1067
We agree with Goesch and colleagues about the risk of transfer of P carinii between immunosuppressed patients. Moreover, there is laboratory evidence to support such transfer.4 Despite the lack of official guidelines5 patients with active PCP should be isolated. TH. BENSOUSAN B. GARO S. ISLAM B. BOURBIGOT J. CLEDES M. GALE
Departments of Intensive Care and Infectious Diseases and Nephrology and Renal Transplantation, University Hospital of Brest, 29285 Brest, France
1.Gajdusek
DC.
Pneumocystis carinii: aetiologic agent with interstitial plasma cell
pneumonia of premature and young infants. Pediatrics 1957; 19: 543-65. 2. Hardy
AP, Wajszczuk CP, Suffredini AF, Hakala TR, Monto HO. Pneumocystis m renal transplant recipients treated with cyclosporine and
carinii pneumonia
steroids. J Infect Dis 1984; 149: 143-47. 3. Ferec C, Bourbigot B, Verlingue C, et al. Circadian variation of lymphocyte subsets after renal transplantation. Transplant Proc 1986; 18: 1308-10. 4. Hughes WT. Natural mode of acquisition for de novo infection with Pneumocystis carinii. J Infect Dis 1982; 145: 842-48. 5. Walzer PD. Pneumocystis carinii: principles and practice of infectious diseases. New York: Churchill Livingstone, 1990: 2103-10.
Infection of human T cells with mycoplasma, inhibition of CD4 expression and HIV-1 gp120 glycoprotein binding, and
infectivity SIR,-Myocoplasma species have been shown to influence HIV-1I growth in human T-cell lines. Introduction of mycoplasma to cultures depresses HIV-1 replication as measured by reverse transcriptase (RT) activity, and treatment of HIV-1 infected T cells with antimycoplasma tetracyclines causes an increase in RT activity.1,2 Mycoplasmas are known to be mitogenic for human T and B cells,3.4 and killed mycoplasma can stimulate HIV-1 p24 antigen production in T-cell lines.s We have shown that a glucose-fermenting mycoplasma, isolated from lymphoid cells of an HIV-1 infected patient with AIDS-related complex, affects the HIV-1 envelope receptor CD46’ expression by T cells. Infection of the human T-cell line CEM8 with this organism led to a specific reduction in the expression of CD4, accompanied by a decrease in binding of the HIV-1 envelope protein gpl20. CEM cells were infected with mycoplasma and observed for 3 weeks during which time mycoplasma infection was monitored by electronmicroscopy and by DNA staining.9 Cell surface expression of CD4 and gp 120 binding were first examined 3 days after mycoplasma infection. Cells were reacted with monoclonal antibodies to CD4 (Leu 3a/3b) or CD5 (Leu 1), a cell surface molecule on CEM unrelated to HIV binding, and compared with uninfected CEM. The expression of each surface antigen was tested in quadruplicate and then analysed in triplicate by flow cytometry (’Facscan’, Becton Dickinson). CD4
and CD5were measured as the median channel of the fluorescence histograms. Differences between these values were assessed with Student’s t-test. To measure gpl20 binding, CEM cells with or without mycoplasma infection were reacted with recombinant gp 120 produced in CHO cells (kindly provided by the MRC AIDS Directed Programme) for 1 h at 37°C. The cells were then incubated with a monoclonal antibody to gpl20, followed by a fluorescein conjugated rabbit antibody to mouse immunoglobulin. The cells were then analysed by flow cytometry. Mycoplasma infection of the CEM line produced a significant reduction in cell surface expression of CD4 (p < 0001); in contrast CD5 values did not change. The inhibition of CD4 expression was accompanied by a 2-log-fold reduction in the binding of gpl20 compared with that on cells lacking mycoplasma infection (figure). These findings remained consistent for 3 weeks. Infection by HIV-1(London Hospital strain X82) was compared in CEM cells with and without mycoplasma infection by means of the Coulter HIV-1p24 antigen assay. Antigen production was reduced by 50% in the presence of mycoplasma at days 6 and 12 after HIV-1infection. Our results show that this mycoplasma significantly reduced CD4 expression with consequent inhibition of gpl20 binding and HIV infection of CEM cells. Together with previously reported data this suggests that mycoplasmas can affect several stages in the life cycle of HIV-1. Departments of Medical Microbiology and Immunology, London Hospital Medical College, London E1 2AD, UK
C. O’TOOLE M. LOWDELL
1. Vasudevachari MB, Mast TC, Saizman NP. Suppression of HIV-1 reverse transcriptase activity by myocoplasma contamination of cell cultures. AIDS Res Hum Retrovirus 1990; 6: 411-16. 2. Lemaître M, Guétard D, Hénin Y, et al. Protective activity of tetracycline analogs against the cytopathic effect of the human immunodeficiency viruses in CEM cells. Res Virol 1990; 141: 5-16. 3. Biberfeld G, Nilsson E. Mitogenicity of Mycoplasma fermentans for human
lymphocytes. Inf Immun 1978; 21: 48-54 H, Brunner H, Ruhl H. Effect of A laidlawii on murine and human lymphocyte cultures. Clin Exp Immunol 1977; 29: 176-80. 5. Chowdhury MIH, Koyanagi Y, Kobayashi S, et al. Mycoplasma and AIDS. Lancet
4. Kirchner
1990; 336: 247-48. 6. Klatzmann D, Champagne E, Chamaret S, et al. T lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature 1984; 312: 767-68. 7. Dalgleish AG, Beverley PCL, Clapham PR, et al. The CD4(T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 1984; 312: 763-67. 8. Folley GE, Lazarus H, Faber S, et al. Continuous culture of human lymphoblasts from peripheral blood of a child with acute leukaemia. Cancer 1965; 18: 522-29. 9. Russell WC, Newman C, Williamson DH. A simple cytochemical technique for demonstration of DNA in cells infected with mycoplasmas and viruses. Nature 1975; 253: 461-62.
