OBJECTIVE

Patients with high titer >>/=100 kIU/ml) of hepatitis C virus (HCV) genotype 1b do not achieve highly sustained virological response rates to combination therapy with interferon plus ribavirin. Non-virological responders (NVRs, namely ultimate resistant cases) who do not achieve HCV-RNA negativity during treatment are also encountered. We investigated the pretreatment virological features of NVRs.

METHODS

We evaluated 50 consecutive Japanese adults with high titer of HCV genotype 1b who received combination therapy for 48 weeks. We investigated the pretreatment substitution patterns in amino acids 1-191 of the core region and amino acids 2209-2248 of NS5A, and early viral kinetics.

RESULTS

Overall, a non-virological response was noted in 12 (24%) patients. Multivariate analysis identified serum albumin <3.9 g/dl, substitutions of amino acid 70 in the core region, and substitutions of amino acid 91 as independent and significant factors associated with a non-virological response. Especially, substitutions of arginine (R) by glutamine (Q) at amino acid 70, and/or leucine (L) by methionine (M) at amino acid 91 were significantly more common in NVRs. The falls in HCV-RNA levels during treatment in patients with specific substitutions in the core region were significantly less than in those without such substitutions.

CONCLUSIONS

Our results suggest that serum albumin and amino acid substitution patterns in the core region in patients with high titers of HCV genotype 1b may have an effect on combination therapy in NVRs. Further large-scale studies are required to examine the role of amino acid substitutions specific to a non-virological response to combination therapy.

The relationships between clinical activity, endoscopic severity, and biological parameters in Crohn's disease have not been thoroughly investigated and a link was therefore sought between these three elements. The following parameters were determined simultaneously in 121 consecutive patients with colonic or ileocolonic Crohn's disease: Crohn's disease activity index, Crohn's disease endoscopic index of severity, and serum albumin, alpha 2-globulin, alpha 1-antitrypsin, orosomucoid, C reactive protein, erythrocyte sedimentation rate, platelets, lymphocyte and polymorphonuclear cell counts, haematocrit, and faecal alpha 1-antitrypsin concentration. The distribution of these parameters was studied and transformation was used so that data matched the normal distribution closely. A weak but significant correlation (r = 0.32; p < 0.001) was found between clinical and endoscopic indices in the whole group of patients and this correlation seemed to be homogenous in various patient subgroups (clinically quiescent or active disease, pure colonic disease, untreated patients). Endoscopic or clinical indices were also found to be weakly linked with biological parameters (r < 0.50). Stepwise linear regression identified C reactive protein as predictive of the clinical index, and, successively, alpha 2-globulin, erythrocyte sedimentation rate, faecal alpha 1-antitrypsin, serum orosomucoid, and alpha 1-antitrypsin as predictive of the endoscopic index. Both predictions were poor--the biological variables accounting for only 22 and 44% respectively of the clinical and endoscopic index variations. In conclusion, Crohn's disease clinical activity seems to be virtually independent of the severity of the mucosal lesions and biological activity.

In our attempt to assess the topology of glucosylceramide biosynthesis, we have employed a truncated ceramide analogue that permeates cell membranes and is converted into water soluble sphingolipid analogues both in living and in fractionated cells. Truncated sphingomyelin is synthesized in the lumen of the Golgi, whereas glucosylceramide is synthesized at the cytosolic surface of the Golgi as shown by (a) the insensitivity of truncated sphingomyelin synthesis and the sensitivity of truncated glucosylceramide synthesis in intact Golgi membranes from rabbit liver to treatment with protease or the chemical reagent DIDS; and (b) sensitivity of truncated sphingomyelin export and insensitivity of truncated glucosylceramide export to decreased temperature and the presence of GTP-gamma-S in semiintact CHO cells. Moreover, subfractionation of rat liver Golgi demonstrated that the sphingomyelin synthase activity was restricted to fractions containing marker enzymes for the proximal Golgi, whereas the capacity to synthesize truncated glucosylceramide was also found in fractions containing distal Golgi markers. A similar distribution of glucosylceramide synthesizing activity was observed in the Golgi of the human liver derived HepG2 cells. The cytosolic orientation of the reaction in HepG2 cells was confirmed by complete extractability of newly formed NBD-glucosylceramide from isolated Golgi membranes or semiintact cells by serum albumin, whereas NBD-sphingomyelin remained protected against such extraction.

