New horizons in the understanding of the causes and management of diabetic foot disease: report from the 2017 Diabetes UK Annual Professional Conference Symposium.

Diabetes-related foot disease remains a common problem. For wounds, classic teaching recommends the treatment of any infection, offloading the wound and ensuring a good blood supply, as well as ensuring that the other modifiable risk factors are addressed and optimized. There remain, however, several questions about these and other aspects of the care of diabetes-related foot disease. Some of these questions are addressed in the present report; in particular, the impact of newer technologies in the identification of any organisms present in a wound, as well as the use of novel approaches to treat infections. The use of new remote sensing technology to identify people at risk of developing foot ulceration is also considered, in an attempt to allow early intervention and prevention of foot ulcers. The psychological impact of foot disease is often overlooked, but with an increasing number of publications on the subject, the cause-and-effect role that psychology plays in foot disease, such as ulcers and Charcot neuroarthropathy, is considered. Finally, because of heterogeneity in diabetic foot studies, comparing results is difficult. A recently published document focusing on ensuring a standardized way of reporting foot disease trials is discussed.

Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients.

A blood donor infected with human immunodeficiency virus-type 1 (HIV-1) and a cohort of six blood or blood product recipients infected from this donor remain free of HIV-1-related disease with stable and normal CD4 lymphocyte counts 10 to 14 years after infection. HIV-1 sequences from either virus isolates or patient peripheral blood mononuclear cells had similar deletions in the nef gene and in the region of overlap of nef and the U3 region of the long terminal repeat (LTR). Full-length sequencing of one isolate genome and amplification of selected HIV-1 genome regions from other cohort members revealed no other abnormalities of obvious functional significance. These data show that survival after HIV infection can be determined by the HIV genome and support the importance of nef or the U3 region of the LTR in determining the pathogenicity of HIV-1.

Constitutive and virus-induced interferon production by peripheral blood leukocytes.

The production of interferon-alpha (IFN-alpha) by normal human peripheral blood mononuclear cells (PBMNC) was studied using polyclonal antipeptide antibodies designed to react either with all IFN-alpha subtypes or with individual subtypes IFN-alpha 2 or IFN-alpha 4. In this study, we demonstrate the detection of intracellular IFN-alpha in PBMNC using immunofluorescence staining and flow-cytometric analysis. Virtually all cells of the PBMNC population were shown to produce IFN-alpha reactive with all three antisera after stimulation with Sendai virus. The immunofluorescence studies also demonstrated that IFN-alpha is produced by PBMNC in the absence of known viral stimulation but is not secreted in detectable levels. Double-labeling with specific monoclonal antibodies to T and B lymphocytes confirmed that the entire populations of these two cell types produce IFN-alpha, both constitutively and after virus induction. Polymorphonuclear cells (PMNC) isolated from Ficoll-Paque pellets were also shown to contain intracellular IFN-alpha, both before and after virus induction. The finding that all PBMNC produce IFN-alpha constitutively suggests that IFN-alpha may have important regulatory functions in situations other than during overt viral infections.

Serological detection of attenuated HIV-1 variants with nef gene deletions.

OBJECTIVE: To investigate whether members of a transfusion-linked cohort (the Sydney Bloodbank Cohort) infected with a nef-deleted strain of HIV-1 could be differentiated from individuals infected with wild-type strains of HIV-1 by characterizing the Nef antibody response of cohort members. DESIGN: Retrospective and prospective analysis of the nef gene sequence and the antibody response to Nef peptides in HIV-infected subjects. METHODS: Plasma was obtained from all individuals of the Sydney cohort, and from a variety of HIV-1-infected and uninfected controls. Antibodies recognizing full-length recombinant HIV-1NL43 Nef protein and synthetic peptide analogues were assessed by enzyme-linked immunosorbent assay. RESULTS: All 34 individuals infected with wild-type HIV-1 had antibodies reacting with full-length Nef protein as well as with a series of synthetic peptides (6-23-mers) spanning most of the Nef protein of HIV-1NL43. Although the HIV-1 quasispecies infecting the Sydney cohort had a consensus deletion of the nef gene corresponding to amino-acids 165-206, HIV-1 strains from individual members of the cohort had additional deletions comprising up to 80% of the nef gene. Members of the cohort had antibodies to peptides homologous to all regions of the Nef protein tested, except for a single peptide (amino-acids 162-177) that lies within the consensus nef deletion for the cohort quasispecies. CONCLUSION: These data show that nef-deleted strains of HIV-1 can be detected serologically. In the Sydney cohort, detection of antibodies to all regions of Nef tested, except that corresponding to amino-acids 162-177, suggests that observed deletions outside this domain occurred after this virus had infected these subjects and stimulated an immune response. A Nef peptide serological assay may be useful for identifying further examples of individuals infected with nef-deleted, attenuated HIV-1 quasispecies and for assessing the evolution of those variants in vivo.

