Antigen-independent cross-talk between macrophages and CD8+ T cells facilitates their cooperation during target destruction

Inflammatory sites associated with tissue destruction often contain a complex mixture of cells including macrophages as well as CD8+ and CD4+ T cells. Here, we have investigated, using islets of Langerhans as targets, if CD8+ T cells and macrophages can cooperate in tissue destruction. CD8+ T cells obtained from the islet inflammatory lesion of non-obese diabetic mice or cloned islet-specific CD8+ T cells were ineffective in destroying islets on their own. Including increasing numbers of macrophages in co-cultures of islets and islet-derived or cloned CD8+ T cells progressively increased and accelerated islet destruction. Macrophages alone were ineffective. Macrophage-depleted islets were not destroyed by islet-derived CD8+ T cells. For cooperative islet destruction to occur, beta cells, but not macrophages, needed to be able to present antigens to CD8+ T cells. CD8+ T cells triggered NO production by macrophages, while macrophages triggered IFN-gamma production by CD8+ T cells. Each of these factors was partially effective, but not sufficient, for maximal islet destruction. Antibodies specific for ICAM-1 and LFA-1 inhibited both cooperative islet destruction and cross-stimulation of CD8+ T cells and macrophages. The data suggest that if CD8+ T cells become only weakly activated by target cells, they are not able to destroy target tissue on their own. However, such CD8+ T cells and local macrophages may still cross-stimulate each other, which then facilitates target destruction. For this to occur, target cells, but not macrophages, need to present antigen to CD8+ T cells.     

Virion-bound ICAM-1 and activated LFA-1: a combination of factors conferring resistance to neutralization by sera from human immunodeficiency virus type 1-infected individuals independently of the disease status and phase

The role of the supplementary interaction between virion-bound host ICAM-1 and LFA-1 on target cells in sensitivity to neutralization of human immunodeficiency virus type 1 (HIV-1) is poorly studied. Serum samples from four long-term nonprogressors (LTNPs) and sequential sera from one progressor were used to assess neutralization sensitivity of isogenic ICAM-1-negative and ICAM-1-bearing HIV-1(NL4-3), a prototype of T-cell-line-adapted viruses. We found that virus neutralization sensitivity to the studied sera is not modified by the additional interaction between virally embedded ICAM-1 and LFA-1 under an inactive state. However, expression on the target cell surface of an activated LFA-1 form renders ICAM-1-bearing virus particles, but not viruses devoid of ICAM-1, more refractory to neutralization by sera from three out of four LTNPs and all sequential sera from the person who has experienced a progression of the HIV-1-associated disease. Although no conclusive correlation could be drawn between virus susceptibility to neutralization and the disease status or stages of HIV-1 infection, these findings demonstrate that other nonspecific virus-cell interactions mediated by virion-anchored host proteins and their normal cognate ligands on target cells represent factors that can affect the mechanism of HIV-1 neutralization.     

Clinical and molecular study in congenital muscular dystrophy with partial laminin alpha 2 (LAMA2) deficiency

Complete laminin alpha2 (LAMA2) deficiency causes approximately half of congenital muscular dystrophy (CMD) cases. Many loss-of-function mutations have been reported in these severe, neonatal-onset patients, but only single missense mutations have been found in milder CMD with partial laminin alpha2 deficiency. Here, we studied nine patients diagnosed with CMD who showed abnormal white-matter signal at brain MRI and partial deficiency of laminin alpha2 on immunofluorescence of muscle biopsy. We screened the entire 9.5 kb laminin alpha2 mRNA from patient muscle biopsy by direct capillary automated sequencing, single strand conformational polymorphism (SSCP), or denaturing high performance liquid chromatography (DHPLC) of overlapping RT-PCR products followed by direct sequencing of heteroduplexes. We identified laminin alpha2 sequence changes in six of nine CMD patients. Each of the gene changes identified, except one, was novel, including three missense changes and two splice-site mutations. The finding of partial laminin alpha2 deficiency by immunostaining is not specific for laminin alpha2 gene mutation carriers, with only two patients (22%) showing clear causative mutations, and an additional three patients (33%) showing possible mutations. The clinical presentation and disease progression was homogeneous in the laminin alpha2-mutation positive and negative CMD patients.     

