Hodjattallah Rabbani

Human papillomavirus type 16 is episomal and a high viral load may be correlated to better prognosis in tonsillar cancer

The aim of our study was to investigate the physical state and the viral load of HPV‐16 in tonsillar cancer and to correlate these findings with clinical outcome. To distinguish between integrated and episomal forms of HPV, 22 fresh‐frozen tonsillar cancer samples were analysed by a method based on restriction enzyme cleavage, ligation and PCR (rliPCR). HPV‐16 was detected in 11/22 and HPV‐33 in 1/22 of the cancers, hence 12/22 (55%) of the tumours were HPV positive. Only extrachromosomal forms of HPV‐16 were observed. Full‐length episomal HPV was detected exclusively in 7/11 of the cancers, whereas both full‐length and deleted forms of episomal HPV‐16 were found in parallel in 2 other tumours. In 1 tumour only a deleted episomal form of HPV‐16 was present. In the remaining HPV‐16 positive tumour both full‐length episomal as well as an 11 kbp PCR product were detected and if the 11 kbp product contained integrated HPV, or was off‐size linearised episomal could not be determined. In 2 cervical cancer controls, HPV‐16 was integrated and could be chromosome located. HPV‐16 was quantified by real‐time PCR and most tonsillar cancers contained between 10 to a few hundred copies of HPV per β‐actin. The 6 patients with tumour sections with ≥190 HPV‐16 copies/β‐actin remained tumour free (p = 0.026) and had a better survival rate (p = 0.039) when compared to the 5 patients with tumours sections with ≤60 HPV‐16 copies/β‐actin. In conclusion, HPV‐16 is mainly episomal in tonsillar cancer. The viral load showed a wide distribution and the clinical outcome in our study was better when the HPV load was higher.

IFN-γ protects short-term ovarian carcinoma cell lines from CTL lysis via a CD94/NKG2A-dependent mechanism

IFN-gamma regulates the immunogenicity of target cells by increasing their expression of HLA class I molecules. This facilitates the T cell receptor-mediated recognition by CD8(+) T cells but decreases target cell sensitivity to lysis by NK cells due to engagement of inhibitory NK receptors. In this study, short-term tumor cell lines from patients with advanced ovarian carcinomas were established. We demonstrate the paradoxical finding that IFN-gamma treatment of these short-term ovarian carcinoma cell lines (OVACs) resulted in resistance of tumor cells to lysis by peptide- and allospecific CD8(+) T cells. Blocking experiments revealed that this phenomenon was dependent on enhanced inhibitory signalling via CD94/NKG2A receptors expressed on the effector cells. This was associated with increased expression of HLA-E mRNA and HLA-G at the protein level in IFN-gamma-treated OVACs. Furthermore, pulsing of untreated OVACs with the leader sequence peptide of HLA-G protected these cells from lysis by CTLs, thus mimicking the inhibitory effect of IFN-gamma. This study provides evidence that CD94/NKG2A receptors play an important role in regulating T cell activity against tumors and shows that IFN-gamma modulation of target cells may shift the balance of triggering and inhibitory signals to T cells, turning off their cytolytic activity.

Artiodactyl IgD: The Missing Link

IgD has been suggested to be a recently developed Ig class, only present in rodents and primates. However, in this paper the cow, sheep, and pig Ig δ genes have been identified and shown to be transcriptionally active. The deduced amino acid sequences from their cDNAs show that artiodactyl IgD H chains are structurally similar to human IgD, where the cow, sheep, and pig IgD H chain constant regions all contain three domains and a hinge region, sharing homologies of 43.6, 44, and 46.8% with their human counterpart, respectively. According to a phylogenetic analysis, the Cδ gene appears to have been duplicated from the Cμ gene >300 million yr ago. The ruminant μCH1 exon and its upstream region was again duplicated before the speciation of the cow and sheep, ∼20 million yr ago, inserted upstream of the δ gene hinge regions, and later modified by gene conversion. A short Sδ (switch δ) sequence resulting from the second duplication, is located immediately upstream of the bovine Cδ gene and directs regular μ-δ class switch recombination in the cow. The presence of Cδ genes in artiodactyls, possibly in most mammals, suggests that IgD may have some as yet unknown biological properties, distinct from those of IgM, conferring a survival advantage.

