LTR compared with 16% in those without 2-LTR (p = 0.06). Detection of 2-LTR in PBMC at baseline was an independent predictor of high plasma HIV-1 RNA levels and subsequent CD4 cell decline in this cohort of patients with predominantly nonsyncytium-inducing (NSI) isolates at baseline. The presence of 2-LTR in PBMC appears to be reflective of ongoing HIV-1 replication, as measured by plasma HIV-1 RNA levels, and identifies persons at risk for immunologic and clinical decline.     

LacZ staining in paraffin-embedded tissue sections

Femora and tibiae of rats carrying leukemia from a LacZ-marked acute promyelocytic leukemia-derived leukemic cell line (LT12NL15) were decalcified using EDTA and routinely embedded in paraffin. Sections were used to develop for the first time an immunostaining method for LacZ, employing catalyzed reporter deposition (CARD) based on the deposition of biotinylated tyramine. This method was used to study homing and adhesion of leukemic cells.     

A theoretical model using mechanical principles to quantify the physical principles of balloon dilatation

RATIONALE AND OBJECTIVES: Balloon dilatation is a mechanical form of controlled injury used to alleviate vascular stenoses. Several factors influence successful angioplasty. Few mechanical models exist to illustrate the physical principles of balloon dilatation. METHODS: We used mechanical analysis of membrane stresses, along with Laplace's law, to determine a relation between balloon inflation and dilating pressures exerted by balloons in stenoses of varying severity, length, and eccentricity. The balloons were assumed to be perfectly inelastic and flexible. We also examined the resultant stresses in the lesion wall of concentric and eccentric stenoses from exertion of dilating pressures. RESULTS: Dilating pressures depend directly on maximal balloon inflation pressure and balloon diameter. Short, focal stenoses experience greater dilating pressures, which often are several multiples of the inflation pressure, than similarly narrowed longer lesions. CONCLUSION: Dilating pressures depend on inflation pressure, balloon diameter, and lesion severity.     

Screening for tyrosinaemia type I

AIMS: To assess the incidence of tyrosinaemia type I in the West Midlands Region, and the value of current neonatal screening programmes for phenylketonuria (PKU) for its detection. METHODS: Retrospective study of results from the PKU neonatal screening programmes in Birmingham (using plasma amino acid chromatography) and in the rest of the West Midlands (using the Guthrie microbiological assay for blood spot phenylalanine) was carried out between January 1985 and March 1994. Patients with tyrosinaemia I born in the region during the same period were identified from a regional database of patients with confirmed inherited metabolic disease. The study was carried out in a specialist children's hospital; the regional centre in the West Midlands for neonatal screening and investigation of inborn errors, and a supraregional centre for liver transplantation and management of paediatric liver disease. RESULTS: Amino acid chromatography showed increased tyrosine in 447 of 145,444 neonates born in Birmingham; this was still increased at 6 weeks of age in six cases. Five had tyrosinaemia I; the sixth had tyrosinaemia type III. Two others in whom amino acid chromatography was considered normal have since presented with tyrosinaemia I. Outside Birmingham, 525,151 children were screened using the Guthrie test. Five have presented clinically with tyrosinaemia I; screening did not contribute to diagnosis in any case. The incidence of tyrosinaemia I was 1 in 20,791 live births within Birmingham and 1 in 105,037 outside. Of the total 12 patients in the West Midlands with tyrosinaemia I, 10 (83%) were of non-oriental Asian ethnicity; the incidence of tyrosinaemia I was 3.7/10(6) head of population in this group and 0.04/10(6) in the rest of the population. CONCLUSIONS: Asians in the West Midlands have a high incidence of tyrosinaemia I. Neonatal PKU screening using amino acid chromatography may contribute to diagnosis and early treatment.     

