Nonlinear ultrafast acoustics at the nano scale.

Pulsed femtosecond lasers can generate acoustic pulses propagating in solids while displaying either diffraction, attenuation, nonlinearity and/or dispersion. When acoustic attenuation and diffraction are negligible, shock waves or solitons can form during propagation. Both wave types are phonon wavepackets with characteristic length scales as short as a few nanometer. Hence, they are well suited for acoustic characterization and manipulation of materials on both ultrafast and ultrashort scales. This work presents an overview of nonlinear ultrasonics since its first experimental demonstration at the beginning of this century to the more recent developments. We start by reviewing the main properties of nonlinear ultrafast acoustic propagation based on the underlying equations. Then we show various results obtained by different groups around the world with an emphasis on recent work. Current issues and directions of future research are discussed.

Chirping of an optical transition by an ultrafast acoustic soliton train in a semiconductor quantum well.

Acoustic solitons formed during the propagation of a picosecond strain pulse in a GaAs crystal with a ZnSe/ZnMgSSe quantum well on top lead to exciton resonance energy shifts of up to 10 meV, and ultrafast frequency modulation, i.e., chirping, of the exciton transition. The effects are well described by a theoretical analysis based on the Korteweg-de Vries equation and accounting for the properties of the excitons in the quantum well.

Targeting of influenza epitopes to murine CR1/CR2 using single-chain antibodies.

Single-chain variable fragment (scFv) antibodies are genetically engineered molecules comprising the variable regions responsible for specific binding. scFv that recognize certain surface molecules on professional antigen presenting cells could therefore be suitable for targeting Ag to these cells. We have produced an scFv that recognizes murine complement receptors 1 and 2 (CR1/CR2) and genetically fused it with different numbers of influenza hemagglutinin peptides which contain both B and T cell epitopes. The CR1/CR2 specific hybridoma 7G6 was used for RT-PCR to obtain the variable regions, which were then combined to create an scFv fragment. The influenza hemagglutinin intersubunit peptide HA317-41 (IP) was engineered to the N terminus of the scFv in one, two or three copies. The so obtained IP(1-3)7G6scFv still bound the complement receptors; the peptides in the construct were recognized by the peptide specific monoclonal IP2-11-1 on Western blots and ELISAs. The CR1/CR2 positive B lymphomas A20 and 2PK3 presented the peptide to an I-Ed restricted IP specific T cell hybridoma more efficiently when incubated with the IP(1)7G6 constructs as compared to the free peptide. The results suggest that scFv could work as targeting devices in subunit vaccines.

Modulation of the humoral immune response by antibody-mediated antigen targeting to complement receptors and Fc receptors.

During an ongoing immune response, immune complexes, composed of Ag, complement factors, and Igs, are formed that can interact with complement receptors (CRs) and IgG Fc receptors (Fc gamma R). The role of CR1/2 and Fc gamma R in the regulation of the immune response was investigated using OVA that was chemically conjugated to whole IgG of the rat anti-mouse CR1/2 mAb 7G6. FACS analysis using the murine B cell lymphoma IIA1.6 confirmed that the 7G6-OVA conjugate recognized CR1/2. Incubating IIA1.6 cells with 7G6-OVA triggered tyrosine phosphorylation and Ag presentation to OVA-specific T cells in vitro. Immunizing mice with 7G6-OVA at a minimal dose of 1 microgram i.p. per mouse markedly enhanced the anti-OVA Ig response, which was primarily of the IgG1 isotype subclass. The 7G6-OVA did not enhance the anti-OVA response in CR1/2-deficient mice. OVA coupled to an isotype control Ab induced a considerably lower anti-OVA response compared with that induced by OVA alone, suggesting inhibition by interaction between the Fc part of the Ab and the inhibitory Fc gamma RIIb on B cells. This findings was supported by the observation that IIA1.6 cells which were incubated with 7G6-OVA lost the ability to present Ag upon transfection with Fc gamma RIIb. In sum, 7G6-conjugated OVA, resembling a natural immune complex, induces an enhanced anti-OVA immune response that involves at least CR1/2-mediated stimulation and that may be partially suppressed by Fc gamma RIIb.

