Proanthocyanidin: a natural crosslinking reagent for stabilizing collagen matrices

While attempting to find a suitable crosslinking reagent for biopolymers, a naturally occurring proanthocyanidin (PA) obtained from grape seeds was selected to fix biological tissues. The cytotoxicity and crosslinking rate, reflected by the in vitro and in vivo degradation of fixed matrices has been studied. The shrinkage temperature of the fixed bovine pericardium increased from 66 to 86 degrees C. A cytotoxicity assay using fibroblast cultures revealed that PA is approximately 120 times less toxic than glutaraldehyde (GA), a currently used tissue stabilizer. In vitro degradation studies showed that fixed tissue was resistant to digestion by bacterial collagenase. Crosslinks between PA and tissues can be stabilized by decreasing the dielectric constant of the solution during storage. After subcutaneous implantation for periods ranging between 3 and 6 weeks, we found no apparent degradation of the GA- or PA-fixed tissues, whereas fresh tissue controls rapidly disintegrated. Beyond 6 weeks PA crosslinks began to degrade. More fibroblasts migrated and proliferated inside the PA-fixed implants compared with GA counterparts. Tissues crosslinked with PA manifested an enhanced collagen expression and deposition and did not calcify after implantation. GA, on the other hand, even after thorough rinsing continued to be cytotoxic, inhibited collagen synthesis and encouraged dystrophic calcification. Collagen matrices crosslinked with PA are expected to be of value in the design of matrices that will encourage cell ingrowth and proliferation, which are temporary in nature, and that are intended to regenerate or replace missing tissues, which can delay the biogradation of collagen. As such they should be of significant value in the emerging field of tissue engineering.     

Analysis of the coxsackievirus B-adenovirus receptor gene in patients with myocarditis or dilated cardiomyopathy

Myocarditis and dilated cardiomyopathy (DCM) are common causes of morbidity and mortality in children and adults, most commonly due to infection with coxsackievirus B or adenovirus. Increased expression of the common human coxsackievirus B-adenovirus receptor (CAR) has been reported in patients with DCM. We investigated the CAR gene in patients with acquired or familial myocarditis/DCM for mutations/polymorphisms. Several polymorphisms or intronic substitutions, distant from the intron-exon boundaries, were identified but no mutations. Based upon these data it appears that CAR gene mutations are not a major host determinant in the development of myocarditis and DCM.     

Oxidative capacity interacts with oxygen delivery to determine maximal O2 uptake in rat skeletal muscles in situ

When intracellular recordings were made from the antral region of murine stomach, cells with three different patterns of electrical activity were detected. One group of cells generated follower potentials, the second group generated pacemaker potentials and the third group generated slow waves that consisted of primary and secondary components. Slow waves recorded in different regions of the gastric antrum had similar amplitudes but different characteristic shapes. At the greater curvature, slow waves had large initial components. Midway between the greater and lesser curvature, the amplitude of the initial component was reduced and at the lesser curvature an initial component was difficult to detect. When the distributions of myenteric (ICC-MY) and intramuscular interstitial cells of Cajal (ICC-IM) were determined, using an antibody to Kit, ICC-MY were found to be present at the greater curvature but were greatly reduced in density at the lesser curvature. In contrast, ICC-IM were found in the circular layer of each region. When recordings were made from the antrum of W/W^V mice, which lack ICC-IM, incomplete slow waves were detected and their amplitudes fell from the greater to the lesser curvature. Again, a corresponding fall in the density of ICC-MY was detected. The observations indicate that the contribution of ICC-MY and ICC-IM to the generation of slow waves varies in different regions of the mouse gastric antrum.     

The transplanted kidney as a source of cytomegalovirus infection.

To determine the incidence of cytomegalovirus infection in renal-transplant recipients we followed 32 prospectively for six months after operation. As judged by serologic change and virus isolation the infection rate for the entire group was 66 per cent (21 of 32 patients) - 59 per cent (13 of 22) for seronegative patients and 80 per cent (eight of 10) for seropositive patients. Of 10 seronegative patients who received kidneys from seronegative donors, only three became infected. However, of 12 seronegative patients who received kidneys from seropositive donors, 10 became infected. Thus, there was a significant correlation between development of infection and seropositivity of the donor (P = 0.03), particularly when the recipient was seronegative (P = 0.02). Five possible and four definite recognizable clinical illnesses were associated with cytomegalovirus infection; all except two were in initially seronegative subjects who received kidneys from seropositive donors. Primary infection and disease in nonimmune recipients may be caused by cytomegalovirus transmitted by the kidneys of latently infected seropositive donors.     