Cyclophosphamide versus ifosfamide in paediatric oncology SIR,-Dr Kushner and Dr Cheung (July 28, p 253) comment on the cyclophosphamide and ifosfamide in paediatric oncology. We
use of
Mycoplasma infected CEM (C).
at
day 3 (A, B) and uninfected CEM
A, stained with monoclonal antibody
to
gp120 (Du Pont) and
FITC-conjugated rabbit antibody to mouse immunoglobulms (Dako). and C, mcubated with
gp120 and
stained
as
in
(A).
B
report similar results from our group. On the basis of previous experience with an intensive cyclophosphamide regimen in the treatment of childhood brain tumours,l leading to great efficacy and to only the expected haematotoxicity, we conducted a phase-11 trial testing the efficacy of ifosfamide.2Fifty-four children received 179 courses of ifosfamide at a dose of 3 g/m2 daily over 3 h for 2 days for recurrent or refractory brain tumours. Haematotoxicity was mild, but 30% of children had neurotoxicity as has been reported for combined ifosfamide and etoposide.3 However, Miser et aP used a different dose and schedule of ifosfamide (1-8 g/m2 daily for 5 days) and neurotoxicity seemed to be less severe than in our patients. Nephrotoxicity seemed also to be a limiting factor in some of our patients. Because of this toxicity and to confirm the efficacy of cyclophosphamide we initiated a trial of cyclophosphamide with etoposide for relapsing or refractory brain tumours and for newly diagnosed brain tumours, alternating with carboplatin/etoposide: two courses of each combination were given before radiotherapy. So far, about forty courses of cyclophosphamide (15 g/m) have been given without any neurotoxicity or nephrotoxicity. Grade 3-4 neutropenia was observed in nearly all patients who had been
We agree with Goesch and colleagues about the risk of transfer of P carinii between immunosuppressed patients. Moreover, there is laboratory evidence to support such transfer.4 Despite the lack of official guidelines5 patients with active PCP should be isolated. TH. BENSOUSAN B. GARO S. ISLAM B. BOURBIGOT J. CLEDES M. GALE
Departments of Intensive Care and Infectious Diseases and Nephrology and Renal Transplantation, University Hospital of Brest, 29285 Brest, France
1.Gajdusek
DC.
Pneumocystis carinii: aetiologic agent with interstitial plasma cell
pneumonia of premature and young infants. Pediatrics 1957; 19: 543-65. 2. Hardy
AP, Wajszczuk CP, Suffredini AF, Hakala TR, Monto HO. Pneumocystis m renal transplant recipients treated with cyclosporine and
carinii pneumonia
steroids. J Infect Dis 1984; 149: 143-47. 3. Ferec C, Bourbigot B, Verlingue C, et al. Circadian variation of lymphocyte subsets after renal transplantation. Transplant Proc 1986; 18: 1308-10. 4. Hughes WT. Natural mode of acquisition for de novo infection with Pneumocystis carinii. J Infect Dis 1982; 145: 842-48. 5. Walzer PD. Pneumocystis carinii: principles and practice of infectious diseases. New York: Churchill Livingstone, 1990: 2103-10.