Murine IgG1 complexes trigger immune effector functions predominantly via Fc gamma RIII (CD16).

Previously, we have demonstrated that phagocytosis of IgG1-coated particles by macrophages in vitro is impaired by deletion of Fc gamma RIII in mice, suggesting that IgG1 may interact preferentially with Fc gamma RIII. In the present study, the biologic relevance of this observation was addressed by triggering various effector functions of the immune system in Fc gamma RIII(-/-) mice, using panels of mAbs of different IgG subclasses. Both binding and phagocytosis of IgG1-coated sheep or human erythrocytes by Fc gamma RIII(-/-) macrophages in vitro were strongly impaired, indicating that the impaired ingestion of complexed IgG1 by Fc gamma RIII(-/-) macrophages is due to a defect in binding. An in vivo consequence of the defective phagocytosis was observed by resistance of Fc gamma RIII-deficient mice to experimental autoimmune hemolytic anemia, as shown by a lack of IgG1-mediated erythrophagocytosis in vivo by liver macrophages. Furthermore, trapping of soluble IgG1-containing immune complexes by follicular dendritic cells in mesenteric lymph nodes from Fc gamma RIII(-/-) mice was abolished. Whole blood from Fc gamma RIII(-/-) mice was unable to induce lysis of tumor cells in the presence of IgG1 antitumor Abs. Finally, IgG1 mAbs proved unable to mount a passive cutaneous anaphylaxis in Fc gamma RIII(-/-) mice. Together, these results demonstrate that IgG1 complexes, either in particulate or in soluble form, trigger in vitro and in vivo immune effector functions in mice predominantly via Fc gamma RIII.

Concepts for a model of good medical laboratory services.

Several international standards and corresponding interpretation documents for quality management systems have been published. Although these standards are found useful to some extent, they are considered to be insufficient in several areas important for medical laboratories particularly in the pre- and post-examinational phases. The normative document for accreditation of laboratories (ISO/IEC Guide 25) is presently being revised and a document for medical laboratories (ISO/TC 212, CD 15189) is at draft stage. Both aim to include aspects of total quality management. The concept of total quality management is rather vague. Generally, its goal has been defined as "business excellence". This term, however, needs some explanation if applied to medical laboratories. Therefore, a project group of the European Confederation of Laboratory Medicine (ECLM) has developed a model for total quality management, which is based on a comprehensive management concept issued by the European Foundation for Quality Management. In the case of a medical laboratory, the term "business excellence" should be replaced by "good medical laboratory services". The proposed model could serve as a basis for future developments of total quality management standards in laboratory medicine. The goal of the "journey" should be clarified before it starts. To the best of our knowledge, this is the first attempt to develop a model of a good medical laboratory.

XPA-deficiency in hairless mice causes a shift in skin tumor types and mutational target genes after exposure to low doses of U.V.B.

Xeroderma pigmentosum (XP) patients with a defect in the nucleotide excision repair gene XPA, develop tumors with a high frequency on sun-exposed areas of the skin. Here we describe that hairless XPA-deficient mice also develop skin tumors with a short latency time and a 100% prevalence after daily exposure to low doses of U.V.B. Surprisingly and in contrast to U.V.B.-exposed repair proficient hairless mice who mainly develop squamous cell carcinomas, the XPA-deficient mice developed papillomas with a high frequency (31%) at a U.V. dose of 32 J/m2 daily. At the highest daily dose of 80 J/m2 mainly squamous cell carcinomas (56%) and only 10% of papillomas were found in XPA-deficient hairless mice. p53 gene mutations were examined in exons 5, 7 and 8 and were detected in only 3 out of 37 of these skin tumors, whereas in tumors of control U.V.B.-irradiated wild type littermates this frequency was higher (45%) and more in line with our previous data. Strikingly, a high incidence of activating ras gene mutations were observed in U.V.B.-induced papillomas (in 11 out of 14 tumors analysed). In only two out of 14 squamous cell carcinomas we found similar ras gene mutations. The observed shift from squamous cell carcinomas in wild type hairless mice to papillomas in XPA-deficient hairless mice, and a corresponding shift in mutated cancer genes in these tumors, provide new clues on the pathogenesis of chemically- versus U.V.B.-induced skin carcinogenesis.