We report the establishment of human-human hybridomas producing monoclonal antibody of predefined antigenic specificity. The U-266 human myeloma cell line was incubated in the presence of 8-azaguanine, and a rapidly growing, 8-azaguanine-resistant, hypoxanthine/amethopterin/thymidine (HAT) medium-sensitive mutant line, U-266AR1, was selected. These cells were fused with lymphoid cells from uninvolved spleens removed at staging laparotomy from patients with untreated Hodgkin's disease who had been previously sensitized to the chemical allergen 2,4-dinitrochlorobenzne. Hybrid cell cultures growing in HAT medium were screened for IgG production. Positive cultures were selected and their supernatants were tested in a solid-phase radioimmunoassay for reactivity with dinitrophenyl hapten coupled to bovine serum albumin. Cultures producing specific antibody were subcloned and expanded, and their antibody products were shown to be monoclonal by biosynthetic labeling and sodium dodecyl sulfate/polyacrylamide gel electrophoresis.

OBJECTIVE

To assess the consistency of caloric intake with American College of Chest Physicians (ACCP) recommendations for critically ill patients and to evaluate the relationship of caloric intake with clinical outcomes.

METHODS

Prospective cohort study.

METHODS

Adult ICUs at two teaching hospitals.

METHODS

Patients with an ICU length of stay of at least 96 h.

RESULTS

On ICU admission, severity of illness (ie, simplified acute physiology score II) and markers of nutritional status (ie, serum albumin level and body mass index) were recorded. The route of feeding (ie, enteral or parenteral), actual caloric intake (ie, percentage of ACCP recommendations: 0 to 32% [tertile I]; 33 to 65% [tertile II];>>/==" BORDER="0"> 66% [tertile III]), and evidence of GI intolerance (ie, gastric aspirate levels,>>/==" BORDER="0"> 100 mL) were recorded daily. The following outcomes were assessed: status on hospital discharge (alive vs dead); spontaneous ventilation before ICU discharge (yes vs no); and ICU discharge without developing nosocomial sepsis (yes vs no). The average caloric intake among 187 participants was 50.6% of the ACCP targets and was similar in both hospitals. Caloric intake was inversely related to the mean number of gastric aspirates>>/==" BORDER="0"> 100 mL/d (Spearman rho = -0.04; p = 0.06), but not to severity of illness, nutritional status, or route of feeding. After accounting for the number of gastric aspirates>>/==" BORDER="0"> 100 mL, severity of illness, nutritional status, and route of feeding, tertile II of caloric intake (vs tertile I) was associated with a significantly greater likelihood of achieving spontaneous ventilation prior to ICU discharge. Tertile III of caloric intake (vs tertile I) was associated with a significantly lower likelihood of both hospital discharge alive and spontaneous ventilation prior to ICU discharge.

CONCLUSIONS

Study participants were underfed relative to ACCP targets. These targets, however, may overestimate needs, since moderate caloric intake (ie, 33 to 65% of ACCP targets; approximately 9 to 18 kcal/kg per day) was associated with better outcomes than higher levels of caloric intake.

