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POSSIBLE AIRBORNE SPREAD OF SERUM-HEPATITIS VIRUS WITHIN A HÆMODIALYSIS UNIT
849
cells were used. Even more convincing evidence of a role for activated macrophages was the result in experiment 2 (table n), when irradiated donor peritoneal cells were transferred into total body irradiated
Preliminary Communications POSSIBLE AIRBORNE SPREAD OF SERUM-HEPATITIS VIRUS WITHIN A HÆMODIALYSIS UNIT
growth was effectively suppressed in these animals, although tumour inhibition was not achieved in the similar group in experi(500 r) recipients;
tumour
ment 1. It seems, in this system, that
functionally intact in either donor or recipient cell populalymphocytes for tumour are not essential resistance. However, tions the recipients’ lymphocytes may not have been completely eradicated by the irradiation 9; alternatively, some of the numerous soluble lymphocyte factors 12 might play a role in interaction with macrophages. The in-vitro experiment showed that macrophages activated by peptone or parasite engulf and subsequently destroy most tumour cells within 4-6 hours. In this in-vitro culture system all adhering cells seemed to be macrophages, but again the presence of a small proportion of lymphocytes cannot be ruled out. Therefore, at this stage, we have not eliminated the possibility that the activated macrophages act on the tumour cells by cooperation with lymphocytes in a specific immunological event, as postulated by Evans and Alexander.6 We have been unable to detect, however, an antigenic relationship between N. brasiliensis and Walker carcinosarcoma, and have no indication of a possible virus infection in rats which might result in shared antigens with the tumour cells. Thus, because the activated macrophages used in our experiments have a phagocytic activity directed against an apparently antigenically unrelated target (the tumour cells), the mechanism is more likely to be analogous to that described by Mackaness.13 After day 10 of a primary infection with N. brasiliensis, rats have in their serum a factor which enhances tumour growth. In vitro these antisera prevent the engulfment of tumour cells by activated macrophages, irrespective of whether either the tumour cells or the activated macrophages are first mixed with the antiserum and then washed, or whether the antiserum is simply present in the culture medium. The factor in these antisera which prevents engulfment in vitro is heat stable and is found in fractions containing only rat IgG2 and no other serum-proteins. No indication for the antigenic specificity of this immunoglobulin fraction has been detected, so it may either contain an antibody, or it may bind to immunoglobulin-receptor sites on the activated macrophages. If this second hypothesis were correct, the IgG2 would prevent the receptors on macrophages from reacting with antibody on the tumour cells and engulfing them. 3
This work was supported by the Swiss National Foundation for Scientific Research (grant 5200.3).
G. D. CHISHOLM J. D. ALMEIDA A. E. KULATILAKE A. B. MACGREGOR D. H. MACKAY E. P. N. O’DONOGHUE R. SHACKMAN A. P. WATERSON Hammersmith Hospital, London W.12 An outbreak of Australia (Au) antigenpositive hepatitis in a hæmodialysis unit seems to have been due to a specific incident, when a considerable amount of Au-antigen-positive blood was spilled. If the reasoning presented is correct, then hæmodialysis units will have to take precautions against the airborne spread of the serum-hepatitis virus.
Sum ary
INTRODUCTION
antigen (Au-Ag) positive hepatitis is a well-recognised hazard in hxmodialysis units, 1,2 but we do not know exactly how the virus spreads within a unit once it has gained admittance. We describe here an outbreak of Au-Ag positive hepatitis in the renal dialysis unit, Hammersmith Hospital, which seems to have stemmed from one specific incident-the spilling, in the ward, of contaminated blood. AUSTRALIA
OUTBREAK
October, 1969, the renal dialysis unit had an outbreak Au-Ag positive hepatitis which lasted for 3 months 3; by January, 1970, none of the patients or staff could be In
of
shown to have retained the antigen. Patients and staff were screened regularly, and the unit remained clear until May 21, 1971, when a 46-year-old female patient from Guyana was reported to be Au positive. 2 days before this finding this patient had had a clotted venous line while on dialysis, and in coping with this emergency a lot of blood had been spilled. The patient died on June 10 from uraemia and because of lack of further sites for access to the circulation. On July 30, 10 weeks after the blood had been spilled, two patients were found to be Au-Ag positive, and in the next 2 weeks three further patients and a member of the staff also became Au-Ag positive. No further positives have occurred since then, so that there remains a cluster of six cases within a 2-week period. On reviewing the dialysis records, we found that four of the positive patients were regularly dialysed on the same evenings with the Guyanan patient. However, on the night of the emergency all five of the subsequently positive patients were present, and this was the only night when they were all dialysed together. The affected member of staff was also on duty that night, and in fact dealt with the emergency.