Spontaneous liver tumors and benzo[a]pyrene-induced lymphomas in XPA-deficient mice.

Defects in the xeroderma pigmentosum complementation group A-correcting (XPA) gene, which encodes a component of the nucleotide excision repair (NER) pathway, are associated with the cancer-prone human disease xeroderma pigmentosum. We previously generated mice lacking the XPA gene, which develop normally but are highly sensitive to ultraviolet-B and 7,12-dimethylbenz[a] anthracene-induced skin tumors. Here we report that XPA-deficient mice spontaneously developed hepatocellular adenomas at a low frequency as they aged. Furthermore, oral treatment of XPA-deficient mice with the carcinogen benzo[a]pyrene (B[a]P) resulted in the induction of mainly lymphomas. These tumors appeared earlier and with a higher incidence than in B[a]P-treated wild-type and heterozygous mice. Our results show for the first time that XPA-deficient mice also displayed an increased sensitivity to developing tumors other than tumors of the skin.

Generation of HER-2/neu-specific cytotoxic neutrophils in vivo: efficient arming of neutrophils by combined administration of granulocyte colony-stimulating factor and Fcgamma receptor I bispecific antibodies.

Abs are able to induce inflammatory antitumor responses by recruiting IgG Fc receptor (FcgammaR)-bearing cytotoxic effector cells. We recently described the capacity of the high affinity FcgammaRI (CD64) to trigger cytotoxic activity of neutrophils (PMN) during granulocyte CSF (G-CSF) treatment. To take advantage of FcgammaRI as a cytotoxic trigger molecule on PMN, two Ab constructs were prepared. We show that a chimeric human IgG1 Ab (Ch520C9) and an anti-FcgammaRI bispecific Ab (BsAb; 22x520C9), both directed to the proto-oncogene product HER-2/neu, interact with FcgammaRI. In addition, both Ab constructs mediate enhanced lysis of HER-2/neu-expressing tumor cells by G-CSF-primed PMN. However, engagement of FcgammaRI by Ch520C9 was inhibited by human serum IgG, thereby abrogating the enhanced Ch520C9-mediated cytotoxicity. BsAb 22x520C9, which binds FcgammaRI outside the ligand binding domain, effectively recruits the cytotoxic potential of FcgammaRI on G-CSF-primed PMN regardless of the presence of human serum. These results indicate that under physiologic conditions, serum IgG impairs activation of FcgammaRI-mediated cytotoxicity by conventional antitumor Abs. The IgG blockade can be circumvented with anti-FcgammaRI BsAbs. Using human FcgammaRI transgenic mice we demonstrate that BsAb 22x520C9 is able to engage FcgammaRI in vivo. BsAb 22x520C9 injected i.v. was readily detected on circulating PMN of G-CSF-treated transgenic animals. In addition, we showed that PMN remain "armed" with BsAb 22x520C9 during migration to inflammatory sites, and that after isolation such PMN specifically lyse HER-2/neu-expressing tumor cells. These results point to the possibility of targeting anti-FcgammaRI BsAbs to G-CSF-primed PMN in vivo, endowing them with specific anti-tumor activity.

Induction of DNA adducts and mutations in spleen, liver and lung of XPA-deficient/lacZ transgenic mice after oral treatment with benzo[a]pyrene: correlation with tumour development.

