Leukocyte-endothelial cell recognition: evidence of a common molecular mechanism shared by neutrophils, lymphocytes, and other leukocytes

The interaction of leukocytes with endothelial cells is intrinsic to the process of leukocyte extravasation, whether during the entry of blood polymorphonuclear leukocytes and monocytes into sites of acute and chronic inflammation, or during the homing of lymphocytes to lymphoid organs. A lymphocyte surface glycoprotein, defined by monoclonal antibody MEL-14, has been described that appears to mediate lymphocyte recognition of postcapillary venules in peripheral lymph nodes, and to control the migration of lymphocytes from the blood into these lymphoid organs. We now report that the antigenic determinant recognized by MEL-14 is present at high levels on other leukocytes as well, including neutrophils, monocytes, and eosinophils; and we demonstrate involvement of the MEL-14 antigen in neutrophil-endothelial cell interactions. MEL-14 immunoprecipitates a neutrophil surface protein of Mr approximately 100,000, similar in m.w. to the 80,000 to 90,000 dalton lymphocyte surface MEL-14 antigen, and it blocks the interaction of neutrophils with endothelial cells in an in vitro model of adhesion to postcapillary venules in lymph node frozen sections. Neutrophil binding to lymph node venules is also inhibited by PPME, a mannose-6-phosphate-rich yeast polysaccharide that is thought to mimic the endothelial cell ligand for the MEL-14-defined lymphocyte receptor. Interestingly, neither MEL-14 nor PPME exhibit a major effect on neutrophil binding to postcapillary venules in Peyer's patches, suggesting that as for lymphocytes, the neutrophil MEL-14 antigen is involved in recognition of tissue-specific endothelial determinants. Finally, we show that MEL-14 inhibits the capacity of neutrophils to migrate from the blood into sites of acute inflammation in the skin. These observations lead us to propose that receptors for tissue-specific endothelial determinants are utilized by neutrophils and lymphocytes and probably other leukocytes during the physiologic process of leukocyte extravasation in vivo.     

A membrane-bound human placental protein kinase activated by endogenous polypeptides

A protein kinase (PPdPK) was purified from plasma membranes of human placenta. Phosphorylation of casein , but not of phosvitin or lactalbumin, by [γ-³²P]ATP in the presence of PPdPK was stimulated about 10-fold by naturally occurring polypeptides prepared from avariety of sources similar to the procedure of Roberts et al. (Proc. Natl. Acad. Sci. U.S.A.77, 3494–3498, 1980). The amino acid phos-phorylated on casein was serine. According to gel exclusion chromatography the mol.wt, of PPdPK was 95 000. In autoradiograms, following polyacrylamide-gel electrophoresis, the autophosphorylation of PPdPK was greatly enhanced by the polypeptide activators.     

Permeabilization of Ehrlich ascites tumor cells by human serum

Human serum rapidly permeabilized Ehrlich ascites tumor cells to inorganic cations such as Rb⁺ and Ca²⁺; serum from several other species showed little or no activity. The effect of human serum was not reversed by washing the cells. Human serum, deficient in specific complement proteins, had no activity, but was reactivated by the addition of the missing complement component. Since Ca²⁺ was not required for the permeabilization, the alternative pathway of complement activation was implicated. Human serum deficient in Factor B of the alternative pathway was ineffective, but permeabilizing activity was restored by addition of Factor B. Rb⁺ uptake of several other cells was not inhibited by human serum. We conclude that an interaction between human complement and Ehrlich ascites tumor cells is responsible for the membrane lesion observed.     