Genetic studies have demonstrated the involvement of the complement regulator factor H in nondiarrheal, nonverocytotoxin (i.e., atypical) cases of hemolytic uremic syndrome. Different factor H mutations have been identified in 10%-30% of patients with atypical hemolytic uremic syndrome (aHUS), and most of these mutations alter single amino acids in the C-terminal region of factor H. Although these mutations are considered to be responsible for the disease, the precise role that factor H plays in the pathogenesis of aHUS is unknown. We report here the structural and functional characterization of three different factor H proteins purified from the plasma of patients with aHUS who carry the factor H mutations W1183L, V1197A, or R1210C. Structural anomalies in factor H were found only in R1210C carriers; these individuals show, in their plasma, a characteristic high-molecular-weight factor H protein that results from the covalent interaction between factor H and human serum albumin. Most important, all three aHUS-associated factor H proteins have a normal cofactor activity in the proteolysis of fluid-phase C3b by factor I but show very low binding to surface-bound C3b. This functional impairment was also demonstrated in recombinant mutant factor H proteins expressed in COS7 cells. These data support the hypothesis that patients with aHUS carry a specific dysfunction in the protection of cellular surfaces from complement activation, offering new possibilities to improve diagnosis and develop appropriate therapies.

Bacterial lipopolysaccharide (LPS) was demonstrated to have the capacity in mice to enhance the response to soluble bovine serum albumin (BSA) and to interfere with the induction of tolerance to human gamma-globulin (HGG). These adjuvant activities were shown to occur under conditions in which LPS could also function as a B cell mitogen. This positive correlation was established by utilizing two experimental situations in which LPS was non-mitogenic for spleen cells. Thus, on the one hand, it was found that LPS did not function as an adjuvant in C3H/HeJ mice, a unique strain whose spleen cells were also unresponsive to LPS-induced mitogenesis. On the other hand, in strains which did respond to LPS mitogenically, LPS failed to function as an adjuvant when it was chemically altered to reduce its in vitro mitogenic activity. A correlation was also observed between mitogenesis and the capacity of LPS to function as a specific immunogen i mice. In contrast to the sustained and prolonged plaque-forming cell response that was observed in mice whose spleen cells were also responsive to LPS-induced mitogenesis, the response was relatively transient in the C3H/HeJ strain. These results are discussed in view of the possible in vivo modes of action of LPS.

The human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell strain provides a unique and useful tool for studies of human adipocyte biology. The cells originate from an adipose tissue specimen of a patient with SGBS. They are neither transformed nor immortalized, and provide an almost unlimited source due to their ability to proliferate for up to 50 generations with retained capacity for adipogenic differentiation. So far, the cells have been used for a number of studies on adipose differentiation, adipocyte glucose uptake, lipolysis, apoptosis, regulation of expression of adipokines, and protein translocation. The cells are efficiently differentiated in the presence of PPARgammaagonists and in the absence of serum and albumin. SGBS adipocytes respond to insulin stimulation by increasing glucose uptake several-fold (EC50 approximately 100 pmol/l), and by very effectively inhibiting (IC50 approximately 10 pmol/l) catecholamine-stimulated lipolysis.

OBJECTIVE

Low birth weight (LBW), resulting from intrauterine growth retardation (IUGR) or prematurity, is a risk factor for adult hypertension and chronic kidney disease. LBW is associated with reduced nephron endowment and increased glomerular volume; however, the development of secondary focal segmental glomerulosclerosis (FSGS) has not been reported previously.

METHODS

The authors describe six patients with clinical and pathologic findings suggesting a secondary form of FSGS, in whom a history of prematurity and very LBW was obtained. No other known causes of secondary FSGS were identified.

RESULTS

The cohort consisted of two women and four men with a mean age of 32 yr. Patients were born at 22 to 30 wk gestation with mean birth weight of 1054 g (range 450 to 1420 g). Mean 24-h urine protein was 3.3 g/d (range 1.3 to 6.0 g/d), mean creatinine clearance 89 cc/min (range 71 to 132 cc/min), mean creatinine 1.2 mg/dl (range 0.9 to 1.5 mg/dl), and mean serum albumin 4.1 g/dl (range 3.4 to 4.8 g/dl). No patient had full nephrotic syndrome. Renal biopsy revealed FSGS involving a minority (mean 8.8%) of glomeruli, with a predominance of perihilar lesions of sclerosis (five of six patients), glomerulomegaly (all six patients), and only mild foot process effacement (mean 32%), all features typical of postadaptive FSGS.