We were interested to study the relationship between DNA lesions, DNA repair, mutation fixation, and tumour development. Therefore, mice harbouring lacZ reporter genes and being either wild-type or defective in the DNA excision repair gene XPA, were treated with the genotoxic carcinogen benzo[a]pyrene at an oral dose of 13 mg/kg b.w. (3 times/week). At different time points, i.e. 1, 5, 9 or 13 weeks after start of the oral administration, levels of BPDE-N2-dG adducts (the major formed DNA adduct by benzo[a]pyrene in mice), and lacZ mutation frequencies were measured both in target (spleen) and non-target (lung and liver) tissues. Both in wild-type and XPA-deficient mice, benzo[a]pyrene treatment resulted in increased BPDE-N2-dG adduct levels in all three tissues analysed. In XPA-deficient mice, BPDE-N2-dG adduct levels still increased up to 13 weeks of oral benzo[a]pyrene treatment, whereas in DNA repair proficient mice steady-state levels were reached after 5 weeks of treatment. After 13 weeks, the BPDE-N2-dG adduct levels observed in XPA-/- mice, were 2- to 3-fold higher than the steady state levels observed in XPA+/+ mice in the same tissues. Mutation frequencies in the lacZ reporter gene were the same in wild-type and XPA-deficient mice that were treated with the solvent only. Oral benzo[a]pyrene treatment resulted in an increase in mutation frequency in the lacZ marker gene in all three tissues, but this increase was most profound in the spleen. After 13 weeks of treatment, a 7-fold increase in lacZ mutation frequency was detected in the spleen of wild-type mice as compared to mutation frequencies in control mice. At the same time point, a 15-fold increase in lacZ mutation frequency was observed in the spleen of XPA-deficient mice. The data presented here show, that a defect in NER mainly results in enhanced mutation frequencies in lymphocytic cells after oral treatment with the genotoxic compound benzo[a]pyrene. Interestingly, as we established in a previously performed carcinogenicity assay, the same oral treatment with benzo[a]pyrene induced lymphomas residing in the spleen of XPA-deficient mice.

Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc gamma RIII (CD16) deficient mice.

The family of receptors for IgG (Fc gamma R) plays an essential role in antibody-mediated effector functions of the immune system. However, the specific contribution of each of the Fc gamma R classes to in vivo immune reactions is still unclear. Here, we demonstrate that mice deficient for the ligand-binding alpha chain of Fc gamma RIII lack NK cell-mediated antibody-dependent cytotoxicity and phagocytosis of IgG1-coated particles by macrophages. Strikingly, these mice lack IgG-mediated mast cell degranulation, are resistant to IgG-dependent passive cutaneous anaphylaxis, and exhibit an impaired Arthus reaction. These results indicate a prominent role for Fc gamma RIII in inflammatory and anaphylactic responses, making this receptor a potential target in immunotherapy.

FcR gamma-chain is essential for both surface expression and function of human Fc gamma RI (CD64) in vivo.

Most Ig receptors exist as hetero-oligomeric complexes with separate ligand binding (alpha) and signal transducing (beta, gamma, or zeta) subunits. For Fc gamma RIIIa and Fc epsilon RI, association with the FcR gamma-chain is essential for surface expression. However, the human high affinity IgG receptor, hFc gamma RI, was found to be surface-expressed by itself in transient transfection models. We have now analyzed the integrity of hFc gamma RI expression in more detail in stable transfectants. In vitro we noted that, in the absence of FcR gamma-chain, surface expression of hFc gamma RI rapidly declined to background levels, in both IIA1.6 B cells and NIH3T3 fibroblasts. The effect of FcR gamma-chain on hFc gamma RI surface expression in vivo was evaluated by using two newly generated transgenic mouse lines, selectively expressing hFc gamma RI on myeloid cells. These transgenic mice were crossed with FcR gamma-chain-deficient mice. Analysis of blood monocytes and peritoneal macrophages showed that surface expression of hFc gamma RI was reduced by approximately 80%. The remaining approximately 20% of receptors were still capable of binding IgG-opsonized RBC, suggesting FcR gamma-chain not to be critical for hFc gamma RI ligand-binding capacity. Importantly, however, hFc gamma RI signaling capacity was lost in FcR gamma-chain-deficient cells. No phagocytosis could be observed using either ligand sensitized (EA-IgG2a) or CD64-targeted erythrocytes (using a bispecific antibody) in both hFc gamma RI transgenic lines. This documents the FcR gamma-chain to be indispensable for both surface membrane expression and function of human Fc gamma RI in vivo.

