Prophylactic immunization against experimental leishmaniasis. IV. Subcutaneous immunization prevents the induction of protective immunity against fatal Leishmania major infection

Durable immunity against fatal L. major infection in genetically susceptible mice can be induced by immunization with 150,000-rad irradiated or heat-killed promastigotes administered i.v. or to a lesser extent i.p. Conversely, subcutaneous (s.c.) and intramuscular (i.m.) injections are not only totally ineffective but generally increase susceptibility to and enhance the progression of the disease, leading to earlier mortality. This detrimental effect is particularly evident with lower infecting challenge doses. Disease exacerbation is apparent in mice given 4 X s.c. injections of as few as 2 X 10(4) irradiated promastigotes, but it appears most potent after doses of 2 X 10(7). When mice given 4 X s.c. injections were subsequently immunized i.v. with 2 X 10(7) irradiated promastigotes, they failed to develop any evidence of protection against infection with 2 X 10(5) promastigotes, whereas mice given i.v. immunization alone were strongly protected. Thus, s.c. injections are capable of blocking the prophylactic effect of i.v. immunization with irradiated parasites. This inhibitory effect can be achieved with a single s.c. injection, although rather less potently than with four, and is even effective against four repeated weekly i.v. immunizations. Once induced, the effect persists undiminished after 100 days. A weaker effect is also inducible by s.c. injection given after i.v. immunization. The blocking effect of s.c. injection is not dependent on continuing viability of the promastigotes, as it can be induced equally readily with heat-killed, formalin-fixed, or sonicated parasites. The phenomenon extends to mouse strains genetically resistant as well as susceptible to L. major infection and, in congenic mice of BALB background, is independent of the major histocompatibility (H-2) gene complex.     

Therapeutic potential of rat monoclonal antibodies: isotype specificity of antibody-dependent cell-mediated cytotoxicity with human lymphocytes

The ability of rat monoclonal antibodies to promote antibody-dependent cell-mediated cytotoxicity with human effector cells was tested by using a variety of antibodies against different human and mouse leukocyte antigens. It was found that only IgG2b antibodies were effective. This isotype has already been shown to be efficient in fixing human complement, which suggests that among rat monoclonal antibodies, the IgG2b subclass might be a good choice for attempts at serotherapy. Further studies with other antibody-mediated effector mechanisms as well as suitable clinical trials are merited.     

Isolation and characterization of calcium-accumulating matrix vesicles from chondrocytes of chicken epiphyseal growth plate cartilage in primary culture

Matrix vesicles (MV) can be readily isolated from culture media of chicken growth plate hypertrophic chondrocytes grown in primary culture. The chondrocytes maintain normal morphology and synthesize type II collagen throughout the culture period. The culture-derived MV are morphologically indistinguishable from MV seen in situ and are rich in alkaline phosphatase. Formation of alkaline phosphatase-rich MV is strongly influenced by the stage of culture: large numbers are released shortly after cell seeding; marked decline is seen during cell spreading and rapid cell division; notable resurgence in alkaline phosphatase-rich MV production occurs as the cells attain confluency. Increasing the initial chondrocyte seeding density proportionately increases MV production. Cells derived from the hypertrophic region are much more capable of forming alkaline phosphatase-rich MV than those from the proliferating zone, indicating that MV formation is dependent on cellular differentiation. MV released by the cultured chondrocytes were compared in protein and phospholipid composition and in their ability to accumulate mineral ions, with plasma membrane fractions and collagenase-released MV obtained from the same tissue. Electrophoretic patterns of proteins, and the phospholipid profiles, suggest that significant modification of the plasma membrane occurs during MV formation. The vesicles are capable of accumulating large amounts of mineral ions from a metastable synthetic cartilage lymph when supplied with alkaline phosphatase substrates. This culture system thus appears to be a useful model for isolating native MV and characterizing factors required for vesicle formation and mineralization.     

