Method for histological preparation of bone sections containing titanium implants

A thin sectioning technique involving hand grinding has been developed to produce 20-40-microns-thick sections of bone-titanium implant sites. Components include: 1) surface staining of sections prior to mounting on slides so bone labels (oxytetracycline-HCl and 2,4-bis(N,N-dicarbomethyl)aminomethylfluorescein (DCAF] can be seen in sections viewed with transmitted light, 2) a pneumatic sample press for bonding sections to slides with a thin, uniform glue line and without trapped air bubbles, and 3) bonding methyl methacrylate embedded sections to clear acrylic slides with methyl methacrylate monomer to provide enhanced bond strength and grinding properties compared to those obtainable with glass slides. Sample cracking and distortion is minimized and the tissue-implant interface can be kept intact. The expense of start-up equipment for this technique is minimal.     

Conversion of phosphatidylglycerol to lyso(bis)phosphatidic acid by alveolar macrophages

We report here studies of the synthesis of lyso(bis)phosphatidic acid [L(b)PA] by normal and BCG-elicited rabbit alveolar macrophages. This study was prompted by our earlier observations that 1) alveolar macrophages did not synthesize L(b)PA de novo despite its abundance in these cells, 2) BCG-elicited cells contained only one-quarter the amount of L(b)PA as normal cells, and 3) the turnover of arachidonate in L(b)PA led to hydroxyeicosatetraenoic acid and leukotriene synthesis. We found that exogenous phosphatidylglycerol (PG) was specifically converted to L(b)PA by both types of cells although BCG-elicited cells had only one-quarter the synthetic capacity of normal cells. Other phospholipids were found to become cell associated but were not significantly metabolized. Both glycerol moieties and the phosphate were incorporated into the product L(b)PA. However, substitution of the ester with an alkyl linkage in position 1 blocked the conversion of PG to L(b)PA. Most of the alkylphosphatidylglycerol was converted to phosphatidylcholine and phosphatidylethanolamine. This result implied that catabolism of the acyl group in position 1 was essential for L(b)PA synthesis. Because alveolar macrophages are present in a surfactant-rich milieu, we suggest that surfactant provides a source of PG for macrophage synthesis of L(b)PA in situ. It is interesting that the surfactants from rabbits challenged with BCG have a significant decrease in PG content.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Immunopathological features of human pulmonary tumors following low-dose interleukin-2

Summary We administered preoperative low-dose interleukin-2 (IL-2) to 10 patients undergoing thoracotomy for pulmonary tumors. The in vivo effect of IL-2 on tumor-associated lymphocyte activity was assessed in the resected specimens by immunohistochemistry and compared with observations in 45 patients who did not receive IL-2. H & E evaluation revealed an increase in intra- and peritumoral lymphocyte infiltration in the IL-2-treated patients. Immunopathological evaluation with monoclonal antibodies revealed that this lymphocyte infiltration was predominantly CD5-positive T cells. The amount of intra-and peritumoral lymphocyte activity correlated with the dose of IL-2 administered (6000–90 000 international units/kg every 8 h for 48 h. IL-2-treated patients showed increases in T-cell-associated activation markers (IL-2 -receptor, transferrin receptor and HLA-DR) on peritumoral lymphocytes, but not on intratumoral lymphocytes. We previously reported that low-dose IL-2 increases the intrinsic natural killer cell cytotoxicity of intratumoral lymphocytes and suggest that this lymphocyte infiltration is further evidence that low-dose IL-2 can augment in vivo lymphocyte activity at the tumor site.This work was supported in part by USPHS grants CA 44 352 (S. H. G.) and 43 658 (A. J. C.). S. G. S. was supported by NIH Surgical Oncology Training Grant CA 09 010     

The daily timing of lamprey attacks

Synopsis Laboratory and field data suggest that several species of parasitic lampreys forage primarily at night, but data forPetromyzon marinus are equivocal. Nocturnal foraging may minimize predation risk. This may be particularly important to those species of lampreys that inhabit shallow streams. Nocturnal foraging also may enhance foraging efficiency if potential hosts at night are quiescent and more easily approached. Future experiments should be designed to avoid several potential sources of bias.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Location of Homologous DNA Sequences Interspersed at Five Regions in the Baculovirus AcMNPV Genome

