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Herpesvirus-like DNA sequences in non-Kaposi's sarcoma skin lesions of transplant patients
Herpesvirus-like DNA sequences in non-Kaposi’s sarcoma skin lesions of transplant patients
Herpesvirus-like DNA sequences (KSHV) have been reported to be associated with various forms of Kaposi’s sarcoma (KS). To determine if KSHV was associated with skin lesions from non-AIDS proliferative immunocompromised patients, 33 skin lesions (basal cell carcinomas, squamous cell carcinomas, actinic keratoses, verruca vulgaris, atypical squamous proliferations, and seborrhoeic keratosis) from 4 organ-transplant patients receiving immunosuppressive therapy were tested for other
KSHV by PCR. KSHV sequences were detected in 82% of these skin lesions. Our results suggest that KSHV is associated with lesions other than KS in non-AIDS immunocompromised patients, and may also be involved in the pathogenesis of the various forms of proliferative skin lesions from organ-transplant patients. Lancet 1995; 345: 1339-40 New herpesvirus-like sequences (KSHV) have been found to be associated with AIDS-related Kaposi’s sarcomas.’1 The sequences were designated KS330 and were found to be highly homologous to capsid-protein coding genes of herpesvirus saimiri and Epstein-Barr virus. A high prevalence of KSHV in classic, endemic, as well as AIDSassociated KS biopsies was also found,2-4 suggesting that this viral with associated might be agent in We looked for KSHV immunodeficiency general. sequences in lesions other than KS, and in people with immunodeficiency not due to AIDS. 33 fresh skin lesions biopsied from 4 HIV-negative organtransplant patients (table) were analysed. Patients were receiving prednisone and cyclosporin; and patients 2 and 4 were also receiving azathioprine. Specimens were collected at the Department of Dermatology, Baylor College of Medicine,
Houston, TX, USA and stored at -70°C until processed. One fresh AIDS-KS as positive control was biopsied at University of Texas Medical Branch, Galveston, TX, USA. DNA and total RNA from the fresh samples was isolated by use of Tri reagent solution (Molecular Research Center, Cincinnati, OH, USA). The primers and conditions of the PCR analysis of 233 bp-sized KSHV sequences (KS330233) were similar to those described earlier.’ Precautions were observed to avoid contamination in all PCR procedures, including aliquoting all reagents, use of positive-displacement pipettes, filtered pipette
tips, and a Template Tamer UV cabinet (Gaithersburg, MD, USA). In addition positive, negative, internal (&bgr;-globin), and reagent controls were included. Amplimers for the (3-globin sequences were obtained from Perkin-Elmer Co (Norwalk, CT, USA) and PCR amplification was carried out as described.5 KSHV (KS330233) expression was analysed by RT-PCR from 3 mg RNA digested with deoxyribonuclease I, amplification grade (Gibco-BRL, Gaithersburg, MD, USA), eliminating possible DNA contamination. RNA samples were reverse transcribed with mRNA capture kit for RT-PCR (Amersham-USB, Cleveland, with random hexamers. Aliquots of the RT mixtures were then amplified through 30 cycles.’ From the same RT an
OH, USA)
*See figure; BCC=basal cell carcinoma, SCC=squamous cell carcinoma, ASP=atypical squamous proliferation, AK=actinic keratosis, SK=seborrhoeic keratosis, VV=verucca
vulgaris. Table: Detection of herpesvirus-like DNA sequences in skin lesions from transplant recipients
mixtures, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) reference-gene sequences were amplified through 30 cycles with G3PDH amplimers (Clontech Laboratories, Palo Alto, CA, USA), and PCR analysis was performed under conditions recommended by the manufacturer. Fragment fractionation of the PCR mixtures
