Applied anatomy of the latissimus dorsi free flap for refinement in one-stage facial reanimation.

BACKGROUND: The face can be reanimated after long-term paralysis by free microneurovascular tissue transfer. Flaps from gracilis and pectoralis minor usually require a two-stage procedure with a cross-face nerve graft. Latissimus dorsi has a much longer muscular nerve, the thoracodorsal nerve, which could avoid the need for a second cross-face nerve graft. Our hypothesis is that the neurovascular pedicles of small segments of latissimus dorsi would be long enough to reach the opposite side of the face and to provide a reliable blood and nerve supply to the flaps. METHOD: To test this hypothesis the thoracodorsal pedicle and its primary branches were dissected in eleven embalmed cadavers. The segmental vessels and nerves were then traced in a series of simulated flaps approximately 8-10 cm × 2-3 cm by micro-dissection, tissue clearing and histology. RESULTS: The thoracodorsal pedicle is 10-14 cm long to where it enters the muscle, and with intra-muscular dissection small chimeric muscle segments 8-10 cm × 2-3 cm can be raised with a clearly defined neurovascular supply. Using micro-dissection the neurovascular pedicle can be lengthened to reach across the face. Segmental arteries and nerves extended to the distal end of all the flaps examined. Artery, vein and nerve run together and are of substantial diameter. CONCLUSION: Small muscle segments of latissimus dorsi can be raised on long neurovascular pedicles. The vessels and nerves are substantial and the likelihood of surgical complications such as flap necrosis and functional disuse on transplantation appear low. Although in our opinion the use of cross-face nerve grafts and transfer of smaller muscle flaps remains the gold standard in facial reanimation in straightforward cases, the micro-dissected latissimus dorsi flap is a useful option in complex cases of facial reanimation. CLINICAL APPLICATION: Facial reanimation using micro-dissected segments of latissimus dorsi has been performed in four complex cases of facial paralysis.

Role for CD40-CD40 ligand interactions in the immune response to solid tumours.

CD40-mediated interactions play an important role in the response to infections, transplantation, and cancer by affecting the development, activation, proliferation and differentiation of a variety of immune cells. In the current study we examined the role of CD40-mediated interactions in immune responses to bladder, pancreatic and breast carcinomas as well as melanoma cell lines using soluble human CD40L (rhCD40L) or anti-CD40 mAb in vitro. CD40 expression was readily detected in a large proportion of the cell lines and was augmented but not induced de novo by treatment with IFNgamma. Treatment of CD40-positive cell lines with rhCD40L or anti-CD40mAb enhanced cell surface expression of ICAM-1 and FAS and stimulated the production of IL-6, IL-8, GROalpha, GM-CSF and TNFalpha but not IL-4, IL-10, TGFbeta, MCP-1, RANTES, MIP-1beta, or IP-10. In addition, incubation of CD40+ tumour cell lines with immobilised rhCD40L or anti-CD40 mAb in vitro resulted in significant inhibition of proliferation and a corresponding decrease in viability. This CD40-mediated inhibition of cell growth was due, at least in part, to alterations in cell cycle and the induction of apoptosis. Transfection of CD40-negative tumour cell lines with the cDNA for CD40 conferred responsiveness to rhCD40L and anti-CD40 antibody. Finally, the presence of CD40 on the surface of carcinoma lines was found to be an important factor in the generation of tumour-specific T cell responses.

Gene transfer to primary acute myeloid leukaemia blasts and myeloid leukaemia cell lines.

The transfer of genes encoding co-stimulatory molecules and/or cytokines to leukaemia cells in order to create autologous tumour vaccines represents a potential immunotherapeutic strategy for treating acute myeloid leukaemia (AML). One of the essential requirements for this strategy if it is to be applicable in a clinical setting is a high efficiency of gene transfer to primary human AML blasts. Using green fluorescent protein (GFP) as a reporter gene, we have systematically evaluated a variety of physical, chemical and viral vector-based gene transfection systems in order to determine which gave the highest gene transfer efficiency to myeloid leukaemia cell lines and primary AML blasts. Transfection efficiency was low for all the physical and chemical transfection methods tested. Retroviral vector-based infection gave a high efficiency of gene transduction in two of the four leukaemia cell lines (KG1a and U937), but was low in primary AML blasts. An adenoviral vector gave a high transduction efficiency in all of the leukaemia cell lines with the exception of the HL60. In primary AML blasts, derived from 19 patients, gene transduction efficiency was variable, ranging from 1.1% to 67.1% (mean 12.1%). Following culture in cytokines GM-CSF/IL-4/CD40L, which induced differentiation of AML blasts to dendritic-like cells, transduction efficiency was increased between two- and eightfold in 6 out of the 15 cases that underwent differentiation.