CONCLUSIONS

Our findings support that very LBW and prematurity promote the development of secondary FSGS. Because birth history is often not obtained by adult nephrologists, this risk factor is likely to be underrecognized.

BACKGROUND

Fibrosis is the common end stage of most liver diseases, for which, unfortunately, there is no effective treatment available currently. It has been shown that mesenchymal stem cells (MSCs) from bone marrow (BM) could engraft in the lung after bleomycin exposure and ameliorate its fibrotic effects. This study was designed to evaluate the effect of Flk1 MSCs from murine BM (termed here Flk1 mMSCs) on fibrosis formation induced by carbon tetrachloride (CCl4).

METHODS

A CCl(4)-induced hepatic fibrosis model was used. Flk1 mMSCs were systemically infused immediately or 1 week after mice were challenged with CCl(4). Control mice received only saline infusion. Fibrosis index and donor-cell engraftment were assessed 2 or 5 weeks after CCl(4) challenge.

RESULTS

We found that Flk1 mMSCs transplantation immediately, but not 1 week after exposure to CCl(4), significantly reduced CCl(4)-induced liver damage and collagen deposition. In addition, levels of hepatic hydroxyproline and serum fibrosis markers in mice receiving immediate Flk1 mMSCs transplantation after CCl(4) challenge were significantly lower compared with those of control mice. More importantly, histologic examination suggested that hepatic damage recovery was much better in these immediately Flk1 mMSCs-treated mice. Immunofluorescence, polymerase chain reaction, and fluorescence in situ hybridization analysis revealed that donor cells engrafted into host liver, had epithelium-like morphology, and expressed albumin, although at low frequency.

CONCLUSIONS

These results suggest that Flk1 mMSCs might initiate endogenous hepatic tissue regeneration, engraft into host liver in response to CCl(4) injury, and ameliorate its fibrogenic effects.

BACKGROUND

The development of de novo diabetes mellitus is a serious complication of kidney transplantation. This study examined the cardiovascular risk profile of patients with post-transplant diabetes (PTDM) and assessed the impact of PTDM on patient survival.

METHODS

This analysis included 1811 adult, renal allograft recipients, transplanted in a single institution between 1983 and 1998. Patient survival was analyzed by univariable and multivariable Cox regression considering PTDM as a time dependent variable.

RESULTS

After a follow-up period of 8.3 +/- 4.5 years, 293 patients (20%) developed PTDM, 14% lost their graft, and 20% died. Compared to patients without DM (NoDM, N = 1186) patients with PTDM were significantly older (40 +/- 14 vs. 48 +/- 12 years, P < 0.001), heavier (76 +/- 23 vs. 86 +/- 25 kg, P < 0.001), and included more African Americans (18 vs. 28%, P = 0.001). In addition, the incidence of PTDM was significantly higher in patients who were transplanted after 1995 than prior to that year. In contrast, there were no significant differences between PTDM and patients who had DM before the transplant (DM; N = 332). Compared to NoDM, patients with PTDM had significantly higher total serum cholesterol and triglycerides (TG), higher systolic blood pressure and higher pulse pressure throughout the post-transplant period. Of interest, all of these abnormalities preceded the development of PTDM. Hypertriglyceridemia was particularly pronounced in PTDM and elevated TG levels correlated with the subsequent development of PTDM, independent of other risk factors (P = 0.001 by multivariate Cox). Compared to NoDM (16% mortality) a significantly higher percent of DM (31%, P < 0.001) and PTDM (22%, P = 0.005) patients died. By Cox regression, PTDM correlated with reduced patient survival (hazard ratio = 1.80, CI 1.35 to 2.41, P = 0.001), and that relationship was independent of other correlates of reduced survival that included: increasing age; transplant year; reduced serum albumin; and male sex.

CONCLUSIONS

s: PTDM is associated with an unfavorable cardiovascular risk profile that precedes the development of hyperglycemia. PTDM is an independent predictor of reduced survival in renal allograft recipients.