was
carried
out on a
2% Seakem GTG
gel (FMC Rockland, ME, USA), stained with ethidium bromide, and visualised with UV. Specificity of the amplification was confirmed by Southern blot. Following transfer to a Hybond N (Amersham Inc, Arlington Heights, IL, USA) in 0-4 mol/L NaOH, membranes were prehybridised in Rapid Hybridization buffer (Amersham). Oligonucleotide probes specific for KSHV,’ &bgr;-globin,5 and G3PDH (sequence position; nts: /5’-3’/: 658-639, EMBL databank, accession number M33197) sequences were located within the amplified gene fragments and radiolabelled with &ggr;-32P dATP (Amersham) using a T4 kinase reaction (Gibco BRL) following the vendors’ recommendations. Membranes were hybridised with radiolabelled gene-specific probes in Rapid Hybridization buffer (Amersham), washed and used to expose X-ray films (Hyperfilm-MP, Amersham). agarose
To assess the integrity of genomic DNA extracted from 33 biopsies, a region of the &bgr;-globin gene was amplified. The 268 bp-sized (3-globin fragments could be detected in all of these samples, indicating the absence of possible inhibitors of PCR (data not shown). 33 skin-tumour biopsies were analysed for the presence of KS330233 sequences by PCR. Agarose-gel electrophoresis of the and Southern-blot PCR subsequent products the revealed of the 233 bp presence hybridisation amplicons of the KS330 viral DNA region in 82% (figure and table). The two most common lesions biopsied were actinic keratosis (AK) and squamous cell carcinoma 1339
neoplastic processes, KSHV may also be involved in neoplastic transformation, possibly together with other pathogens and/or cellular genetic changes. References 1
Chang Y, Cesarman E, Pessin MS, et al. Identification of herpes virus like sequences in AIDS-associated Kaposi’s sarcomas. Science 1994; 266: 1865-69.
2
Dupin N,
3
Huang YQ, Kaplan MH, Poiesz B, et al. Human herpesvirus-like nucleic acid in various forms of Kaposi’s sarcoma. Lancet 1995; 45:
4
IJ, Hsu Y-S, Chang Y-C, Wang I-W. Herpesvirus-like DNA sequence in Kaposi’s sarcoma from AIDS and non-AIDS patients in Taiwan. Lancet 1995; 345: 722-23.
5
Resnick RM, Cornelissen MTE, Wright DK, et al. Detection and typing of human papillomavirus in archival cervical cancer specimens by DNA amplification with consensus primers. J Natl Cancer Inst 1990; 82: 1477-84.
6
Euvrard
Grandadam M, Calvez V, et al. sequences in patients with Mediterranean
Herpesvirus-like DNA Kaposi’s sarcoma. Lancet
1995; 345: 761-62.
759-61.
7
Southern blot hybridisation of the KS330233 PCR products with isotope labelled KS330233 sequence specific
C=negative control; normal skin sample from an otherwise healthy patient with basal cell carcinoma (BCC). P=positive control; cutaneous AIDS-KS sample. 1-33=specimens from transplant recipients (see table).
S, Chardonnet Y, Pouteil-Noble C, et al. Association of skin malignancies with various and multiple carcinogenic and non carcinogenic human papillomaviruses in renal transplant recipients. Cancer 1993; 72: 2198-206. McGregor JM, Crook T, Yu CC, et al. Posttransplant skin cancer: possible role for p53 gene mutation but not for oncogenic human papillomaviruses. J Am Acad Dermatol 1994; 30: 701-06.
a
8
Brash DE, Rudolph JE, Simon JA, et al. A role for sunlight in skin cancer: UV induced p53 mutations in squamous cell carcinoma. Proc Natl Acad Sci USA 1991; 101: 24-28.
9
Rady P, Scinicariello F, Wagner RF, Tyring SK. p53 mutations in basal
Figure:
oligonucleotide probe
Su
cell carcinomas. Cancer Res 1992; 52: 3804-06.