Antigen targeting to myeloid-specific human Fc gamma RI/CD64 triggers enhanced antibody responses in transgenic mice.

Besides their phagocytic effector functions, myeloid cells have an essential role as accessory cells in the induction of optimal humoral immune responses by presenting captured antigens and activating lymphocytes. Antigen presentation by human monocytes was recently found to be enhanced in vitro through the high-affinity Fc receptor for IgG (Fc gamma RI; CD64), which is exclusively present on myeloid cells. To evaluate a comparable role of Fc gamma RI in antigen presentation in vivo, we generated human Fc gamma RI transgenic mice. Under control of its endogenous promoter, human Fc gamma RI was selectively expressed on murine myeloid cells at physiological expression levels. As in humans, expression was properly regulated by the cytokines IFN-gamma, G-CSF, IL-4, and IL-10, and was up-regulated during inflammation. The human receptor expressed by murine macrophages bound monomeric human IgG and mediated particle phagocytosis and IgG complex internalization. To evaluate whether specific targeting of antigens to Fc gamma RI can induce enhanced antibody responses, mice were immunized with an anti-human Fc gamma RI antibody containing antigenic determinants. Transgenic mice produced antigen-specific antibody responses with high IgG1 titers and substantial IgG2a and IgG2b responses. These data demonstrate that human Fc gamma RI on myeloid cells is highly active in mediating enhanced antigen presentation in vivo, and show that anti-Fc gamma RI mAbs are promising vaccine adjuvants.

Skewed distribution of IgG Fc receptor IIa (CD32) polymorphism is associated with renal disease in systemic lupus erythematosus patients.

OBJECTIVE: Fc gamma receptors of class IIa (Fc gamma RIIa) occur in 2 allelic forms, with either a low (IIa-R131) or a high (IIa-H131) affinity for complexed IgG2 and IgG3. This polymorphism might have implications for the handling of immune complexes. Therefore, we determined the distribution of the Fc gamma RIIa allotypes in patients with systemic lupus erythematosus (SLE), with or without a history of lupus nephritis. METHODS: We studied 95 unrelated white European patients with SLE, as defined by the American College of Rheumatology criteria, 50 of whom had a history of lupus nephritis, and 69 healthy white European control subjects. Fc gamma RIIa allotypes were determined by immunophenotyping of blood monocytes. RESULTS: It was found that lupus nephritis was significantly associated with the "low affinity" Fc gamma RIIa R/R131 allotype and with the R131 allele, compared with healthy controls. No significant association was found upon comparison of groups with and without nephritis. CONCLUSION: SLE patients with a history of lupus nephritis have an abnormal distribution of Fc gamma RIIa allotypes. Fc gamma RIIa may well play a role in the pathogenesis of lupus nephritis, since IIa-R/R131 SLE patients seem to have a higher incidence of developing this complication.

Functional association between the human myeloid immunoglobulin A Fc receptor (CD89) and FcR gamma chain. Molecular basis for CD89/FcR gamma chain association.