Two hundred forty-one patients with a monoclonal protein in the serum but initially no evidence of multiple myeloma, macroglobulinemia, amyloidosis or lymphoma were followed up for more than five years. At the conclusion of the studies the patients were classified as follows: Group 1, patients without significant increase in monoclonal protein, 57 per cent; group 2, patients with more than 50 per cent increase in monoclonal serum protein or development of monoclonal urine protein, 9 per cent; group 3, patients who died without five-year serum studies, 23 per cent; and group 4, patients in whom myeloma, macroglobulinemia or amyloidosis developed, 11 per cent. Initially, the hemoglobin level, size of serum monoclonal protein peak, number of plasma cells in the bone marrow and levels of normal immunoglobulins were not significantly different among the four groups. The median interval from recognition of the monoclonal protein to diagnosis of multiple myeloma was 64 months, of macroglobulinemia 103 months and of amyloidosis 92 months. A significant increase of the monoclonal protein or development of myeloma, macroglobulinemia or amyloidosis occurred in 18 per cent of the patients with monoclonal immunoglobulin G(IgG), in 28 per cent with immunoglobulin A (IgA) and in 25 per cent with immunoglobulin M (IgM). Retrospective analysis of age, sex, presence of organomegaly, hemoglobin level, size and type of serum monoclonal protein peak, presence of small amounts monoclonal light chain in the urine, serum albumin level, levels of uninvolved immunoglobulins, IgG subclass and level of plasma cells in the bone marrow did not show how to distinguish initially between stable benign disease and progressive disease. Therefore, periodic reexamination of patients with monoclonal gammopathy is essential.

In the randomized Hemodialysis (HEMO) Study, chronic high-flux dialysis, as defined by higher beta-2 microglobulin (beta(2)M) clearance, compared with low-flux dialysis did not significantly alter all-cause mortality in the entire cohort but was associated with lower mortality in long-term dialysis patients. This analysis examined the determinants of serum beta(2)M levels and the associations of serum beta(2)M levels or dialyzer beta(2)M clearance with mortality. In a multivariable regression model that examined 1704 patients, baseline residual kidney urea clearance and dialyzer beta(2)M clearance were strong predictors of predialysis serum beta(2)M levels at 1 mo of follow-up, with regression coefficients of -7.21 (+/-0.69 SE) mg/L per ml/min per 35 L urea volume (P < 0.0001) and -1.94 (+/-0.30) mg/L per ml/min (P < 0.0001),respectively. In addition, black race and baseline years on dialysis correlated positively whereas age, diabetes, serum albumin, and body mass index correlated negatively with serum beta(2)M levels (P < 0.05). In time-dependent Cox regression models, mean cumulative predialysis serum beta(2)M levels but not dialyzer beta(2)M clearance were associated with all-cause mortality (relative risk = 1.11 per 10-mg/L increase in beta(2)M level; 95% confidence interval 1.05 to 1.19; P = 0.001), after adjustment for residual kidney urea clearance and number of prestudy years on dialysis. This association is supportive of the potential value of beta(2)M as a marker to guide chronic hemodialysis therapy.

The effect of high-flux hemodialysis membranes on patient survival has not been unequivocally determined. In this prospective, randomized clinical trial, we enrolled 738 incident hemodialysis patients, stratified them by serum albumin < or = 4 and >4 g/dl, and assigned them to either low-flux or high-flux membranes. We followed patients for 3 to 7.5 yr. Kaplan-Meier survival analysis showed no significant difference between high-flux and low-flux membranes, and a Cox proportional hazards model concurred. Patients with serum albumin < or = 4 g/dl had significantly higher survival rates in the high-flux group compared with the low-flux group (P = 0.032). In addition, a secondary analysis revealed that high-flux membranes may significantly improve survival of patients with diabetes. Among those with serum albumin < or = 4 g/dl, slightly different effects among patients with and without diabetes suggested a potential interaction between diabetes status and low serum albumin in the reduction of risk conferred by high-flux membranes. In summary, we did not detect a significant survival benefit with either high-flux or low-flux membranes in the population overall, but the use of high-flux membranes conferred a significant survival benefit among patients with serum albumin < or = 4 g/dl. The apparent survival benefit among patients who have diabetes and are treated with high-flux membranes requires confirmation given the post hoc nature of our analysis.