Lanes:
of the KSHV was 78% in AKs and was 93% in SCCs. In other biopsied skin lesions (basal cell carcinomas, atypical squamous proliferations, seborrhoeic keratoses, verruca vulgaris) KSHV sequences were also detected. Two KSHV DNA-positive SCC biopsies were analysed by RT-PCR from patient 4. In both SCCs, RT-PCR and subsequent Southern-blot analysis detected expression of KS330233 viral mRNA transcripts (data not shown), suggesting active virus replication in these lesions. KSHV sequences have been found in classic, endemic, and AIDS-associated KS. We have now found this supposedly viral sequence in various proliferative skin lesions, particularly in SCCs which are common tumours in immunocompromised organ-transplant patients. Other reports suggest that human papillomaviruses6 or ultraviolet-light-related cellular genetic changes, such as p53 mutations,’-9 can have a part in post-transplant skin carcinogenesis. In a previous report, KSHV sequences were found not only in KS lesions but also in low copy number in clinically normal adjacent skin.2 We have found KSHV in a clinically normal skin sample from an organ transplant patient and also detected the viral sequences in low copy numbers (data not shown). However, neither study could exclude the possibility of microscopic lesions. The presence of KSHV in lesions other than KS suggests it is a widespread latent virus activated by immunosuppressive conditions and associated with proliferating lesions of immunosuppressed patients. Because KSHV has homology to Epstein-Barr and herpesvirus saimiri viruses which are also related to
(SCC). The prevalence
1340
Department of Microbiology and Immunology (S K Tyring MD, Hughes PhD, P L Rady MD), and Department of Dermatology (Yen MD), University of Texas Medical Branch, Galveston, Texas, USA; and Department of Dermatology (I Orengo MD, J L Rollefson MD, S Bruce MD), Baylor College of Medicine, Houston, Texas, USA T K
Correspondence to: Dr Stephen K Tyring, Department of Microbiology and Immunology, J-19, University of Texas Medical Branch, Galveston, TX 77555-1019, USA
Preconceptional natural-killer-cell activity as a predictor of miscarriage
There is no immunological test for the prospective identification of alloimmune causes of miscarriage. We investigated whether activity of natural killer cells was predictive of subsequent abortion in women who had had unexplained recurrent abortions and had received no treatment. 24 women with high preconceptional NK activity, defined as mean plus 1 SD of NK activity of 47 controls, had a significantly higher abortion rate in the next pregnancy than 44 women with normal levels of NK activity (71 vs 20%; relative risk 3.5; 95% CI 1.8-6-5). The preconceptional evaluation of NK activity in women with recurrent miscarriages may thus be predictive of the risk of pregnancy loss at the next conception. Lancet 1995; 345: 1340-42
Herpesvirus-like DNA sequences (KSHV) have been reported to be associated with various forms of Kaposi’s sarcoma (KS). To determine if KSHV was associated with skin lesions from non-AIDS proliferative immunocompromised patients, 33 skin lesions (basal cell carcinomas, squamous cell carcinomas, actinic keratoses, verruca vulgaris, atypical squamous proliferations, and seborrhoeic keratosis) from 4 organ-transplant patients receiving immunosuppressive therapy were tested for other
KSHV by PCR. KSHV sequences were detected in 82% of these skin lesions. Our results suggest that KSHV is associated with lesions other than KS in non-AIDS immunocompromised patients, and may also be involved in the pathogenesis of the various forms of proliferative skin lesions from organ-transplant patients. Lancet 1995; 345: 1339-40 New herpesvirus-like sequences (KSHV) have been found to be associated with AIDS-related Kaposi’s sarcomas.’1 The sequences were designated KS330 and were found to be highly homologous to capsid-protein coding genes of herpesvirus saimiri and Epstein-Barr virus. A high prevalence of KSHV in classic, endemic, as well as AIDSassociated KS biopsies was also found,2-4 suggesting that this viral with associated might be agent in We looked for KSHV immunodeficiency general. sequences in lesions other than KS, and in people with immunodeficiency not due to AIDS. 33 fresh skin lesions biopsied from 4 HIV-negative organtransplant patients (table) were analysed. Patients were receiving prednisone and cyclosporin; and patients 2 and 4 were also receiving azathioprine. Specimens were collected at the Department of Dermatology, Baylor College of Medicine,