Requests for reprints should be addressed to R. K., ImmunoResearch Group, Schonleinstrasse 22, CH-8032 Zurich,
biology
Switzerland. REFERENCES 1. 2. 3. 4. 5.
Yashphe, D. J. Israel J. med. Sci. 1971, 7, 90. Hellström, K. E., Hellström, I. Adv. Cancer Res. 1969, 12, 167. Keller, R., Ogilvie, B. M., Simpson, E. Lancet, 1971, i, 678. Ogilvie, B. M. Immunology, 1967, 12, 113. Keller, R. Int. Archs Allergy, 1970, 37, 197,
6. Evans, R., Alexander, P. Nature, 1970, 228, 620. 7. Keller, R. Unpublished. 8. Jones, V. E. Immunology, 1969, 16, 589. 9. Uhr, J. W., Finkelstein, M. S. Progr. Allergy, 1967, 10, 37. 10. Perkins, E. H., Nettesheim, P., Morita, T. Reticuloendoth. Soc. 1966, 3, 71. 11. Schrek, R., Elrod, L. M. Radiation Res. 1965, 24, 657. 12. Dumonde, D. C., Maini, R. N. Clin. Allergy, 1971, 1, 123. 13. Mackaness, G. B. J. exp. Med. 1969, 129, 973.
850 The 10 beds in the unit were arranged in two rows of 5, and when the antigen-positive blood was spilled the position was as follows, with the emergency in bed 4:
IMMUNOGLOBULINS ON THE SURFACE OF HUMAN LYMPHOCYTES
M. PAPAMICHAIL J. C. BROWN E. J. HOLBOROW The two remaining patients, shown as negative, have remained negative for Au-Ag, as have sixteen further patients dialysed on different nights of the week.
Summary
DISCUSSION
Au-Ag positive,
or
serum,
infected serum, and in
is spread by transmission is via
hepatitis
most cases
the parenteral route, but it is now accepted that ingestion of infected serum can also transmit the disease.4 Whatever the route, the incubation period of the disease is 50-150 days. In this outbreak, if the six cases of hepatitis within the space of 2 weeks were due to a single incident, only one transfer cycle could have been involved. It is most unlikely that all the patients were transfused with contaminated blood within a short period of time, and the unit has used only Au-Ag screened blood since Feb. 1, 1971. Since the previous outbreak in 1969, all patients on the unit have had their own individual Kiil dialysers, and spread by contaminated equipment is not likely. Because of the close time distribution, and also the close spatial distribution at a time when Au-Ag positive blood was spilled, we suggest that the outbreak was caused by airborne distribution of infected blood. Airborne droplets, containing virus, could be taken up either by ingestion or by a respiratory route-we cannot say which. If we are right, then heemodialysis units will have to take care to prevent the airborne spread of blood or serum droplets, as well as avoiding skin or parenteral contact with potentially infectious blood. This work was supported by a research grant to A. P. W. from the Department of Health and Social Security. J. D. A. is supported by a grant from the Medical Research Council. Requests for reprints should be addressed to J. D. A. REFERENCES 1. London, W. T., DiFiglia, M., Sutnick, A. I., Blumberg, B. S. New Engl. J. Med. 1969, 281, 571. 2. Hawe, B. J., Goldsmith, H. J., Jones, P. O. Br. med. J. 1971, i, 540. 3. Almeida, J. D. Postgrad. med. J. 1971, 47, 484. 4. Krugman, S., Giles, J. P., Hammond, J. J. Am. med. Ass. 1967, 200, 365.
" The introduction of a BILL into our legislature for amending ’ thepoor laws ’ of England, has given rise to a discussion ... The principle which has been advocated in Parliament by the supporters of this measure, is neither more nor less than this,-that the present generation being the owners of the soil, have it in their power to decree... that a portion of the succeeding generation shall be deprived of the means of subsistence. Whether such a horrible principle as this can be worked out into detail, events, possibly of a sanguinary character, will determine... the labouring population should demand a revival of that statute which in olden times required that every landowner should till a third of his estate, or in failure thereof forfeit that third to the public ... It should be remembered that the strength of the labourer is his property, and that whengoaded to desperation, the combined multitude of labourers may feel disposed to inquire, why their wealth is the only property in the state which is denied the protection of the law."-Lancet, 1834, ii, 666 ,
M.R.C. Rheumatism Research Unit, Canadian Red Cross Memorial Hospital, Taplow, Maidenhead, Berkshire
By a membrane staining technique, fluorescein-conjugated antisera to im-
munoglobulins were reacted with purified human blood lymphocyte suspensions. A proportion of peripheral-blood and lymph-node lymphocytes were shown to have immunoglobulins on their surfaces. The clinical observations suggest that they belong to the bonemarrow-derived " B "-cell population. Patients with active rheumatoid arthritis show an increased proportion in their peripheral blood, and the lymphocytes from three patients with chronic lymphocytic leukæmia had both IgG and IgM on their surfaces. INTRODUCTION
RECENT evidence suggests the existence of a distinct subpopulation of lymphocytes bearing immunoglobulins on their surfaces. 1-7 Most of this evidence stems from animal experiments which identify such immunoglobulin-bearing cells as " marrowed-derived" bursaequivalent " " B " cells. Whether they are antigenreactive cells, plasma-cell precursors, or circulating memory cells remains an open question. We describe here the presence and numbers of lymphocytes bearing immunoglobulins on their surfaces in human blood and lymphoid tissue. MATERIALS AND METHODS
Peripheral-blood Lymphocytes Laboratory staff, newborn cord-blood, and fetal blood were the source of normal human lymphocytes. Lymphocytes from patients with definite active rheumatoid arthritis, none of whom were on steroids or cytotoxic therapy, were also studied, and cells from 3 patients with chronic lymphocytic leukaemia, 3 with multiple myeloma, and 1 with acquired primary hypogammaglobulinsemia were
also included.
