Khalid B. Islam

Effects of intramyocardial injection of phVEGF‐A165 as sole therapy in patients with refractory coronary artery disease – 12‐month follow‐up: Angiogenic gene therapy

Abstract. Sarkar N, Rück A, Källner G, Y‐Hassan S, Blomberg P, Islam KB, van der Linden J, Lindblom D, Nygren AT, Lind B, Brodin L‐Å, Drvota V, Sylvén C (Karolinska Institute, Huddinge University Hospital, Novum, Stockholm, Sweden). Effects of intramyocardial injection of phVEGF‐A165 as sole therapy in patients with refractory coronary artery disease: 12‐month follow‐up. Angiogenic gene therapy. J Intern Med 2001; 250: 373–381.

Gene Combination Raises Broad Human Immunodeficiency Virus-Specific Cytotoxicity

DNA plasmid immunization has the important advantage over traditional vaccines of making it possible to combine selected genes into one vaccine. The efficacy of a combination of DNA plasmids encoding the nef, rev, and tat HIV-1 regulatory genes in inducing cellular immune responses was analyzed in asymptomatic HIV-1-infected patients. Patients initially selected for having low or no detectable immune responses to Nef, Rev, or Tat antigens developed MHC class I-restricted cytolytic activities as well as enhanced bystander effects. The induction of memory cells against target cells infected with the whole HIV-1 genome was analyzed by using a pseudovirus HIV-1/murine leukemia virus (MuLV), and target cells infected with vaccinia virus carrying the respective gene. The most remarkable change observed after immunization with the gene combination was an increase in cytotoxic T lymphocyte (CTL) precursors to target cells infected with the whole HIV-1 genome. Infection by the pseudotype HIV-1/MuLV virus should result in a multitude of HIV-1 peptides presented on the target cell surface, representative of the in vivo situation. An in vitro assessment of the expression of the single and combined gene products showed that this was consistent with the induction of CTL responses in vivo. No clinical advantage or adverse effects were noted. Therapeutic effects of such immunization may become measurable by structured therapy interruption.

Electroporation in combination with a plasmid vector containing SV40 enhancer elements results in increased and persistent gene expression in mouse muscle.

Gene transfer into muscle upon injection of plasmid DNA is feasible but occurs with low frequency. However, by using electroporation after injection of plasmid DNA into mouse muscle it has been demonstrated that gene expression can be increased more than 150-fold. In this communication, we have used this technique in combination with plasmids containing a tandem repeat of three 72-bp DNA elements from the SV40 enhancer to study gene expression. Our results show that the combination of electroporation and a plasmid vector carrying these DNA elements results in increased and more persistent gene expression of the luciferase reporter gene in BALB/c mouse muscle. At 14 days after gene delivery, the gene expression was 16-fold higher in muscles injected and electroporated with the plasmid carrying the SV40 enhancers than with control plasmid. We have also studied the effects of the vehicle in which the plasmid was delivered, and the DNase inhibitor aurintricarboxylic acid (ATA), on gene expression. By combining ATA with 150 mM sodium phosphate buffer we were able to obtain a 2-fold increase in gene expression compared to delivery of the plasmid in physiological saline. These results are of importance for the development of efficient delivery techniques for naked DNA.

Humoral Immunity in scid Mice Reconstituted with Cells from Immunoglobulin-Deficient or Normal Humans

The complexity of the immune system makes it difficult to study the influence of genetic variation in humans by the use of conventional in vitro or experimental animal models. Since genetic traits often determine predisposition to immunopathies of various forms, the development of technologies that permit the direct, long-term study of a selected human immune system under experimental conditions is warranted. Several different models are available for such an analysis, each having its particular limitations (Table I). Thus, as an example, the inbred mouse strains that exist today originate from a small number of founders (Klein 1986), making it unlikely that a particular genetic background, corresponding to a selected gene abnormality in a patient with an inherited immunopathy, would be represented. Recently, several difTerent methods have been developed where components of a human immune system are transferred to mice. Thus, in particular, the adoptive transfer of human lymphoid organs (McCune et al. 1988) or lymphocytes (Mosier et al. 1988) to severe combined itnmunodeficient C.B-17 scid/scid (scid) mice has demonstrated the general applicability of such an approach, scid mice are severely deficient in functional lymphocytes due to the impairment of antigen receptor gene recombination (Bosma et al. 1983, Bosma & Carroll 1991), which facilitates repopulation with xenogeneic cells, since their capacity to reject foreign tissue is decreased.

Aberrant expression of α‐Gal on primary human endothelium does not confer susceptibility to NK cell cytotoxicity or increased NK cell adhesion

The contribution of Galα1,3Gal (α‐Gal) to cell‐mediated organ xenograft rejection is controversial. We have used recombinant lentiviruses encoding a porcine α1,3 galactosyltransferase (α1,3GalT) to obtain α‐Gal‐expressing primary human aortic endothelial cells (HAEC) at a frequency of 70–90%. These cells were compared to non‐transduced and mock‐transduced HAEC with regard to their susceptibility to human NK cell‐mediated lysis, ability to stimulate IFN‐γ production by NK cells, and support of NK cell adhesion under static and dynamic conditions. Using green fluorescent protein (GFP) as a reporter gene, it was shown that the frequency of green fluorescent HAEC increased until day 5 post‐transduction, and at a multiplicity of infection of 2.5, it reached98%. Lentiviral transduction did not result in activation of HAEC, and transduced HAEC responded as expected to TNF‐α and IFN‐γ stimulation. No differences were detected between non‐α‐Gal‐ and α‐Gal‐expressing HAEC in terms of their susceptibility to NK cell‐mediated lysis, ability to stimulate IFN‐γ production by NK cells, or ability to support NK cell adhesion under static and dynamic conditions. In conclusion, these data argue against an important role for the α‐Gal epitope in the direct interaction between endothelium and NK cells and prove that recombinant lentiviruses are efficient gene carriers for primary human endothelial cells.