Developmental changes in the adenine nucleotide translocase in the guinea pig

Investigations of developmental changes in energy metabolism in guinea pig liver mitochondria showed that mitochondria from the newborn were well coupled, with respiratory control ratios and membrane energy potentials similar to those obtained with mitochondria from the 1-day-old and the adult. In contrast, there was a 3-fold increase in the rate of mitochondrial respiration and a 2-fold increase in adenine nucleotide content during the first 24 h of extrauterine life. There was no significant change in the ATP/ADP ratio and only a 30% increase in the uncoupled rate of respiration during this same time period. Titrations of the adenine nucleotide translocase with the specific inhibitor, carboxyatractyloside, showed that the newborn had only 50% of the adenine nucleotide translocase activity of the adult. Furthermore, by applying flux control theory to these inhibitor titrations, it was possible to demonstrate that the adenine nucleotide translocase exerted greater control over respiration in the newborn than in the adult, and at maximal rates of coupled respiration the translocase had a control strength of 0.98. The consequences of this finding on cellular energy metabolism are discussed in relation to adaptation of the newborn to extrauterine life.     

A prostaglandin omega-hydroxylase cytochrome P-450 (P-450PG-omega) purified from lungs of pregnant rabbits

Cytochrome P-450-dependent prostaglandin omega-hydroxylation is induced over 100-fold during late gestation in rabbit pulmonary microsomes (Powell, W.S. (1978) J. Biol. Chem. 253, 6711-6716). Purification of cytochromes P-450 from lung microsomes of pregnant rabbits yielded three fractions. Two of these fractions correspond to rabbit lung P-450I (LM2) and P-450II (LM5), which together constitute 70-97% of total cytochrome P-450 in lung microsomes from nonpregnant rabbits. The third form, which we designate rabbit cytochrome P-450PG-omega, regioselectively hydroxylates prostaglandins at the omega-position in reconstituted systems with a turnover of 1-5 min-1. Titration with purified pig liver cytochrome b5, demonstrated a 4-fold maximum stimulation at a cytochrome b5 to a P-450 molar ratio of 1-2. Rabbit lung P-450PG-omega formed a typical type I binding spectrum upon the addition of prostaglandin E1 with a calculated K8 of 1 microM, which agreed reasonably well with the kinetically calculated Km of 3 microM. Cytochrome P-450PG-omega was isolated as a low-spin isozyme with a lambda max (450 nm) in the CO-difference spectrum distinguishable from P-450I (451 nm) and P-450II (449 nm). Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis demonstrated that although purified P-450PG-omega had a relatively low specific content (12.1 nmol mg-1), it appeared homogeneous with a calculated minimum Mr of 56,000, intermediate between rabbit LM4 and LM6. When lung microsomes from pregnant and nonpregnant rabbit were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a protein band, with a Mr identical to P-450PG-omega, was observed in the pregnant rabbit, whereas this band appeared to be very faint or absent in microsomes from the nonpregnant rabbit. Purification of cytochromes P-450 from nonpregnant rabbit lung yielded only P-450I and P-450II. P-450PG-omega appears to be a novel rabbit P-450, possessing high activity towards omega-hydroxylation of prostaglandins, and is greatly induced during pregnancy in rabbit lung.     

REGIONAL ISOLATION PERFUSION IN THE TREATMENT OF ADVANCED MALIGNANT DISEASE

Regional isolation perfusion with an alkylating agent was carried out in a small community hospital, using readily available equipment, ten times in nine cases of advanced malignant disease. Visible regression of tumor, either grossly or histologically, occurred in seven patients and decided subjective improvement occurred in five patients.The long range effect of the procedure could not be determined in the present series, not enough time having elapsed.Side effects included jaundice in four patients, massive necrosis of the tumor in one patient, nausea and vomiting in three patients, postoperative edema in five patients, wound infection in two patients, Horner''s syndrome in one patient, temporary alopecia in three patients, and depression of hemoglobin level in seven patients, of the leukocyte count in four patients and of the platelet count in four patients.A feature of the procedure used in the present series was a system of monitoring the escape of alkylating agent into the systemic circulation.     

Analysis of nonmethylated GATC sites in the Escherichia coli chromosome and identification of sites that are differentially methylated in response to environmental stimuli.

Seven GATC sites that are nonmethylated in logarithmic growth phase cells using glycerol as a carbon source were isolated from the Escherichia coli chromosome. Three of these GATC sites are located upstream of the operons gut, mtl, and ppiA, whereas DNA sequences adjacent to three other nonmethylated GATC sites are not homologous to previously identified genes. The seventh nonmethylated GATC site is located downstream of uspA. The protection of this site from DNA methylation requires leucine-responsive regulatory protein and is leucine responsive. The carbon source and the growth phase influenced the protection of the GATC site 5' of the ppiA gene. The other five sites were protected under all the environmental conditions examined.