Infection of human T cells with mycoplasma, inhibition of CD4 expression and HIV-1 gp120 glycoprotein binding, and
infectivity SIR,-Myocoplasma species have been shown to influence HIV-1I growth in human T-cell lines. Introduction of mycoplasma to cultures depresses HIV-1 replication as measured by reverse transcriptase (RT) activity, and treatment of HIV-1 infected T cells with antimycoplasma tetracyclines causes an increase in RT activity.1,2 Mycoplasmas are known to be mitogenic for human T and B cells,3.4 and killed mycoplasma can stimulate HIV-1 p24 antigen production in T-cell lines.s We have shown that a glucose-fermenting mycoplasma, isolated from lymphoid cells of an HIV-1 infected patient with AIDS-related complex, affects the HIV-1 envelope receptor CD46’ expression by T cells. Infection of the human T-cell line CEM8 with this organism led to a specific reduction in the expression of CD4, accompanied by a decrease in binding of the HIV-1 envelope protein gpl20. CEM cells were infected with mycoplasma and observed for 3 weeks during which time mycoplasma infection was monitored by electronmicroscopy and by DNA staining.9 Cell surface expression of CD4 and gp 120 binding were first examined 3 days after mycoplasma infection. Cells were reacted with monoclonal antibodies to CD4 (Leu 3a/3b) or CD5 (Leu 1), a cell surface molecule on CEM unrelated to HIV binding, and compared with uninfected CEM. The expression of each surface antigen was tested in quadruplicate and then analysed in triplicate by flow cytometry (’Facscan’, Becton Dickinson). CD4
and CD5were measured as the median channel of the fluorescence histograms. Differences between these values were assessed with Student’s t-test. To measure gpl20 binding, CEM cells with or without mycoplasma infection were reacted with recombinant gp 120 produced in CHO cells (kindly provided by the MRC AIDS Directed Programme) for 1 h at 37°C. The cells were then incubated with a monoclonal antibody to gpl20, followed by a fluorescein conjugated rabbit antibody to mouse immunoglobulin. The cells were then analysed by flow cytometry. Mycoplasma infection of the CEM line produced a significant reduction in cell surface expression of CD4 (p < 0001); in contrast CD5 values did not change. The inhibition of CD4 expression was accompanied by a 2-log-fold reduction in the binding of gpl20 compared with that on cells lacking mycoplasma infection (figure). These findings remained consistent for 3 weeks. Infection by HIV-1(London Hospital strain X82) was compared in CEM cells with and without mycoplasma infection by means of the Coulter HIV-1p24 antigen assay. Antigen production was reduced by 50% in the presence of mycoplasma at days 6 and 12 after HIV-1infection. Our results show that this mycoplasma significantly reduced CD4 expression with consequent inhibition of gpl20 binding and HIV infection of CEM cells. Together with previously reported data this suggests that mycoplasmas can affect several stages in the life cycle of HIV-1. Departments of Medical Microbiology and Immunology, London Hospital Medical College, London E1 2AD, UK
C. O’TOOLE M. LOWDELL
1. Vasudevachari MB, Mast TC, Saizman NP. Suppression of HIV-1 reverse transcriptase activity by myocoplasma contamination of cell cultures. AIDS Res Hum Retrovirus 1990; 6: 411-16. 2. Lemaître M, Guétard D, Hénin Y, et al. Protective activity of tetracycline analogs against the cytopathic effect of the human immunodeficiency viruses in CEM cells. Res Virol 1990; 141: 5-16. 3. Biberfeld G, Nilsson E. Mitogenicity of Mycoplasma fermentans for human
lymphocytes. Inf Immun 1978; 21: 48-54 H, Brunner H, Ruhl H. Effect of A laidlawii on murine and human lymphocyte cultures. Clin Exp Immunol 1977; 29: 176-80. 5. Chowdhury MIH, Koyanagi Y, Kobayashi S, et al. Mycoplasma and AIDS. Lancet
4. Kirchner
1990; 336: 247-48. 6. Klatzmann D, Champagne E, Chamaret S, et al. T lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature 1984; 312: 767-68. 7. Dalgleish AG, Beverley PCL, Clapham PR, et al. The CD4(T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 1984; 312: 763-67. 8. Folley GE, Lazarus H, Faber S, et al. Continuous culture of human lymphoblasts from peripheral blood of a child with acute leukaemia. Cancer 1965; 18: 522-29. 9. Russell WC, Newman C, Williamson DH. A simple cytochemical technique for demonstration of DNA in cells infected with mycoplasmas and viruses. Nature 1975; 253: 461-62.
Cyclophosphamide versus ifosfamide in paediatric oncology SIR,-Dr Kushner and Dr Cheung (July 28, p 253) comment on the cyclophosphamide and ifosfamide in paediatric oncology. We
use of
Mycoplasma infected CEM (C).
at
day 3 (A, B) and uninfected CEM
A, stained with monoclonal antibody
to
gp120 (Du Pont) and
FITC-conjugated rabbit antibody to mouse immunoglobulms (Dako). and C, mcubated with
gp120 and
stained
as
in
(A).
B
report similar results from our group. On the basis of previous experience with an intensive cyclophosphamide regimen in the treatment of childhood brain tumours,l leading to great efficacy and to only the expected haematotoxicity, we conducted a phase-11 trial testing the efficacy of ifosfamide.2Fifty-four children received 179 courses of ifosfamide at a dose of 3 g/m2 daily over 3 h for 2 days for recurrent or refractory brain tumours. Haematotoxicity was mild, but 30% of children had neurotoxicity as has been reported for combined ifosfamide and etoposide.3 However, Miser et aP used a different dose and schedule of ifosfamide (1-8 g/m2 daily for 5 days) and neurotoxicity seemed to be less severe than in our patients. Nephrotoxicity seemed also to be a limiting factor in some of our patients. Because of this toxicity and to confirm the efficacy of cyclophosphamide we initiated a trial of cyclophosphamide with etoposide for relapsing or refractory brain tumours and for newly diagnosed brain tumours, alternating with carboplatin/etoposide: two courses of each combination were given before radiotherapy. So far, about forty courses of cyclophosphamide (15 g/m) have been given without any neurotoxicity or nephrotoxicity. Grade 3-4 neutropenia was observed in nearly all patients who had been