Detection of cells producing murine interferon-alpha using antipeptide antibodies.

The purpose of this study was to produce antibodies which could be used to investigate the expression of murine (Mu)IFN-alpha. Rabbits were immunized with a peptide, corresponding to the 15 COOH-terminal amino acids of MuIFN-alpha-1, conjugated to keyhole limpet haemocyanin (KLH), and the resulting antipeptide antibodies were identified by indirect ELISA. Antipeptide antibodies were purified from rabbit immune sera by immunoadsorption to peptide immobilized on nitrocellulose and any remaining antibodies to KLH removed by immunoadsorption to KLH-Sepharose. The characterization of the antipeptide antibodies by ELISA, immunoprecipitation, affinity chromatography and immunofluorescence demonstrated that the antibodies recognize the peptide immunogen and the native IFN-alpha molecule. Using these antibodies for immunofluorescence staining and flow cytometric analyses of stained cells, we have shown that unstimulated murine spleen cells produce IFN-alpha. This finding is in agreement with the recent demonstration of constitutive IFN-alpha production by unstimulated human leucocytes and has important implications for the functions of interferons. The production, characterization and use of antipeptide antibodies as described herein may also have broader application for studies of the expression of other cytokines.

Simian immunodeficiency virus and human immunodeficiency virus type 1 nef proteins show distinct patterns and mechanisms of Src kinase activation.

The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases.

Anomalies in Nef expression within the central nervous system of HIV-1 positive individuals/AIDS patients with or without AIDS dementia complex.

In determining levels of expression of HIV-1 Nef protein within the central nervous system (CNS) we assessed antibody responses to the protein both peripherally and in CNS. Antibodies to Nef were not detected within the CNS despite detection of antibodies to both gp41 and Nef in peripheral blood and representative virus isolates derived from CNS and peripheral blood (PB) samples containing full length nef sequence and virus-infected cells expressing Nef protein. We conclude from this that expression of Nef within the CNS is such that little or no antibody production occurs and that these differences indicate that Nef protein may not be directly contributing to the AIDS dementia complex. Expression of Nef protein in PHA-activated peripheral blood mononuclear cells from CNS derived isolates was different to that of coincidental PB derived isolates in that partial surface expression was observed for the latter. The results suggest that antigenic presentation of Nef within the CNS is anomalous and that Nef protein expression, at least for the limited number of in vitro derived isolates tested, has a different localization pattern.

Post-transcriptional regulation of interferon-alpha 4 subtype production by lymphoblastoid cells.

The constitutive production of interferon-alpha (IFN-alpha) subtypes by the lymphoblastoid cell lines, Namalwa, Daudi and Raji, was investigated using sensitive and semi-quantitative flow cytometric techniques. Further, we sought to determine whether the previously described failure of these cell lines to produce IFN-alpha-4 was a result of the deletion of the IFN A4 gene. Cytoplasmic production of IFN-alpha-2 and IFN-alpha-4 was assessed using IFN-alpha subtype-specific antipeptide antibodies and FITC-labelled secondary antibodies in indirect immunofluorescence-flow cytometry studies. The constitutive production of IFN-alpha-2 was detected in all three cell lines. Significant increases in fluorescence representing increased production of IFN-alpha-2 and possibly other IFN-alpha subtypes were detected after induction by Sendai virus. Approximately 100 per cent of cells in the Namalwa, Daudi and Raji cell populations contained IFN-alpha-2 before and after induction. However, no cells from the same cell populations contained the IFN-alpha-4 subtype. Analysis of genomic DNA isolated from the lymphoblastoid cells using the Polymerase Chain Reaction (PCR) and oligonucleotide primers specific for IFN A2 or IFN A4 confirmed the presence of the genes encoding both IFN-alpha subtypes. Furthermore, using reverse transcriptase-PCR amplification, mRNAs for both IFN-alpha-2 and IFN-alpha-4 were detected. Therefore, in contrast to some leukaemias and derived cell lines where IFN A genes have been deleted, these cell lines of B cell lineage exhibit selective expression of IFN A genes, as a result of altered transcriptional/translational control of IFN-alpha expression.

Immunolocalisation of interferon-alpha in hepatitis C patients and its correlation with response to interferon-alpha therapy.