Tissue-culture Fluid Medium 199 (BDH Chemicals Ltd., Poole, England) with 5% fetal calf-serum was used as washing and diluting fluid throughout the experiments.
Lymphocyte Separation 25 ml. peripheral blood was collected by venepuncture into heparinised plastic tubes. The buffy-coat" leucocyte-rich cell layer was obtained by light centrifugation, layered carefully on to 10 ml. of a mixture of’Ficoll’ (Pharmacia, Sweden) 6% and triosyl 13-3% (’ 75 Glaxo Laboratories, Greenford, England), and centrifuged at 2000 g. for 30 minutes at laboratory temperature. The resultant lymphocyte-rich layer above the interface was aspirated and washed three times in 199. The cells thus obtained consisted of 95% lymphocytes and 5% monocytes with an occasional polymorph. In some cases, contaminating red cells were lysed by exposure to 0-83% ammonium "
chloride for 10 minutes washing twice in 199.
at room
This
temperature followed by the
procedure facilitated
cells were used. Even more convincing evidence of a role for activated macrophages was the result in experiment 2 (table n), when irradiated donor peritoneal cells were transferred into total body irradiated
Preliminary Communications POSSIBLE AIRBORNE SPREAD OF SERUM-HEPATITIS VIRUS WITHIN A HÆMODIALYSIS UNIT
growth was effectively suppressed in these animals, although tumour inhibition was not achieved in the similar group in experi(500 r) recipients;
tumour
ment 1. It seems, in this system, that
functionally intact in either donor or recipient cell populalymphocytes for tumour are not essential resistance. However, tions the recipients’ lymphocytes may not have been completely eradicated by the irradiation 9; alternatively, some of the numerous soluble lymphocyte factors 12 might play a role in interaction with macrophages. The in-vitro experiment showed that macrophages activated by peptone or parasite engulf and subsequently destroy most tumour cells within 4-6 hours. In this in-vitro culture system all adhering cells seemed to be macrophages, but again the presence of a small proportion of lymphocytes cannot be ruled out. Therefore, at this stage, we have not eliminated the possibility that the activated macrophages act on the tumour cells by cooperation with lymphocytes in a specific immunological event, as postulated by Evans and Alexander.6 We have been unable to detect, however, an antigenic relationship between N. brasiliensis and Walker carcinosarcoma, and have no indication of a possible virus infection in rats which might result in shared antigens with the tumour cells. Thus, because the activated macrophages used in our experiments have a phagocytic activity directed against an apparently antigenically unrelated target (the tumour cells), the mechanism is more likely to be analogous to that described by Mackaness.13 After day 10 of a primary infection with N. brasiliensis, rats have in their serum a factor which enhances tumour growth. In vitro these antisera prevent the engulfment of tumour cells by activated macrophages, irrespective of whether either the tumour cells or the activated macrophages are first mixed with the antiserum and then washed, or whether the antiserum is simply present in the culture medium. The factor in these antisera which prevents engulfment in vitro is heat stable and is found in fractions containing only rat IgG2 and no other serum-proteins. No indication for the antigenic specificity of this immunoglobulin fraction has been detected, so it may either contain an antibody, or it may bind to immunoglobulin-receptor sites on the activated macrophages. If this second hypothesis were correct, the IgG2 would prevent the receptors on macrophages from reacting with antibody on the tumour cells and engulfing them. 3
This work was supported by the Swiss National Foundation for Scientific Research (grant 5200.3).