Salvador E. Luria (1912–1991), a unique human being

In Memory of Salvador E. Luria     

Monoclonal antibody L6-daunomycin conjugates constructed to release free drug at the lower pH of tumor tissue

Summary Measurements in cancer patients showed that the pH of tumors averages 0.8 unit lower than that of the surrounding normal tissues, confirming published work. Based on this, the anti-carcinoma monoclonal antibody (mAb) L6 was used to prepare immunoconjugates with daunomycin (DM), the drug being released at the acidic pH of the tumor. A direct linking of the aconitic derivative of DM (AcoDM) to mAb L6 led to conjugates that either had a low drug/antibody ratio (<5:1) or precipitated in vitro. In order to increase the drug load and avoid precipitation, several biopolymers were tested as spacers between the drug and the L6. To attach the polymer derivative to the mAb, the former was maleimidized and the mAb was thiolated. The AcoM/mAb ratio obtained was 20, and the mAb retained its highly specific binding to tumor cells. At pH 6 the AcoDM-L6 conjugate was toxic to cultured C-3347 carcinoma cells with an inhibitory concentration (IC₅₀) of 5 µg/ml. The conjugate was less effective than the free DM with an IC₅₀ of 0.2 µg/ml. The L6 alone was not toxic. At a tumor pH of 6.5, 15% of the AcoDM was released. The amount of released drug reached a maximum 24–48 h after exposure to the acidic medium.In vivo localization studies demonstrated a similar tumor uptake of the conjugate and mAb L6 with 18% of the injected dose/g tumor and a maximum uptake in tumor 48 h after injection. Our data indicate that it is possible to construct conjugates based on a pH-sensitive linker that can be targeted successfully to a tumor with release of a portion of the drug at the tumor site, but testing is needed to establish whether such release has anti-tumor activity in vivo and offers an advantage over treatment with unconjugated drug.     

Leukotriene C4 is an essential 5-lipoxygenase intermediate in A23187-induced macrophage cytostatic activity against P815 tumor cells

Resident peritoneal macrophages incubated with 3.5 x 10(-7) M Calcium ionophore A23187 in tumor cell growth medium (TGM) release large amounts of leukotriene (LT)E4 and an unidentified 5-lipoxygenase product, whereas A23187-stimulated macrophages produce in serum free medium LTD4, predominately. LTC4 and 3H-LTC4 incubated for 20 min at 37 degree C in serum containing TGM, convert into LTE4 and 3H-LTE4, respectively. Thus, LTC4 released from A23187-stimulated macrophages is an intermediate in TGM which rapidly converts into LTE4, probably because of the presence of gamma-glutamyl transpeptidase and cystenylglycinase in TGM. Macrophages express antitumor cytostatic activity towards P815 cells (49-53%) in a cocultured ratio (macrophage: tumor cell) 2:1 when stimulated with 3.5 x 10(-7) M A23187 in TGM. The 5-lipoxygenase inhibitor AA861 reverses the cytostatic activity by 42-58% and it inhibits also the formation of A23187-induced 5-lipoxygenase products from macrophages. Restoration of 38% macrophage- antitumor cytostatic activity by exogenous LTC4 (10(-8) M) indicates that LTC4 is an essential 5-lipoxygenase intermediate in the pathway of required signals underlying A23187-induced macrophage antitumor cytostatic activity. Macrophages not stimulated by A23187 do not express cytostatic activity in the presence of LTC4. This implies that besides LTC4, increased cytosolic [Ca2+] is required for A23187 induction of macrophage cytostatic activity.     

Host specialization,genetic variability,and differentiation in three species of Tomoplagia from Brazilian rupestrian grasslands

In this study, the geographic and host plant-associated genetic structure of Tomoplagia spp. (Diptera: Tephritidae) was investigated. The study was developed in the rupestrian montane grasslands of the Espinhaço Mountain Range, Brazil, where these flies’ larvae develop within flower heads of Asteraceae. The three species of Tomoplagia investigated have different host spans within the subtribe Vernoniinae: one is strictly monophagous (Tomoplagia grandis Prado, Norrbom & Lewinsohn) and two are oligophagous [Tomoplagia incompleta (Williston) and Tomoplagia bicolor Prado, Norrbom & Lewinsohn]. The genetic variability and differentiation throughout their geographic distribution were investigated at the local scale (sympatric, between different host species in the same locality) and the regional scale (allopatric, on the same or different host species in different localities). Allele richness was greater for populations of the two oligophagous species. Moreover, population genetic structure was low in all species both on the local and regional scales, and the attribution of individuals of the three species to different genetic groups was not strongly associated with either locality or host plant. These results indicate the absence of simple geographic and host-associated genetic structure in these specialized herbivores, suggesting that females of T. bicolor and T. incompleta have low host philopatry, using various host species indiscriminately within their distribution range. Therefore, the use of host plants by fruit flies of the genus Tomoplagia is a plastic character that can shift temporally and spatially among phylogenetically related and chemically similar plants. Contrary to expectations, given the specialized and intimate nature of their host association, the studied Tomoplagia species showed no clear host-associated genetic differentiation; instead, genetic variation presents a mosaic pattern across hosts and localities.     