Houston, TX, USA and stored at -70°C until processed. One fresh AIDS-KS as positive control was biopsied at University of Texas Medical Branch, Galveston, TX, USA. DNA and total RNA from the fresh samples was isolated by use of Tri reagent solution (Molecular Research Center, Cincinnati, OH, USA). The primers and conditions of the PCR analysis of 233 bp-sized KSHV sequences (KS330233) were similar to those described earlier.’ Precautions were observed to avoid contamination in all PCR procedures, including aliquoting all reagents, use of positive-displacement pipettes, filtered pipette
tips, and a Template Tamer UV cabinet (Gaithersburg, MD, USA). In addition positive, negative, internal (&bgr;-globin), and reagent controls were included. Amplimers for the (3-globin sequences were obtained from Perkin-Elmer Co (Norwalk, CT, USA) and PCR amplification was carried out as described.5 KSHV (KS330233) expression was analysed by RT-PCR from 3 mg RNA digested with deoxyribonuclease I, amplification grade (Gibco-BRL, Gaithersburg, MD, USA), eliminating possible DNA contamination. RNA samples were reverse transcribed with mRNA capture kit for RT-PCR (Amersham-USB, Cleveland, with random hexamers. Aliquots of the RT mixtures were then amplified through 30 cycles.’ From the same RT an
OH, USA)
*See figure; BCC=basal cell carcinoma, SCC=squamous cell carcinoma, ASP=atypical squamous proliferation, AK=actinic keratosis, SK=seborrhoeic keratosis, VV=verucca
vulgaris. Table: Detection of herpesvirus-like DNA sequences in skin lesions from transplant recipients
mixtures, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) reference-gene sequences were amplified through 30 cycles with G3PDH amplimers (Clontech Laboratories, Palo Alto, CA, USA), and PCR analysis was performed under conditions recommended by the manufacturer. Fragment fractionation of the PCR mixtures
was
carried
out on a
2% Seakem GTG
gel (FMC Rockland, ME, USA), stained with ethidium bromide, and visualised with UV. Specificity of the amplification was confirmed by Southern blot. Following transfer to a Hybond N (Amersham Inc, Arlington Heights, IL, USA) in 0-4 mol/L NaOH, membranes were prehybridised in Rapid Hybridization buffer (Amersham). Oligonucleotide probes specific for KSHV,’ &bgr;-globin,5 and G3PDH (sequence position; nts: /5’-3’/: 658-639, EMBL databank, accession number M33197) sequences were located within the amplified gene fragments and radiolabelled with &ggr;-32P dATP (Amersham) using a T4 kinase reaction (Gibco BRL) following the vendors’ recommendations. Membranes were hybridised with radiolabelled gene-specific probes in Rapid Hybridization buffer (Amersham), washed and used to expose X-ray films (Hyperfilm-MP, Amersham). agarose
To assess the integrity of genomic DNA extracted from 33 biopsies, a region of the &bgr;-globin gene was amplified. The 268 bp-sized (3-globin fragments could be detected in all of these samples, indicating the absence of possible inhibitors of PCR (data not shown). 33 skin-tumour biopsies were analysed for the presence of KS330233 sequences by PCR. Agarose-gel electrophoresis of the and Southern-blot PCR subsequent products the revealed of the 233 bp presence hybridisation amplicons of the KS330 viral DNA region in 82% (figure and table). The two most common lesions biopsied were actinic keratosis (AK) and squamous cell carcinoma 1339
neoplastic processes, KSHV may also be involved in neoplastic transformation, possibly together with other pathogens and/or cellular genetic changes. References 1
Chang Y, Cesarman E, Pessin MS, et al. Identification of herpes virus like sequences in AIDS-associated Kaposi’s sarcomas. Science 1994; 266: 1865-69.
2
Dupin N,
3
Huang YQ, Kaplan MH, Poiesz B, et al. Human herpesvirus-like nucleic acid in various forms of Kaposi’s sarcoma. Lancet 1995; 45:
4
IJ, Hsu Y-S, Chang Y-C, Wang I-W. Herpesvirus-like DNA sequence in Kaposi’s sarcoma from AIDS and non-AIDS patients in Taiwan. Lancet 1995; 345: 722-23.
5
Resnick RM, Cornelissen MTE, Wright DK, et al. Detection and typing of human papillomavirus in archival cervical cancer specimens by DNA amplification with consensus primers. J Natl Cancer Inst 1990; 82: 1477-84.