In vivo expression of human immunoglobulin germ-line mRNA in normal and in immunodeficient individuals.

Previous in vitro studies suggest that transcription of the unrearranged immunoglobulin switch region and its 5′ flanking region precedes isotype switching. These transcripts, which arc devoid of a variable region, contain unique exons and are called germ‐line (GL) mRNA. A crucial point in this regard is whether such transcripts could be detected in vivo, and if their expression correlates with immunoglobulin class switching in health and disease. To understand the in vivo role of this transcriptional activity we have adapted the reverse transcription‐polymerase chain reaction (PCR) to analyse the GL transcripts from unstimulated peripheral blood mononuclear cells (PBMC) in healthy individuals and in different immunological diseases. Furthermore, mononuclear cells from different human organs were also analysed. We report here that GL (Iα. Iγ and Iɛ used lo designate the GL mRNA for IgA. IgG and IgE, respectively) nRNA are expressed differentially during ontogeny of B cells. Unexpectedly, no difference of Iα mRNA expression between the PBMC and the secondary lymphoid organs was detected. Rather. the levels of GL transcripts were correlated to ihe number of sIgM+ cells. GL mKNA of all three isotypes could be detected in PBMC from healthy donors, whereas there was a decrease of specific GL transcript synthesis in individuals with Immunoglobulin deficiency. Furthermore, during the in vivo immune response in a parasitic infection, we could demonstrate an induction of GL k mRNA during in vivo immune response. Concomitantly. there was also increased synthesis of productive F. transcripts. These findings implicate a potential role of GL transcription during in vivo immunoglobulin class switching.

Nonsurgical direct delivery of plasmid DNA into rat heart: time course, dose response, and the influence of different promoters on gene expression.

Transfer of genes encoding therapeutic proteins into the myocardium shows great potential for treatment of coronary artery disease. To quantitatively elucidate the behavior of plasmid DNA following cardiac gene transfer, time kinetics, dose-response relationship, systemic spread to the liver, and the influence of different promoters on plasmid DNA gene expression in rat hearts were examined using a novel nonsurgical direct delivery method that enables testing of large numbers of animals. Plasmids encoding either vascular endothelial growth factor A 165 or a fusion protein between enhanced green fluorescent protein (EGFP) luciferase were injected directly in rat hearts under echocardiographic guidance. The results show that gene expression is dose related and that the duration of gene expression is transient. These findings underscore the necessity to explore other efficient vectors or alternative methods of gene delivery to achieve increased and prolonged gene expression.

Studies of the molecular basis of IgA production, subclass regulation and class-switch recombination in IgA nephropathy patients

IgA nephropathy (IgAN), the most common form of glomerulonephritis, is characterized by normal to elevated levels of serum IgA. In order to understand the molecular mechanism(s) involved in the production of IgA in IgAN, peripheral blood mononuclear cells (PBMC) from these patients were analysed in this study. IL‐10, transforming growth factor‐beta 1 (TGF‐β1) and CD40 have previously been shown to be involved in IgA production. We show here that CD40L expression was increased three‐fold in these patients. However, expression of TGF‐β1 in serum levels was comparable to controls. In vitro stimulation of PBMC with a polyclonal activator resulted in a three‐fold increase in synthesis of both IgA subclasses, with a preference for IgA1 RNA. Insitu hybridization studies also showed a three‐fold increase in the numbers of IgA1‐ and IgA2‐producing cells, but the subclass distribution was similar to the controls. Furthermore, using the nested primer polymerase chain reaction (PCR) for amplifying switch (Sμ/Sα) breakpoints we could demonstrate that in unstimulated PBMC the switch frequency did not differ from that of control donors. Sequence analysis of the amplified switch breakpoints and the Iα regulatory region from patients showed no structural abnormality. Although we have previously demonstrated a correlation to in vivo germ‐line RNA expression and class switching, no Iα transcripts were detected in unstimulated PBMC from these patients. However, stimulation of PBMC with TGF‐β1 resulted in Iα production. Taken together, results from in vivo and in vitro studies suggest that increased cytokine production and hyperresponsiveness to polyclonal stimulation may play an important role in the increased synthesis of IgA. The preference for IgA1 is due to increased production of IgA1 per cell, and the absence of Iα RNA indicates that additional defect(s) in immune regulation may play an important role in the pathogenesis of IgAN.

Subtypes A, C, G, and recombinant HIV type 1 are circulating in Bangladesh.

We analyzed the genetic diversity of HIV-1 circulating in Bangladesh by direct sequencing and subsequent phylogenetic analysis of the V3 region of the env gene and p17 fragment of the gag gene from nine unrelated patients. The sequences from one sample grouped into subtype A, five samples grouped into subtype C, and one grouped into subtype G. In addition, two patients appeared to be infected with different recombinant viruses consisting of subtype A and unclassifiable viral sequences. Epidemiological analysis revealed heterosexual transmission in the majority of cases. Furthermore, most subjects had a history of traveling, either to India or to the Arabian Peninsula. This study shows that several HIV-1 subtypes are circulating in Bangladesh, and we conclude that there must have been several introductions of HIV-1 into the Bangladeshi population.

BTK mediated apoptosis, a possible mechanism for failure to generate high titer retroviral producer clones.

It has been shown previously that mutations in the cytoplasmic protein kinase, Brutons tyrosine kinase (BTK) lead to X‐linked agammaglobulinemia, an inherited primary immunodeficiency, thus making it a potential candidate for gene therapy.