Localised interferon-alpha production was investigated in hepatitis C patients entered into a trial of interferon-alpha-2a therapy. Antibodies capable of reacting specifically with interferon-alpha-2, interferon-alpha-4 or with all interferon-alpha subtypes were used as immunohistochemical and immunofluorescence probes to study interferon-alpha production in liver biopsy tissue, and peripheral blood mononuclear cells prior to and after stimulation with Sendai virus. Measurement of cytoplasmic interferon-alpha, specifically interferon-alpha-2 and interferon-alpha-4, in peripheral blood mononuclear cells isolated from the hepatitis C patients and of total interferon-alpha secreted into culture supernatants by these cells showed interferon-alpha production similar to that of peripheral blood mononuclear cells isolated from normal individuals. Interferon-alpha-positive cells were observed in the infiltrating mononuclear cells of the liver biopsy tissue obtained from 8 of the 14 patients. Lymphocytes, fibroblasts, Kupffer cells, polymorphonuclear cells and monocytes stained positive for interferon-alpha, and specifically interferon-alpha-4, in all of the eight patients. The cytoplasm of hepatocytes also stained weakly positive in three of these patients. Interferon-alpha positive cells showed a good correlation with the degree of histological damage observed in the liver biopsies but not with presence of antibodies towards hepatitis C virus or levels of serum alanine aminotransferase measured prior to interferon-alpha-2a therapy. Interestingly, response to therapy seemed linked to local interferon-alpha production status. Those patients who responded best to therapy displayed no or only low levels of interferon-alpha positive cells in liver biopsy tissue. Thus patients with a lower activation of their endogenous interferon-alpha system may benefit from administration of exogenous interferon-alpha.

Nef 27, but not the Nef 25 isoform of human immunodeficiency virus-type 1 pNL4.3 down-regulates surface CD4 and IL-2R expression in peripheral blood mononuclear cells and transformed T cells.

Continuing controversy surrounds the cellular effects of the Nef protein of HIV-1, a nonstructural protein expressed by most isolates. Highly purified protein isoforms of MW 27 kDa (Nef 27) and 25 kDa (Nef 25), produced in Escherichia coli by translation from the first and second start codons of HIV-1 nef clone pNL4.3, respectively, were introduced into cells by a sophisticated electroporation technique which uses electric field rather than electric charge to transfer macromolecules across cell membranes. Electroporation of Nef 27 reduced the expression of cell surface CD4 by 30-50%, as measured by flow cytometry, on phytohemagglutinin (PHA)-activated PBMC as well as on a variety of CD4+ T-cell lines (MT-2, CEM, and Jurkat). Reduction in surface CD4 was observed in all cells of the CD4+ T-cell lines but only in the CD4+ cells of the mixed PBMC population. Electroporation of Nef 27 into MT-2 cells and PHA-activated PBMC also reduced the expression of IL-2R to background levels. Other cell surface antigens analyzed such as CD2, CD7, or transferrin receptor (TfR) were not affected by the introduction of HIV-1 Nef 27. In contrast to the effects of Nef 27, electroporation of Nef 25 into cells at equivalent concentrations did not affect the surface expression of CD4 and IL-2R. These data show that the HIV-1 clone pNL4.3 Nef 27 but not the Nef 25 isoform specifically decreases expression of two cell surface receptors important for antigen recognition of MHC class II antigens and for cell proliferation. Production of Nef 27 during HIV-1 infection of cells of the immune system may contribute to immunodeficiency even in the absence of direct viral cytopathic effects.

Selective production of interferon-alpha subtypes by cultured peripheral blood mononuclear cells and lymphoblastoid cell lines.

The biological significance of the existence of multiple interferon-alpha (IFN-alpha) subtypes is unknown but may represent a finely tuned mechanism whereby different subtypes are produced in response to different stimuli. To investigate the expression of individual IFN-alpha subtypes, polyclonal antipeptide antisera designed to react with all IFN-alpha subtypes, or with a particular subtype, IFN-alpha 2 or IFN-alpha 4, have been produced. In this study we demonstrate the utility of these antisera for the detection, using indirect immunofluorescence staining, of intracellular IFN-alpha produced by human peripheral blood mononuclear cells (PBMC) and lymphoblastoid cells. Secreted IFN-alpha was also investigated by bioassay and a sandwich radioimmunoassay (RIA), using two monoclonal antibodies (mAb) and specific for IFN-alpha 4. The PBMC were shown to produce IFN reactive with all three polyclonal antisera, after stimulation with Sendai virus. The lymphoblastoid cells also produced IFN, including IFN-alpha 2, but IFN-alpha 4 was not detected either intracellularly, by immunofluorescence, or in the medium, by sandwich RIA. The immunofluorescence studies also demonstrate that in the absence of viral stimulation IFN-alpha is found in the cytoplasm of PBMC and lymphoblastoid cells but not secreted in detectable levels. The finding that two lymphoblastoid cell lines do not produce the subtype IFN-alpha 4 raises important questions as to whether other cell lines and cell types produce IFN-alpha subtypes selectively, and whether individual IFN-alpha subtypes have different roles in human physiology and pathology.