BRCA1 and BRCA2 mutations in Russian familial breast cancer

We have screened index cases from 25 Russian breast/ovarian cancer families for germ‐line mutations in all coding exons of the BRCA1 and BRCA2 genes, using multiplex heteroduplex analysis. In addition we tested 22 patients with breast cancer diagnosed before age 40 without family history and 6 patients with bilateral breast cancer. The frequency of families with germline mutations in BRCA was 16% (4/25). One BRCA1 mutation, 5382insC, was found in three families. The results of present study, and those of a separate study of 19 breast‐ovarian cancer families, suggest that BRCA1 5382insC is a founder mutation in the Russian population. Three BRCA2 mutations were found in patients with breast cancer without family history: two in young patients and one in patients with bilateral breast cancer. Four novel BRCA2 mutations were identified: three frameshift (695insT, 1528del4, 9318del4) and one nonsense (S1099X). © 2002 Wiley‐Liss, Inc.     

Lack of directed Vα14-Jα281 rearrangements in NK1+ T cells

NK1.1⁺ T cells are an unusual subset of TCRαβ cells distinguished by their highly restricted Vβ repertoire and predominant usage of an invariant Vα14-Jα281 chain. To assess whether a directed rearrangement mechanism could be responsible for this invariant α chain, we have analyzed Vα14 rearrangements by polymerase chain reaction and Southern blot in a panel of cloned T-T hybrids derived from thymic NK1.1⁺ T cells. As expected a high proportion (17/20) of the hybrids had rearranged Vα14 to Jα281. However, Vα14-Jα281 rearrangements always occurred on only one chromosome and were accompanied by other Vα-Ja rearrangements (not involving Vα14) on the homologous chromosome. These data argue that rigorous ligand selection rather than directed rearrangement is responsible for the high frequency of Vα14-Jα281 rearrangements in NK1.1⁺ T cells.     

Ehlers-Danlos syndrome type IV caused by Gly400Glu, Gly595Cys and Glyl003Asp substitutions in collagen III: clinical features, biochemical screening, and molecular confirmation

Three patients with Ehlers-Danlos syndrome type IV (EDS IV) and biochemical evidence of structural defects in collagen III were investigated for mutations within the collagen III gene ( COL3A1 ). Single strand conformation polymorphism analysis of α1(III) cDNA indicated the presence of different heterozygous sequence changes in each of the patients. Nucleotide sequencing revealed mutations leading to the substitution of glycine 400 with glutamic acid, glycine 595 with cysteine, and glycine 1003 with aspartic acid. EDS IV is a life-threatening disorder which, as the clinical histories of our patients and their families show, still often escapes diagnosis. Biochemical and molecular studies can clarify the diagnosis and help provide appropriate management and counselling.     

Evidence for a glycerol pathway through aquaporin 1 (CHIP28) channels

Permeabilities to glycerol and small non-electrolytes of three Aquaporin 1 CHIP (AQP1) water channels were measured in AQP1 cRNA-injected Xenopus laevis oocytes and in human AQP1 channels reconstituted in proteoliposomes. By an osmotic swelling assay, significant increases of ethylene glycol, glycerol and 1,3-propanediol apparent permeability coefficients (P_solutes) were found in oocytes expressing human, rat and frog AQP1. p-Chloromercuribenzene sulphonate (PCMBS) and CuSO₄ inhibited, by 95% and 58% respectively, apparent glycerol permeability (P _gly) in oocytes expressing human AQP1. pCMBS inhibition was reversed by -mercaptoethanol and CuSO₄ inhibition was partly reversed by the Cu²⁺-binding peptide Gly-Gly-His. Tritiated glycerol uptakes confirmed the augmented P _gly value of AQP1 cRNA-injected oocytes. In contrast, no increases of urea, meso-erythritol, D- or L-threitol, xylitol and mannitol uptakes were detected. Stopped-flow light scattering experiments performed with human AQP1 proteoliposomes also revealed a much greater increase of P _gly than did those with protein-free liposomes; the initial rate of proteoliposomes also swelling was inhibited by 96.2% with HgCl₂ and by 72.5% with CuSO₄. In AQP1 cRNA-injected oocytes and in proteoliposomes, the value of the glycerol reflection coefficient was 0.74–0.80, indicating that water and glycerol share the same pathway. All these results provide strong evidence that water and certain small solutes permeate the AQP1 channels expressed at the surface of X. laevis oocytes or reconstituted in proteoliposomes. The urea exclusion suggests that the selectivity of the AQP1 channels not only depends on the size of the solutes but probably also on their flexibility and their ability to form H-bonds.     