Prevalence, genetics and clinical presentation of chronic granulomatous disease in Sweden

To estimate the prevalence of chronic granulomatous disease (CGD) in Sweden, an inquiry asking for known and possible CGD cases was mailed to paediatric, internal medicine and infectious disease departments all over Sweden. The detected patients were characterized as to genetics and the clinical presentation. Twenty–one patients (belonging to 16 different families) were found, corresponding to a prevalence of ∼ 1/450 000 individuals. The patients with X–linked disease, lacking a functional gp91phox protein (n= 12), comprised 57% and 43% of the patients had an autosomal recessive (AR) disease lacking p47phox (n= 7) or p67phox (n=1), respectively. All unrelated patients with X–linked disease displayed different gene abnormalities such as point mutations predicting nonsense (n= 3), missense (n= 1) or splice site mutations (n = 2), but also a total deletion and a unique 40 base pair duplicature insertion. The patients with p47phox–deficiency showed a GT deletion at a GTGT tandem repeat, and the p67phox–deficient patient displayed a heterozygous in–frame deletion of AAG combined with a large deletion in the other allele. Three patients died during the study period, two from Pseudomonas cepacia infections. Patients with X–linked disease had more frequent infections (mean of 1.7 per year), than the patients with AR inheritance (0.5 infections per year). The most common infections were dermal abscesses (n=111), followed by lymphadenitis (n=82) and pneumonias (n=73). Inflammatory bowel disease–like symptoms, mimicking Crohns disease of the colon, was seen in three CGD patients.

Mapping of the chicken immunoglobulin heavy-chain constant region gene locus reveals an inverted α gene upstream of a condensed υ gene

Chicken antibodies are increasingly being used as diagnostic and therapeutic tools. As only the genomic organization of the µ encoding gene was previously known, we analysed the chicken immunoglobulin Y (IgY) and IgA (υ and α chain) immunoglobulin heavy‐chain constant region (IGHC) genes and the organization of the chicken IGHC locus. The α gene is encoded by four separate exons, whereas, surprisingly, there is no intervening DNA sequence between the CH1 and CH2 domains of the IgY heavy chain, which is thus encoded by three exons separated by two introns. DNA sequence analysis shows that the exon boundaries of the chicken IGHC genes are not consistent with published domain borders. Furthermore, differences in DNA sequence confirm the existence of IgY, IgA and IgM allotypes in chickens. Finally, our results show that the IGHC genes of chicken (IgY, IgA and IgM) are all colocated on chromosome E18C15W15, where the α gene is located upstream of the υ gene in an inverted transcriptional orientation. The distances between the µ and α genes and the α and υ genes are about 18 and 15 kilobases, respectively, and thus, the size of the whole chicken IGHC locus is approximately 67 kilobases.

Signalling molecules and cytokine production in T cells of multiple myeloma‐increased abnormalities with advancing stage

T‐cell immune dysfunction in patients with malignant tumours has been attributed to the altered expression of components of the T‐cell receptor (TCR)/CD3 complex and their associated intracellular protein tyrosine kinases. In this study, four‐colour flow cytometry was applied to study the surface bound molecules TCRαβ, CD28, CD152 and CD154 involved in T‐cell signalling and the signal transduction molecules CD3ζ, p56lck, p59fyn, ZAP‐70 and phosphatidyl‐inositol‐3 kinase (PI3‐k) as well as the intracellular cytokines interferon‐γ (IFN‐γ), interleukin (IL)‐4 and IL‐2 as a functional read‐out of non‐stimulated and superantigen (staphylococcus enterotoxin B)‐stimulated blood T cells of multiple myeloma (MM) patients at different stages of the disease. Multiple abnormalities were demonstrated in the CD4 and CD8 populations, both under non‐stimulated and superantigen‐stimulated conditions. There was a marked reduction, particular in advanced stage MM, in the proportion of CD4 and CD8 cells expressing CD28, CD152, CD3ζ, p56lck, ZAP‐70 and PI3‐k. The level of intracellular T‐cell cytokines (IFN‐γ, IL‐2 and IL‐4) was normal or increased in non‐stimulated cells but activation‐induced cytokine production was impaired. These results illustrated profound and multiple T‐cell signalling defects, from the surface and down‐stream, consistent with involvement of a master T‐cell function, especially in advanced stage MM. These data should be taken into consideration when developing immune‐based therapeutic approaches and when applying new emerging technologies that aim to restore T‐cell functions.

Long-term idiotype vaccination combined with interleukin-12 (IL-12), or IL-12 and granulocyte macrophage colony-stimulating factor, in early-stage multiple myeloma patients