An examination of Autographa californica nuclear polyhedrosis virus DNA revealed the presence of five interspersed regions, rich in EcoRI restriction sites, which shared homologous sequences. These homologous regions (hr), designated hr₁ to hr₅, occur at or near the following EcoRI fragment junctions: hr₁EcoRI-B—EcoRI-I (0.0 map units); hr₂, EcoRI-A—EcoRI-J (19.8 map units); hr₃, EcoRI-C—EcoRI-G (52.9 map units); hr₄, EcoRI-Q—EcoRI-L (69.8 map units); and hr₅, EcoRI-S—EcoRI-X (88.0 map units). Four of these regions were identified, by cross-blot hybridization of HindIII-restricted A. californica nuclear polyhedrosis virus DNA, to be within the HindIII-A/B, -F, -L, and -Q fragments. The location of these regions and the identification of a fifth homologous region were confirmed, and their characterization was facilitated, by using two plasmids with HindIII-L or -Q fragment insertions, which contained the homologous regions hr₂ and hr₅, respectively. The sizes of the homologous regions were about 800 base pairs for hr₂, 500 base pairs for hr₅, and less than 500 base pairs for hr₁, hr₃, and hr₄. A set of small EcoRI fragments (EcoRI minifragments) which ranged in size from 225 to 73 base pairs were detected in A. californica nuclear polyhedrosis virus DNA and HindIII-L and -Q fragments by polyacrylamide gel analysis. Some of the minifragments in viral DNA were present in extramolar amounts and corresponded in size to some of the minifragments present in HindIII-L and -Q. Clones of some of the EcoRI minifragments were used as probes in hybridizations to digests of viral DNA and of HindIII-L and -Q. The hybridization data, obtained under various levels of stringency, suggested that there was a degree of mismatching between the sequences which were responsible for the homology.     

Postoperative Depression of Tumour-directed Cell-mediated Immunity in Patients with Malignant Disease

Leucocytes from 46 melanoma patients, 45 breast carcinoma patients, and 95 control donors were tested by the leucocyte migration test against the supernatants of homogenates of malignant melanomas, breast carcinomas, simple breast tumours, and breasts showing simple cystic disease. By comparison with controls inhibition of migration occurred significantly more frequently when tumour patients'' leucocytes were exposed to extracts of histogenetically similar tumours.Cell-mediated immunity to tumour-associated antigens was measured in 12 patients with breast carcinoma and 12 with malignant melanoma immediately before surgical operation and in the postoperative period. All patients tested before operation showed significant inhibition of migration on contact with extracts of histogenetically similar tumours. Postoperatively the degree of leucocyte migration inhibition was reduced in all patients with melanoma and breast carcinoma. Significant inhibition of leucocyte migration returned in most patients 6-22 days after operation.     

Intratumoral interleukin-2 immunotherapy: Activation of tumor-infiltrating and splenic lymphocytes in vivo

Direct intratumoral injection of interleukin-2 (IL-2) was evaluated in a murine model. Balb/c mice received 5 × 10⁴ Line 1 alveolar carcinoma cells (L1C2) by subcutaneous injection. On the third day following tumor implantation, mice received injections of IL-2 (5 × 10³–5 × 10⁴ units) or diluent twice daily, either by i. p. or intratumoral injection, 5 days/week for 3 weeks. Intratumoral injection of 5 × 10⁴ units IL-2 significantly reduced tumor volume (P <0.05 versus control), increased median survival time (P = 0.0001), and resulted in a 23.5% cure rate (P = 0.008). There were no long-term survivors in the other treatment groups. Both tumor-infiltrating lymphocytes (TIL) and splenic lymphocytes isolated directly from IL-2-treated mice demonstrated enhanced cytolytic activity compared to diluent-treated controls. To determine whether non-T-cell-mediated antitumor responses were active in our model, intratumoral immunotherapy was evaluated in athymic Balb/cnu/nu mice. In order to decrease the recruitment of lymphocyte precursors, nude mice were splenectomized and received cyclophosphamide prior to tumor injection and IL-2 therapy. Intratumoral IL-2 immunotherapy also significantly decreased tumor volume in these immunodeficient mice (P <0.02), but did not lead to long-term survival. We conclude that both TIL and splenic lymphocytes are activated in vivo in response to intratumoral IL-2 immunotherapy, suggesting that intratumoral therapy with IL-2 activates both local and systemic antitumor responses.Supported by the Tobacco-Related Disease Research Program of the University of California, the Cancer Research Coordinating Committee, the Jonsson Cancer Center Foundation, and Veterans Administration Medical Research Funds