6
Euvrard
Grandadam M, Calvez V, et al. sequences in patients with Mediterranean
Herpesvirus-like DNA Kaposi’s sarcoma. Lancet
1995; 345: 761-62.
759-61.
7
Southern blot hybridisation of the KS330233 PCR products with isotope labelled KS330233 sequence specific
C=negative control; normal skin sample from an otherwise healthy patient with basal cell carcinoma (BCC). P=positive control; cutaneous AIDS-KS sample. 1-33=specimens from transplant recipients (see table).
S, Chardonnet Y, Pouteil-Noble C, et al. Association of skin malignancies with various and multiple carcinogenic and non carcinogenic human papillomaviruses in renal transplant recipients. Cancer 1993; 72: 2198-206. McGregor JM, Crook T, Yu CC, et al. Posttransplant skin cancer: possible role for p53 gene mutation but not for oncogenic human papillomaviruses. J Am Acad Dermatol 1994; 30: 701-06.
a
8
Brash DE, Rudolph JE, Simon JA, et al. A role for sunlight in skin cancer: UV induced p53 mutations in squamous cell carcinoma. Proc Natl Acad Sci USA 1991; 101: 24-28.
9
Rady P, Scinicariello F, Wagner RF, Tyring SK. p53 mutations in basal
Figure:
oligonucleotide probe
Su
cell carcinomas. Cancer Res 1992; 52: 3804-06.
Lanes:
of the KSHV was 78% in AKs and was 93% in SCCs. In other biopsied skin lesions (basal cell carcinomas, atypical squamous proliferations, seborrhoeic keratoses, verruca vulgaris) KSHV sequences were also detected. Two KSHV DNA-positive SCC biopsies were analysed by RT-PCR from patient 4. In both SCCs, RT-PCR and subsequent Southern-blot analysis detected expression of KS330233 viral mRNA transcripts (data not shown), suggesting active virus replication in these lesions. KSHV sequences have been found in classic, endemic, and AIDS-associated KS. We have now found this supposedly viral sequence in various proliferative skin lesions, particularly in SCCs which are common tumours in immunocompromised organ-transplant patients. Other reports suggest that human papillomaviruses6 or ultraviolet-light-related cellular genetic changes, such as p53 mutations,’-9 can have a part in post-transplant skin carcinogenesis. In a previous report, KSHV sequences were found not only in KS lesions but also in low copy number in clinically normal adjacent skin.2 We have found KSHV in a clinically normal skin sample from an organ transplant patient and also detected the viral sequences in low copy numbers (data not shown). However, neither study could exclude the possibility of microscopic lesions. The presence of KSHV in lesions other than KS suggests it is a widespread latent virus activated by immunosuppressive conditions and associated with proliferating lesions of immunosuppressed patients. Because KSHV has homology to Epstein-Barr and herpesvirus saimiri viruses which are also related to
(SCC). The prevalence
1340
Department of Microbiology and Immunology (S K Tyring MD, Hughes PhD, P L Rady MD), and Department of Dermatology (Yen MD), University of Texas Medical Branch, Galveston, Texas, USA; and Department of Dermatology (I Orengo MD, J L Rollefson MD, S Bruce MD), Baylor College of Medicine, Houston, Texas, USA T K
Correspondence to: Dr Stephen K Tyring, Department of Microbiology and Immunology, J-19, University of Texas Medical Branch, Galveston, TX 77555-1019, USA
Preconceptional natural-killer-cell activity as a predictor of miscarriage
There is no immunological test for the prospective identification of alloimmune causes of miscarriage. We investigated whether activity of natural killer cells was predictive of subsequent abortion in women who had had unexplained recurrent abortions and had received no treatment. 24 women with high preconceptional NK activity, defined as mean plus 1 SD of NK activity of 47 controls, had a significantly higher abortion rate in the next pregnancy than 44 women with normal levels of NK activity (71 vs 20%; relative risk 3.5; 95% CI 1.8-6-5). The preconceptional evaluation of NK activity in women with recurrent miscarriages may thus be predictive of the risk of pregnancy loss at the next conception. Lancet 1995; 345: 1340-42