Purpose and Experimental Design: Twenty-eight patients with immunoglobulin G myeloma stages I to II were immunized i.d. over 110 weeks with autologous M protein combined with interleukin-12 (IL-12; n = 15) or with IL-12 and granulocyte macrophage colony-stimulating factor (GM-CSF; n = 13). Idiotype-specific T-cell responses were assessed by [3H]thymidine incorporation, enzyme-linked immunospot assay, and delayed-type hypersensitivity reaction. Results: Based on these three assays, idiotype-specific immune responses were noted in 5 of 15 (33%) patients in the IL-12 group and in 11 of 13 (85%) patients in the GM-CSF/IL-12 group (P < 0.01). Immune response was seen only in patients with M-component concentration of <50 g/L. Three of 16 (19%) responders showed a gradually increasing idiotype-specific T-cell response, whereas 11 of 16 (69%) patients showed initial response, which then disappeared rapidly; the latter pattern was frequently associated with subsequent progressive disease. Immune nonresponse was associated with an increase in the numbers of CD4+/CD25+ cells (regulatory T cells), which was absent in responding patients. Median time to progression for immune responders (n = 16) was 108 weeks compared with 26 weeks for nonresponders (n = 12; P = 0.03). Conclusions: These results indicate that idiotype immunization of myeloma patients with GM-CSF and IL-12 may induce specific T-cell response more frequently than with IL-12 alone and that immune response may correlate with time to progression and nonresponse with increased numbers of regulatory T cells.

Autologous T lymphocytes may specifically recognize leukaemic B cells in patients with chronic lymphocytic leukaemia

This study analysed a naturally occurring specific cellular immunity against tumour cells in chronic lymphocytic leukaemia (CLL) patients. Five out of eight patients had blood T lymphocytes able to recognize spontaneously and specifically the autologous tumour B cells (proliferation assay). In these five patients, detection of cytokines by real‐time reverse transcription polymerase chain reaction (RT‐PCR) revealed that granulocyte–macrophage colony‐stimulating factor (GM‐CSF) was the most abundant cytokine gene expressed by the T cells that recognized the autologous tumour B cells. Other activated cytokine genes were γ‐interferon (IFN), interleukin (IL)‐2 and tumour necrosis factor (TNF)‐α, but not IL‐4. This profile suggests a type 1 anti‐B‐CLL T‐cell response. CD80 and CD54 were relatively downregulated on the native tumour B cells compared with control normal B cells. Upregulation of CD80 on the leukaemic cells was mandatory for the induction of such a specific T‐cell response. CD80 and CD54 monoclonal antibodies inhibited the specific T‐cell DNA synthesis proliferation. The proliferative T‐cell response was either MHC class I or class II restricted (inhibition by monoclonal antibodies). The specific cytokine gene expression could be found in isolated CD4, as well as CD8, T‐cell subsets. This study demonstrated the presence of a potential natural specific CD4, as well as a CD8 type 1 T‐cell immunity against the leukaemic CLL tumour B cells in CLL. A further detailed analysis of the spontaneous anti‐CLL T‐cell immunity is warranted that may facilitate the development of effective anti‐tumour vaccines in CLL.

CHARACTERIZATION OF THE BOVINE CALPHA GENE

The complete genomic sequence of a bovine Cα gene is reported here. The genomic sequence was obtained from a Cα phage clone that had been cloned from a genomic EMBL4 phage vector library. The Cα sequence had previously been expressed as a chimeric antibody and identified as IgA using IgA‐specific antibodies. Intron/exon boundaries were determined by comparison of the genomic sequence with an expressed bovine Cα sequence obtained from spleen by reverse transcription‐polymerase chain reaction (RT‐PCR). Analysis of 50 Swedish bovine genomic DNA samples using genomic blots and five different restriction enzymes failed to detect evidence of polymorphism. However, PstI digests of Brown Swiss DNA showed a restriction fragment length polymorphism (RFLP), suggesting that at least two allelic variants of bovine IgA exist. Comparison of the deduced amino acid sequence of bovine IgA with sequences available for other species indicated that the highest homology was with that of swine, another artiodactyl. This was the highest homology observed for all mammalian IgA compared except for that between IgA1 and IgA2 in humans. Bovine IgA shares with rabbit IgA3 and IgA4, an additional N‐linked glycosylation site at position 282. However, the collective data indicate that cattle are like swine and rodents and unlike rabbits in having a single locus of the gene encoding IgA of this species.

Polymorphism of the IGHG3 gene in cattle.

Abstract We cloned and analyzed the gene (IGHG3) encoding cattle IgG3 using a spleen cDNA library and a genomic phage clone. IGHG3 differs most remarkably from IGHG1 and IGHG2 in the structure of its hinge. The IGHG3 hinge is encoded by two exons, one of which encodes a 22 amino acid 3′ extension of the CH1 exon. The IGHG3 gene is highly homologous to the C-gamma pseudogene previously reported. Seventy-one DNA samples from different cattle were tested by pulsed-field gel electrophoresis and Southern blot to detect genetic polymorphisms. This analysis revealed two different alleles which we designated IGHG3a and IGHG3b. The two putative alleles differ from each other by a 6 amino acid substitution in the coding region and an 84 base pair insertion in the intron between the CH2 and CH3 exons. Our data also indicate that the cattle heavy chain locus spans approximately 400 kilobases from IGHM to IGHA and encodes only three IgG subclasses, all of which are transcriptionally active.