Variation in IgG1 heavy chain allotype does not contribute to differences in biological activity of two human anti-Rhesus (D) monoclonal antibodies.

BACKGROUND: Pooled human anti-Rhesus D antiserum is currently administered for the prevention of RhD alloimmunization. Increased demand, and decreased supply, of donated pooled antiserum has led to the investigation of the suitability of human monoclonal anti-RhD antibodies for use in its place. However, it is unclear which biological properties of monoclonal antibodies are important for function in RhD-positive foetal red cell clearance and the prevention of alloimmunization. Various antibodies behave differently in a number of in vitro assays of biological function. OBJECTIVES: To compare the function and structure of two human anti-RhD IgG1 monoclonal antibodies which differ in their ability to promote red cell lysis in vitro. In particular to examine whether the functional differences correlate to differences in the IgG1 heavy chain constant region (allotype). STUDY DESIGN: We report here the cloning, characterization and re-expression in stable myeloma cell transformants of cDNAs coding for two such antibodies, secreted by the heterohybridoma cell lines ESD-1 (THERAD 03) and LHM 70/45.3 (THERAD 06). The cDNAs were then recombined to exchange portions of the Fc encoding regions and the recombinant antibodies were assayed in vitro to determine RhD-positive red cell-dependent activity. RESULTS: Recombinant THERAD 03 and 06 antibodies behaved identically to the parent antibodies. The 'inactive' THERAD 06 did not have biological activity reconstituted by exchange with the THERAD 03 Fc regions, nor was THERAD 03 activity abolished by the reciprocal Fc region exchange. CONCLUSIONS: Human monoclonal anti-RhD antibodies can be cloned and re-expressed in stable cell lines, and exhibit identical properties to the parent antibodies. Differences in biological activity cannot be attributed to differences in IgG1 heavy chain allotype.

A LINE-like transposable element in Drosophila, the I factor, encodes a protein with properties similar to those of retroviral nucleocapsids.

I factors are members of the LINE-like family of transposable elements and move by reverse transcription of an RNA intermediate. Complete I factors contain two open reading frames. The amino acid sequence encoded by the first of these, ORF1, includes the motif CX2CX4HX4C that is characteristic of the nucleocapsid domain of retroviral gag polypeptides followed by a copy of the slightly different sequences CX2CX4HX6C and CX2CX9HX6C. The function of this protein is unknown. We have expressed this protein in Escherichia coli and Spodoptera frugiperda cells and have shown that it binds both DNA and RNA but without any evidence for sequence specificity. The properties of deletion derivatives of the protein indicate that more than one region is responsible for DNA binding and that the CCHC motif is not essential for this. The ORF1 protein expressed in either E. coli or Spodoptera cells forms high molecular weight structures that require the region of the protein including the CCHC motif for their formation. This protein can also accelerate the annealing of complementary single-stranded oligonucleotides. These results suggest that this protein may associate with the RNA transposition intermediates of the I factor to form particles that enter the nucleus during transposition and that it may stimulate both the priming of reverse transcription and integration. This may be generally true for the product of the first open reading frame of LINE-like elements.

Polypeptide components of Drosophila small nuclear ribonucleoprotein particles.

In eukaryotes splicing of pre-mRNAs is mediated by the spliceosome, a dynamic complex of small nuclear ribonucleoprotein particles (snRNPs) that associate transiently during spliceosome assembly and the splicing reaction. We have purified snRNPs from nuclear extracts of Drosophila cells by affinity chromatography with an antibody specific for the trimethylguanosine (m3G) cap structure of snRNAs U1-U5. The polypeptide components of Drosophila snRNPs have been characterized and shown to consist of a number of proteins shared by all the snRNPs, and some proteins which appear to be specific to individual snRNP particles. On the basis of their apparent molecular weight and antigenicity many of these common and particle specific Drosophila snRNP proteins are remarkably conserved between Drosophila and human spliceosomes. By probing western blots of the Drosophila snRNP polypeptides with a number of antisera raised against human snRNP proteins, Drosophila polypeptides equivalent to many of the HeLa snRNP-common proteins have been identified, as well as candidates for a number of U1, U2 and U5-specific proteins.

A prominent serine-rich region in Vmw175, the major transcriptional regulator protein of herpes simplex virus type 1, is not essential for virus growth in tissue culture.