Antimicrobial activities against 84 Listeria monocytogenes isolates from patients with systemic listeriosis at a comprehensive cancer center (1955-1997)

Listeriosis is a serious complication in patients undergoing treatment for cancer. We present antimicrobial susceptibility profiles of 84 clinical Listeria monocytogenes isolates. During 1955 to 1997, in vitro susceptibility for penicillin (97.6%), ampicillin (90.7%), erythromycin (98.8%), tetracycline (96.9%), and gentamicin (98.0%) remained unchanged. All isolates were susceptible to amikacin, ciprofloxacin, imipenem, rifampin, trimethoprim-sulfamethoxazole (TMP-SMX), and vancomycin. High prevalence of clindamycin resistance (96.2%) was unexpected. Ampicillin plus gentamicin is standard therapy for systemic listerosis, and TMP-SMX may be used for patients with beta-lactam intolerance. In vitro susceptibility profiles for carbapenem and fluoronated quinolone are promising, although clinical validation is critically needed before routine use is advocated, especially for listeric patients with severe cellular immune defects.     

Use of immunoblotting to detect Aspergillus fumigatus antigen in sera and urines of rats with experimental invasive aspergillosis.

Immunoblotting was used to detect Aspergillus fumigatus antigen in sera and urines of immunosuppressed rats experimentally infected with A. fumigatus. Organisms were administered by both intravenous and intratracheal injections. Intravenously infected rats developed disseminated aspergillosis, but intratracheally infected rats developed pulmonary disease only. Fungal cultures of blood and urine samples from infected rats were negative. In the urines of intravenously infected rats, antigen was detected 24 to 48 h after infection; in the urines of intratracheally infected animals, antigen was detected on days 4 to 5 after infection. Antigen in serum was detected later than antigen in urine was. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of serum and urine samples, the most strongly reacting antigenic materials were found in the 88-, 40-, 27-, and 20-kilodalton regions. These dominant antigens appeared to be the same as those of control antigens prepared from A. fumigatus grown in vitro. Rabbit antiserum to Aspergillus filtrate antigen was found to be more immunoreactive than antiserum to mycelial or conidial antigen. No mycelium-specific antigens were detected.     

Leukotriene B4 Receptor (BLT-1) Modulates Neutrophil Influx into the Peritoneum but Not the Lung and Liver during Surgically Induced Bacterial Peritonitis in Mice

Leukotriene B₄ (LTB₄) is a rapidly synthesized, early neutrophil chemoattractant that signals via its cell surface receptor, BLT-1, to attract and activate neutrophils during peritonitis. BLT-1-deficient (BLT-1^−/−) mice were used to determine the effects of LTB₄ on neutrophil migration and activation, bacterial levels, and survival after cecal ligation and puncture (CLP). Male BLT-1^−/− or wild-type (WT) BALB/c mice underwent CLP. Tissues were harvested for determination of levels of bacteria, myeloperoxidase (MPO), LTB₄, macrophage inflammatory protein 2 (MIP-2), and neutrophil (polymorphonuclear leukocyte [PMN]) numbers at 4 and 18 h after CLP. PMN activation was determined by an assessment of phagocytosis ability and CD11b expression. Survival was also determined. BLT-1^−/− mice had decreased numbers of PMNs in the peritoneum at both 4 and 18 h after CLP but increased numbers of PMNs in the blood at 18 h compared with WT mice. Liver and lung MPO levels were significantly higher in BLT-1^−/− mice at both 4 and 18 h after CLP, with increased bacterial levels in the blood, the liver, and peritoneal fluid at 4 h. Bacterial levels remained higher in peritoneal fluid at 18 h, but blood and liver bacterial levels at 18 h were not different from levels at 4 h. PMN phagocytosis and CD11b levels were decreased in BLT-1^−/− mice. LTB₄ levels were similar between the groups before and after CLP, but MIP-2 levels were decreased both locally and systemically in BLT-1^−/− mice. Survival was significantly improved in BLT-1^−/− mice (71%) compared with WT mice (14%) at 48 h post-CLP. Thus, LTB₄ modulates neutrophil migration into the mouse peritoneum, but not the lung or liver, after CLP. Despite higher bacterial and PMN levels at remote sites, there was increased survival in BLT-1^−/− mice compared to WT mice. Decreased PMN activation may result in less remote organ dysfunction and improved survival.     

Alzheimer's disease: the relationship between the density of senile plaques, neurofibrillary tangles and A4 protein in human patients.

The numerical density of senile plaques (SP) and neurofibrillary tangles (NFT) as revealed by the Glees silver method was compared with SP and NFT revealed by the Gallyas method and with amyloid (A4) deposits in immunostained sections in 6 elderly cases of Alzheimer's disease. The density of NFT was generally greater and A4 lower in tissue from hippocampus compared with the neocortex suggesting that A4 deposition was less important than the degree of paired helical filament (PHF) related damage in the hippocampus. The density of Glees SP was positively correlated Gallyas SP weakly correlated with A4 deposit number. A stepwise multiple regression analysis which included A4 deposit and Gallyas SP density and accounted for 54% of the variation in Glees SP density. Hence, different populations of SP were revealed by the different staining methods. The results suggested that the Glees method may stain a population of SP in a region of cortex where both amyloid deposition and neurofibrillary changes have occurred.