Inhibition of HIV-1 by modification of a host membrane protease

While it is clear that CD4 Is the receptor for the gp120 envelopeprotein of HIV-1, substantial evidence suggests that other hostcell proteins are required for successful membrane fusion. Studieswere initiated to examine the potential for a protein receptorwhich has an elastase-like character to participate in fusionof HIV-1 with permissive host cells. A synthetic elastase inhibitorwas shown to significantly reduce HIV-1 infectivity when presentduring, but not after, the initial contact between virus andcells. A human T cell elastase-like membrane component was purifiedand shown to be lipid-associated. By competitive Inhibition,the purified protein was shown to bind gp160 within the HIV-1fusion domain. The binding parameters of whole T cell membraneextract, with a hydrophobic pentapeptide representative of thefusion domain, suggested an elastase-like protein is the single,secondary T cell receptor for HIV-1 (K = 1x10³ M^–1). Thepentapeptide interacted with porcine and human (epithelial andpolymorphonuclear leukocyte), but not murine, elastase isoforms,suggesting its participation In the permissiveness of host cellsto infection.     

Restriction fragment length polymorphisms of the CYP11B1 gene in the Japanese population

Summary Restriction fragment length polymorphisms (RFLPs) of the CYP11B1 gene were studied in Japanese using cDNA clone P450c11 as a probe. Genomic DNAs from 60 unrelated Japanese individuals were digested with 8 different restriction enzymes and analyzed by Southern blot hybridization. Two RFLPs were detected inMspI digests of the DNA. One(A) was characterized by polymorphic bands at 3.4 and 2.5 kilobasepairs (kb) and the other (B) by polymorphic bands at 1.7 and 1.2 kb. The third RFLP was observed inPvuII-digested samples and was polymorphic at 5.8 and 4.0 kb bands. Two of the three RFLPs found, RFLP (A) and (C), have not been described in the only previous report which was based on Caucasian samples. We also examined the RFLPs of a 3 generation family of 11-hydroxylase deficiency caused by an abnormality of the CYP11B1 gene. All the family members were homozygous in all three RFLPs and was thus not informative.     

Phenytoin promotes Th2 type immune response in mice

The effects of chronic administration of phenytoin, a common anticonvulsive drug, on immune responses were studied in mice. Anti-keyhole limpet haemocyanin (KLH) IgE antibody response after KLH-immunization was enhanced in phenytoin-treated mice. Proliferative responses of spleen cells induced with KLH, concanavalin A (ConA), lipopolysaccharide and anti-CD3 antibody were reduced in phenytoin-treated mice. Accessory function of spleen adherent cells on ConA-induced T cell proliferative response was reduced in phenytoin-treated mice. KLH-induced IL-4 production of spleen cells was enhanced, while IFN-gamma production was reduced in phenytoin-treated mice. In addition, production of IL-1 alpha, but not IL-6 and IL-12 by spleen adherent cells from phenytoin-treated mice was reduced. Natural killer cell activity was reduced in phenytoin-treated mice. These results suggest that phenytoin treatment preferentially induces a Th2 type response. We also observed that plasma ACTH and corticosterone levels were increased in phenytoin-treated mice, and speculated that phenytoin might act directly and indirectly, through HPA axis activation, on the immune system to modulate Th1/Th2 balance.