OBJECTIVE

To evaluate the efficacy of intraarterial 90yttrium (90Y) microspheres in nonresectable hepatocellular carcinoma (HCC).

METHODS

Patients with nonresectable HCC, but without extrahepatic disease, who also had lung shunting < 15% and tumor-to-normal ratio>> or =2, as determined by simulation using (99m)technetium macroaggregated albumin, were entered into the study. The radiation dose delivered to the lungs, tumor, and normal liver was estimated by a partition model. 90Y microspheres were infused into the hepatic artery at the time of hepatic angiography or through an implanted arterial portacatheter under fluoroscopy. Repeated treatments were given for residual or recurrent tumor. Response to treatment was monitored by serum alpha-fetoprotein or ferritin levels, together with serial computed tomography.

RESULTS

Seventy-one patients, including 20 patients with postoperative recurrence, were initially treated with an activity of 0.8 to 5.0 Giga-Becquerel (GBq) (21.6-135.1 mCi) (median 3.0 GBq or 81.1 mCi) of 90Y microspheres. There was a 50% reduction in tumor volume in 19 (26.7%) patients after the first treatment. However, the overall objective response in terms of changes in alpha-fetoprotein levels was 89% [partial response (PR) 67%, complete response (CR) 22%] among the 46 patients with raised pretreatment levels. The serum ferritin level in the other 25 patients dropped by 34 to 99% after treatment. Treatment was repeated in 15 patients. The maximum number of treatments was 5 and the maximum total activity was 13.0 GBq (351.4 mCi), given in 3 treatments. The estimated radiation doses to the nontumorous liver ranged from 25 to 136 Gy (median 52 Gy) in the first treatment and the highest total radiation dose was estimated to be 324 Gy. For the tumors, the estimated radiation doses ranged from 83 to 748 Gy (median 225 Gy) in the initial treatment and the highest cumulative dose reached was 1580 Gy. The residual tumors were resected in 4 patients. Two of these had complete histological remission, but only occasional viable tumor cells were found in the necrotic centers of the tumors resected from the other 2 patients. The median survival of the 71 patients was 9.4 months (range 1.8 to 46.4 months). Treatment was well tolerated and there was no bone-marrow toxicity, or clinical evidence of radiation hepatitis or pneumonitis.

CONCLUSIONS

Selective internal radiation therapy using 90Y microspheres is effective for selected cases of nonresectable HCC and is well tolerated. The objective response rate in terms of drop in tumor marker levels is higher than that based on reduction in tumor volume shown by computed tomography. The nontumorous liver appears more tolerant to internal radiation than external beam radiation. Selective internal radiation treatment may convert nonresectable tumors to resectable ones.

To assess the significance of macromolecular sequence differences among species, we compared the serum albumins of 81 pairs of vertebrate species capable of producing viable hybrids. Micro-complement fixation experiments showed that the average difference between the albumins within such pairs was only 3 immunological distance units for placental mammals (31 pairs), but 36 units for frogs (50 pairs). Albumin immunological distance is strongly correlated with other measures of genetic distance, including those made with DNA annealing techniques. It therefore seems likely that mammalian species pairs capable of hybridization are far more similar at the macromolecular sequence level than is the case for most hybridizable frogs. We think the most likely explanation for the marked molecular restriction on hybridization among mammals is that the ratio of regulatory evolution to protein evolution is higher for mammals than for frogs. Mammals may have experienced unusually rapid regulatory evolution; indeed, this could be the factor responsible for their unusually rapid anatomical evolution.