Herpes simplex virus type 1 (HSV-1) encodes five immediate early (IE) genes of which at least three are involved in the transcriptional regulation of later classes of viral genes. Perhaps the most important of these regulatory proteins is Vmw175, a nuclear phosphoprotein of 1298 predicted amino acid residues. In the absence of functional Vmw175 the virus fails to activate early or late genes or to repress IE gene expression. All viruses of the sub-family alphaherpes-virinae encode polypeptides that are closely related to Vmw175. Mutational studies have shown that regions of homology within this family of gene regulators are generally of functional importance. One of the most striking conserved stretches of amino acid sequence is a run of serine residues followed by a highly acidic region in the amino-terminal fifth of the polypeptide. We have constructed an HSV-1 virus which lacks this serine-rich run within Vmw175. Surprisingly, the virus was viable in tissue culture cells and expressed apparently normal amounts of viral polypeptides. In plaque assays it was very slightly temperature-sensitive and, depending on the state of the host cells, could generate plaques with a syncytial morphology. The mutant protein was able to bind to DNA in a manner indistinguishable from that of the wild-type polypeptide. We conclude that despite its conservation in all of the alphaherpes-virinae so far sequenced, the serine-rich homology is not important for virus growth in tissue culture.

The major transcriptional regulatory protein of herpes simplex virus type 1 includes a protease resistant DNA binding domain.

Herpes simplex virus type 1 expresses five immediate-early (IE) polypeptides. In the absence of functional Vmw175 (the product of IE gene 3) activation of transcription of later classes of viral genes and repression of IE gene expression does not occur. The recognition of specific DNA sequences by Vmw175 requires, as determined by sensitivity to mutation, a part of the protein highly conserved in the corresponding proteins of related herpes viruses. However, mutations in other parts of the protein can also disrupt specific DNA binding. This paper shows that the DNA binding domain of Vmw175 can be liberated as a functional unit by digestion with proteinase K. Analysis of mutant Vmw175 proteins showed that the proteinase K resistant domain has an amino terminus between amino acid residues 229 and 292, while its carboxy terminus is between residues 495 and 518. Mutations outside this region which affect DNA binding by the intact protein do not eliminate binding of the proteinase K resistant domain. This implies that direct DNA binding by Vmw175 involves a linear subsection of the polypeptide, and that mutations in other parts of the polypeptide which affect DNA binding of the whole protein do so by indirect means.

A mutant of herpes simplex virus type 1 immediate early polypeptide Vmw175 binds to the cap site of its own promoter in vitro but fails to autoregulate in vivo.

Vmw175, the product of herpes simplex virus type 1 immediate early (IE) gene 3, is essential for viral replication. It is required for the activation of transcription from both early and late gene promoters and also for the repression of IE gene expression. Vmw175 is able to bind specifically to certain DNA sequences, some of which (including that at the cap site of IE gene 3) contain the consensus sequence ATCGTC. The presence of this sequence at the cap site has been correlated with the ability of Vmw175 to autoregulate its own promoter. This report describes the characterization of five viruses with temperature-sensitive (ts) lesions in Vmw175. Four of these mutants express Vmw175 which is ts in its ability to bind to DNA in vitro and to autoregulate IE-3 gene expression in the infected cell. Although Vmw175 produced by the remaining mutant, ts1225, fails to autoregulate IE-3 expression at the non-permissive temperature (NPT) its DNA-binding properties are indistinguishable from those of the wild-type protein. This suggests that the ability of Vmw175 to bind to the IE-3 cap site (as measured in vitro) is insufficient for autoregulation (in vivo). All five newly characterized ts mutants are partially permissive for early gene transcription at the NPT, although Vmw175 expressed by four of them is unable to bind to the IE-3 cap site sequence at elevated temperatures. This suggests that binding to one class of recognition sequences by Vmw175, as measured in vitro, is not absolutely required for the activation of early gene promoters during virus infection. The lesions in these five ts mutants lie in the carboxy-terminal third of the polypeptide; three of the mutations (those in ts1219, ts1221 and ts1225) were identified by DNA sequence analysis and were found to affect amino acid residues that are conserved in the homologous proteins from varicella-zoster virus and pseudorabies virus.

Mutational dissection of the HSV-1 immediate-early protein Vmw175 involved in transcriptional transactivation and repression.

Vmw175 is one of five immediate-early (IE) proteins encoded by herpes simplex virus type-1 (HSV-1). It is required for the transcription of later classes of genes and for the accompanying repression of IE expression. Vmw175 has been shown to be a transactivator of transcription and also to autoregulate its own synthesis. We have made a large number of small, in-frame, insertion and deletion mutants of a plasmid-borne copy of the gene encoding Vmw175. Study of the activity of the resultant mutant polypeptides in transient transfection assays has defined the regions of the protein important for the repression of its own promoter, and for the transactivation of an HSV early promoter in synergy with another HSV IE protein, Vmw110. Large stretches of the protein are relatively unimportant for either function, while the regions most sensitive to disruption correlate to sequences conserved between Vmw175 and VZV 140K, the corresponding transactivating protein of Varicella-Zoster virus. The region from amino acids 275 to 490 is particularly important for both repression and transactivation, whereas that from around 840 to 1100 seems to be more important for transactivation than repression. The nuclear localization signal has been mapped to within residues 682-774.