X-linked creatine transporter (SLC6A8) mutations in about 1% of males with mental retardation of unknown etiology

Mutations in the creatine transporter gene, SLC6A8 (MIM 30036), located in Xq28, have been found in families with X-linked mental retardation (XLMR) as well as in males with idiopathic mental retardation (MR). In order to estimate the frequency of such mutations in the MR population, a screening of 478 males with MR of unknown cause was undertaken. All 13 exons of SLC6A8 were sequenced using genomic DNA. Six novel potentially pathogenic mutations were identified that were not encountered in at least 588 male control chromosomes: two deletions (p.Asn336del, p.Ile347del) and a splice site alteration (c.1016+2C>T) are considered pathogenic based on the nature of the variant. A mutation (p.Arg391Trp) should be considered pathogenic owing to its localization in a highly conserved region. Two other missense variants (p.Lys4Arg, p.Gly26Arg) are not conserved but were not observed in over 300 male control chromosomes. Their pathogenicity is uncertain. A missense variant (p.Val182Met), was classified as a polymorphism based on a normal creatine/creatinine (Cr:Crn) ratio and cerebral creatine signal in proton magnetic resonance spectroscopy (H-MRS) in the patient. Furthermore, we found 14 novel intronic and neutral variants that were not encountered in at least 280 male control chromosomes and should be considered as unclassified variants. Our findings of a minimum of four pathogenic mutations and two potentially pathogenic mutations indicate that about 1% of males with MR of unknown etiology might have a SLC6A8 mutation. Thus, DNA sequence analysis and/or a Cr:Crn urine screen is warranted in any male with MR of unknown cause.Amy J. Clark and Efraim H. Rosenberg have contributed equally to this work.     

Pathogen race determines the type of resistance response in the stripe rust –Triticum dicoccoides pathosystem

Wild relatives of crop plants may serve as a promising source for screening for new disease resistance genes that can be utilized in breeding programs. Triticum dicoccoides, the wild progenitor of most cultivated wheats, was shown to harbor many resistance genes against the major diseases attacking cultivated wheat. Stripe rust is a devastating fungal disease that attacks wheat in many regions of the world. New races of Puccinia striiformis Westend. f. sp. tritici, the causative agent of stripe rust, have overcome most of the known Yr resistance genes in wheat. Therefore, there is a need to search for new resistance genes in the T. dicoccoides gene pool. A set of 120 T. dicoccoides accessions, collected from 13 populations representing different habitats in Israel and vicinity, was tested for resistance to three prevalent stripe rust races (38E134, 6E16 and 6E0). Of these 120 accessions, 14, 8 and 12% were resistant to races 38E134, 6E16 and 6E0, respectively, while 57, 2 and 4% were moderately resistant to these races, respectively. A unique resistance was found in the population of Mt Hermon where >80% of the accessions showed resistance to all races. Distribution of infection types (ITs) of race 38E134 showed a normal distribution that can fit a quantitative pattern of response, while the distributions of ITs of races 6E16 and 6E0 had excess of extreme values and therefore showing a qualitative pattern of response. anova testing the main factor effects and interaction showed significant effects of population, race and their interaction on IT. Significant positive correlations were obtained between the resistance to races 6E16 and 6E0 and humidity variables of the collections sites, while resistance to race 38E134 was positively correlated with temperature variables. These results show that the pathogen race can determine the type of resistance response, qualitative or quantitative, in the stripe rust—T. dicoccoides pathosystem. The obtained results also reveal that the distribution of resistance to different pathogen races can be affected by different climatic factors.