Ancient DNA analyses rely on the extraction of the tiny amounts of DNA remaining in samples that are hundreds to tens of thousands of years old. Despite the critical role extraction efficiency plays in this field of research, no study has comprehensively compared ancient DNA extraction techniques to date. There are a wide range of methods currently in use, which rely on such disparate principles as spin columns, alcohol precipitation, or binding to silica. We have compared a number of these methods using quantitative PCR and then optimized each step of the most promising method. We found that most chemicals routinely added to ancient DNA extraction buffers do not increase, and sometimes even decrease, DNA yields. Consequently, our optimized method uses a buffer consisting solely of EDTA and proteinase K for bone digestion and binding DNA to silica via guanidinium thiocyanate for DNA purification. In a comparison with published methods, this minimalist approach, on average, outperforms all other methods in terms of DNA yields as measured using quantitative PCR. We also found that the addition of bovine serum albumin (BSA) to the PCR helps to overcome inhibitors in ancient DNA extracts. Finally, we observed a marked difference in the performance between different types of DNA polymerases, as measured by amplification success.

A hybridoma secreting a monoclonal antibody (H-20) that recognizes the 2,2,7-trimethylguanosine(m3G)-containing cap structure of U snRNAs was derived from a mouse which was immunized with a m3G-containing human serum albumin conjugate. The antibody specifically reacts with intact small nuclear ribonucleoprotein particles, U snRNPs, and allows the snRNPs U1 to U6 to be isolated in one step from nuclear extracts of eucaryotic cells by affinity chromatography on a preparative scale. Antibody-bound snRNPs are desorbed from the affinity column by elution with excess of the cross-reactive nucleoside 7-methylguanosine (m7G), which guarantees maintenance of their native structure. The 20 affinity column also allows the snRNPs U1, U2 and U5 to be separated from U4/U6 RNPs by sequential elution of the particles with m7G under differential salt concentrations. As determined by competitive radioimmunoassay and protein-A--Sepharose immunoprecipitation, mAb H-20 crossreacts with intact m7G cap structures. In particular we could show that non-denatured m7G-capped SP6/beta-globin RNA was precipitated efficiently by the antibody while GpppG-capped or non-capped RNAs did not react. Thus the monoclonal antibody H-20 should have a wide application, not only for studying the molecular biology and immunology of the U snRNPs from diverse organisms, but also for the characterization and isolation of m7G-capped transcripts.

Extravascular transport of fluorescein isothiocyanate-conjugated bovine serum albumin and a graded series of fluorescein isothiocyanate-dextrans from Mr 19,400 to 71,800 were studied in both normal tissue (granulation) and tumor (VX2 carcinoma) grown in a rabbit ear chamber. Sodium fluorescein was used as a representative small molecule. A one-dimensional diffusion model adequately described extravascular transport in both normal and tumor tissue. Measured diffusion coefficients showed a relationship with molecular size which progressively deviates from that of free diffusion in water, with values for albumin being significantly reduced from that for a dextran of equivalent size. Macromolecular transport in tumor tissue was hindered to a lesser extent than in normal tissue, which is consistent with reports of reduced contents of glycosaminoglycans, and markedly large interstitial space in tumors. Diffusion coefficients for dextran were found to vary with molecular weight according to the expression, D = a(Mr)b, in both normal tissue (a = 10(6) and b = -2.96) and tumor (a = 2.51 X 10(-2) and b = 1.14).

Successive rational mutations of human butyrylcholinesterase (BChE) followed by fusion to human serum albumin have yielded an efficient hydrolase that offers realistic options for therapy of cocaine overdose and abuse. This albumin-BChE prevented seizures in rats given a normally lethal cocaine injection (100 mg/kg, i.p.), lowered brain cocaine levels even when administered after the drug, and provided rescue after convulsions commenced. Moreover, it selectively blocked cocaine-induced reinstatement of drug seeking in rats that had previously self-administered cocaine. The enzyme treatment was well tolerated and may be worth exploring for clinical application in humans.