A positive crossmatch in liver transplantation--no effect or inappropriate analysis? A prospective study

Secondary brain damage after transient cerebral hypoxia-ischemia (HI) is caused by a cascade of cellular events. In this study, complementary methods of magnetic resonance imaging and histochemistry were used to investigate the formation of cytotoxic and vasogenic edema during secondary brain damage induced by transient HI in 7-d-old rats. To elicit injury, 21 rats underwent right common carotid artery ligation followed by 1.5 h of 8% O2 exposure. Sequential apparent diffusion coefficient (ADC) and transversal relaxation time (T2) weighted magnetic resonance imaging were recorded for up to 3 d in 13 7-d-old rats. Eight animals were killed at various intervals between the end of HI and 21 h of recovery to perform histochemical assays using neuronal and astrocytic markers. Changes of the ADC revealed a biphasic function for the evolution of cytotoxic edema during the recovery period. At the end of HI, the ADC in the ipsilateral cortex was significantly decreased. Upon reoxygenation, it returned transiently to normal followed by a secondary, although less pronounced, decline after 8-48 h. After this, the ADC rose steadily. From 8 h of recovery, the proportion of vasogenic edema steadily increased as indicated by the T2 prolongation. At 21 h, the majority of glial cells showed immunoreactivity for glial fibrillary acidic protein and were of larger size, whereas the neurons were apoptotic. These results indicate that the delayed cerebral injury is accompanied by late glial swelling in conjunction with an enlarged interstitial space due to cell damage.     

Mitochondrial respiratory chain succinate-cytochrome-c oxidoreductase deficiency and hepatic cirrhosis

OBJECTIVES: The current need to evaluate necessity and cost of diagnostic and therapeutic procedures extends to transplant services. We reviewed our experience over the past 3 years as we have moved away from routine post-transplant nuclear medicine scans, ultrasounds, and cystograms. METHODS: From January 1, 1992 to December 31, 1994, 252 kidney transplants were performed at Virginia Mason Medical Center. There were 74 live donor and 178 cadaver donor kidneys transplanted. The records of these patients were reviewed for the type and number of post-transplant imaging done during their initial hospitalization. RESULTS: During the study period, the number of post-transplant imaging studies per patient decreased from 2.7 to 1.4 (P = 0.000), the percentage of patients discharged without any studies rose from 2.8% to 24.4% (P = 0.001), and the trend in 1-year actual graft survival increased from 84.7% to 93.0% (P = 0.187). CONCLUSIONS: Post-transplant imaging studies can be safely reduced. Many patients with good initial graft function can avoid having any studies.     

Minimizing a binding domain from protein A

We present a systematic approach to minimizing the Z-domain of protein A, a three-helix bundle (59 residues total) that binds tightly (Kd = 10 nM) to the Fc portion of an immunoglobin IgG1. Despite the fact that all the contacts seen in the x-ray structure of the complex with the IgG are derived from residues in the first two helices, when helix 3 is deleted, binding affinity is reduced > 10(5)-fold (Kd > 1 mM). By using structure-based design and phage display methods, we have iteratively improved the stability and binding affinity for a two-helix derivative, 33 residues in length, such that it binds IgG1, with a Kd of 43 nM. This was accomplished by stepwise selection of random mutations from three regions of the truncated Z-peptide: the 4 hydrophobic residues from helix 1 and helix 2 that contacted helix 3 (the exoface), followed by 5 residues between helix 1 and helix 2 (the intraface), and lastly by 19 residues at or near the interface that interacts with Fc (the interface). As selected mutations from each region were compiled (12 in total), they led to progressive increases in affinity for IgG, and concomitant increases in alpha-helical content reflecting increased stabilization of the two-helix scaffold. Thus, by sequential increases in the stability of the structure and improvements in the quality of the intermolecular contacts, one can reduce larger binding domains to smaller ones. Such mini-protein binding domains are more amenable to synthetic chemistry and thus may be useful starting points for the design of smaller organic mimics. Smaller binding motifs also provide simplified and more tractable models for understanding determinants of protein function and stability.