FcR gamma chain has previously been shown to interact with the TCR-CD3 complex, the IgE Fc receptor I (Fc epsilon RI), and the class I and IIIA IgG receptors (Fc gamma RI and Fc gamma RIIIa). Here, we demonstrate that the Fc receptor gamma chain associates with Fc alpha R in transfected IIA1.6 B lymphocytes. Fc alpha R could be expressed at the surface of IIA1.6 B cells by itself, but was devoid of signaling capacity. Upon co-expression of FcR gamma chain, a physical interaction with Fc alpha R could be demonstrated. This association proved crucial for the triggering of both proximal (intracellular calcium increase and tyrosine phosphorylation), as well as distal (IL-2 release), signal transduction responses. We next tested the hypothesis that a positively charged arginine residue (Arg209) within the transmembrane domain of Fc alpha R promotes association with FcR gamma chain. We therefore constructed Fc alpha R molecules where Arg209 was mutated to either a positively charged histidine, a negatively charged aspartic acid, or an uncharged leucine. A functional association between Fc alpha R and FcR gamma chain was observed only with a positively charged residue (Arg209 or His209) present within the Fc alpha R transmembrane domain. These data show that transmembrane signal transduction by the Fc alpha R is mediated via FcR gamma chain, and that Fc alpha R requires a positively charged residue within the transmembrane domain to promote functional association.

Functional differences between two Fc receptor ITAM signaling motifs.

Most Ig receptors exist as multi-subunit complexes with a unique ligand binding alpha chain and a common signaling FcR gamma-chain. The myeloid Fc gamma RIIa (CD32) appears unique among FcR because both ligand-binding and signaling capacity are found in the alpha chain. Within the cytoplasmic tails of Fc gamma RIIa and FcR gamma-chain similar, but not identical, activatory motifs (ITAMs) have been defined, in which tyrosines play an important role. Previously, Fc gamma RIIa-ITAM was shown to be critical for both proximal and distal activatory functions in IIA1.6 B-cell transfectants. Triggering of interleukin-2 (IL-2) release and antigen presentation was absent in Fc gamma RIIa, but not in FcR gamma-chain receptor complexes. We now assessed the capacity of Fc gamma RIIa wild-type and Fc gamma RIIa/gamma chimeric molecules to trigger IL-2 production and antigen presentation by B cells. Both of these functions could solely be triggered by receptors containing the FcRIIa was capable of functional interaction with FcR gamma-chain, thus reconstituting the capacity to trigger IL-2 release and antigen presentation. These data document qualitative differences between Fc receptor ITAMs.

Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA.

Xeroderma pigmentosum patients with a defect in the nucleotide-excision repair gene XPA are characterized by, for example, a > 1,000-fold higher risk of developing sunlight-induced skin cancer. Nucleotide-excision repair (NER) is involved in the removal of a wide spectrum of DNA lesions. The XPA protein functions in a pre-incision step, the recognition of DNA damage. To permit the functional analysis of the XPA gene in vivo, we have generated XPA-deficient mice by gene targeting in embryonic stem cells. The XPA-/-mice appear normal, at least until the age of 13 months. XPA-/-mice are highly susceptible to ultraviolet (UV)-B-induced skin and eye tumours and to 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin tumours. We conclude that the XPA-deficient mice strongly mimic the phenotype of humans with xeroderma pigmentosum.

Structure of the gene for the human myeloid IgA Fc receptor (CD89).

A better understanding of IgA's role in immunity requires insight in IgAR complexity. We have now isolated, characterized, and sequenced the gene encoding the prototypic human myeloid IgA FcR (CD89). The gene consists of five exons and spans approximately 12 kilobase pairs. The leader peptide is encoded by two exons, the second of which is 36 bp long and specifies the predicted peptidase cleavage site. A similar, but shorter (21 bp) mini-exon has been found in the FcR for IgG (Fc gamma R) genes, and the FcR for IgE (Fc epsilon RI alpha) gene (human and rodent). The third and fourth exons code for two homologous Ig-like domains. The final exon encodes a short extracellular region, a hydrophobic transmembrane region, and a short cytoplasmic tail. The sequence of the 5'-flanking region was determined, and one major and several minor transcription initiation sites were mapped by primer extension studies. A putative TATA box was located at an appropriate location relative to the start site. Southern blot analyses of genomic DNA confirm the restriction map generated from cloned DNA. These data define the Fc alpha R gene as a distantly related member of the IgR gene family.