Three-hundred and twenty-four patients were enrolled in an open-label, multicenter, international study in which pre- and post-liver transplantation (LT) patients with recurrent chronic hepatitis B (CHB) and evidence of lamivudine-resistant HBV were treated with adefovir dipivoxil 10 mg once daily. In the pre- and post-LT cohorts, 128 and 196 patients were treated for a median duration of 18.7 and 56.1 weeks, respectively. In patients who received 48 weeks of treatment, 81% of the pre-LT and 34% of the post-LT cohort achieved undetectable serum hepatitis B virus (HBV) DNA (Roche Amplicor Monitor polymerase chain reaction [PCR] assay lower limit of quantification [LLQ] < 400 copies/mL) with a median change in serum HBV DNA from baseline of -4.1 log(10) and -4.3 log(10) copies/mL, respectively. Serum alanine aminotransferase (ALT), albumin, bilirubin, and prothrombin time normalized in 76%, 81%, 50%, and 83% of pre-LT patients and 49%, 76%, 75%, and 20% of post-LT patients. The Child-Pugh-Turcotte (CPT) score improved in over 90% of patients in both cohorts. Genotypic analysis of 122 HBV baseline samples revealed that 98% of these patients had lamivudine-resistant mutant HBV. No adefovir resistance mutations were identified in patients after 48 weeks of therapy. One-year survival was 84% for pre-LT and 93% for post-LT patients (Kaplan-Meier analysis). Treatment-related adverse effects associated with adefovir dipivoxil in this setting were primarily mild to moderate in severity. In conclusion, 48 weeks of adefovir dipivoxil resulted in significant improvements in virologic, biochemical, and clinical parameters in CHB patients pre- and post-LT with lamivudine-resistant HBV.

An in vitro model of cellular immunity in the guinea pig was established. Animals were immunized with tubercle bacilli, bovine gamma globulin, or picrylated human serum albumin in complete Freund's adjuvant. Oil-induced peritoneal exudates from immune and control animals were cultured overnight with and without specific antigen. The cultures were washed and the macrophage monolayers were infected with Listeria monocytogenes. At intervals the monolayers were lysed and the numbers of viable intracellular bacteria were quantitated by pour plate cultures. Random monolayers were also evaluated in sequence by visually counting the intracellular bacteria on Gram-stained plates. Both methods demonstrated that the macrophages from immune animals had markedly enhanced listericidal activity when the peritoneal exudates were cultured with antigen before infection. Macrophage migration inhibition was also demonstrated under these conditions. The experiments reported here describe an in vitro model of cellular immunity which will allow separation and recombination of cell types and direct assay of cell products in efforts to elucidate further the mechanisms of the immunologically mediated enhancement of macrophage bactericidal capacity.

We have developed a new class of magnetic resonance imaging contrast agents with large proton relaxation enhancements and high molecular relaxivities. The reagents are built from the polyamidoamine form of Starburst dendrimers in which free amines have been conjugated to the chelator 2-(4-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid. The dendrimer gadolinium poly-chelates have enhancement factors, i.e., the ratio of the relaxivity per Gd(III) ion to that of Gd(III)-diethylenetriaminepentaacetic acid, of up to 6. These factors are more than twice those observed for analogous metal-chelate conjugates formed with serum albumins, polylysine, or dextran. One of the dendrimer-metal chelate conjugates has 170 gadolinium ions bound, which greatly exceeds the number bound to other macromolecular agents reported in the literature, and has a molecular relaxivity of 5,800 (mM.s)-1, at 25 MHz, 20 degrees C, and pH of 7.4. We observed that these dendrimer-based agents enhance conventional MR images and 3D time of flight MR angiograms, and that those with molecular weights of 8,508 and 139,000 g/mole have enhancement half lives of 40 +/- 10 and 200 +/- 100 min, much longer than the 24 +/- 4 min measured for Gd(III)-diethylenetriaminepentaacetic acid. Our results suggest that this new and powerful class of contrast agents have the potential for diverse and extensive application in MR imaging.