G. D. CHISHOLM J. D. ALMEIDA A. E. KULATILAKE A. B. MACGREGOR D. H. MACKAY E. P. N. O’DONOGHUE R. SHACKMAN A. P. WATERSON Hammersmith Hospital, London W.12 An outbreak of Australia (Au) antigenpositive hepatitis in a hæmodialysis unit seems to have been due to a specific incident, when a considerable amount of Au-antigen-positive blood was spilled. If the reasoning presented is correct, then hæmodialysis units will have to take precautions against the airborne spread of the serum-hepatitis virus.
Sum ary
INTRODUCTION
antigen (Au-Ag) positive hepatitis is a well-recognised hazard in hxmodialysis units, 1,2 but we do not know exactly how the virus spreads within a unit once it has gained admittance. We describe here an outbreak of Au-Ag positive hepatitis in the renal dialysis unit, Hammersmith Hospital, which seems to have stemmed from one specific incident-the spilling, in the ward, of contaminated blood. AUSTRALIA
OUTBREAK
October, 1969, the renal dialysis unit had an outbreak Au-Ag positive hepatitis which lasted for 3 months 3; by January, 1970, none of the patients or staff could be In
of
shown to have retained the antigen. Patients and staff were screened regularly, and the unit remained clear until May 21, 1971, when a 46-year-old female patient from Guyana was reported to be Au positive. 2 days before this finding this patient had had a clotted venous line while on dialysis, and in coping with this emergency a lot of blood had been spilled. The patient died on June 10 from uraemia and because of lack of further sites for access to the circulation. On July 30, 10 weeks after the blood had been spilled, two patients were found to be Au-Ag positive, and in the next 2 weeks three further patients and a member of the staff also became Au-Ag positive. No further positives have occurred since then, so that there remains a cluster of six cases within a 2-week period. On reviewing the dialysis records, we found that four of the positive patients were regularly dialysed on the same evenings with the Guyanan patient. However, on the night of the emergency all five of the subsequently positive patients were present, and this was the only night when they were all dialysed together. The affected member of staff was also on duty that night, and in fact dealt with the emergency.
Requests for reprints should be addressed to R. K., ImmunoResearch Group, Schonleinstrasse 22, CH-8032 Zurich,
biology
Switzerland. REFERENCES 1. 2. 3. 4. 5.
Yashphe, D. J. Israel J. med. Sci. 1971, 7, 90. Hellström, K. E., Hellström, I. Adv. Cancer Res. 1969, 12, 167. Keller, R., Ogilvie, B. M., Simpson, E. Lancet, 1971, i, 678. Ogilvie, B. M. Immunology, 1967, 12, 113. Keller, R. Int. Archs Allergy, 1970, 37, 197,
6. Evans, R., Alexander, P. Nature, 1970, 228, 620. 7. Keller, R. Unpublished. 8. Jones, V. E. Immunology, 1969, 16, 589. 9. Uhr, J. W., Finkelstein, M. S. Progr. Allergy, 1967, 10, 37. 10. Perkins, E. H., Nettesheim, P., Morita, T. Reticuloendoth. Soc. 1966, 3, 71. 11. Schrek, R., Elrod, L. M. Radiation Res. 1965, 24, 657. 12. Dumonde, D. C., Maini, R. N. Clin. Allergy, 1971, 1, 123. 13. Mackaness, G. B. J. exp. Med. 1969, 129, 973.
850 The 10 beds in the unit were arranged in two rows of 5, and when the antigen-positive blood was spilled the position was as follows, with the emergency in bed 4:
IMMUNOGLOBULINS ON THE SURFACE OF HUMAN LYMPHOCYTES
M. PAPAMICHAIL J. C. BROWN E. J. HOLBOROW The two remaining patients, shown as negative, have remained negative for Au-Ag, as have sixteen further patients dialysed on different nights of the week.
Summary
DISCUSSION
Au-Ag positive,
or
serum,
infected serum, and in
is spread by transmission is via
hepatitis
most cases
the parenteral route, but it is now accepted that ingestion of infected serum can also transmit the disease.4 Whatever the route, the incubation period of the disease is 50-150 days. In this outbreak, if the six cases of hepatitis within the space of 2 weeks were due to a single incident, only one transfer cycle could have been involved. It is most unlikely that all the patients were transfused with contaminated blood within a short period of time, and the unit has used only Au-Ag screened blood since Feb. 1, 1971. Since the previous outbreak in 1969, all patients on the unit have had their own individual Kiil dialysers, and spread by contaminated equipment is not likely. Because of the close time distribution, and also the close spatial distribution at a time when Au-Ag positive blood was spilled, we suggest that the outbreak was caused by airborne distribution of infected blood. Airborne droplets, containing virus, could be taken up either by ingestion or by a respiratory route-we cannot say which. If we are right, then heemodialysis units will have to take care to prevent the airborne spread of blood or serum droplets, as well as avoiding skin or parenteral contact with potentially infectious blood. This work was supported by a research grant to A. P. W. from the Department of Health and Social Security. J. D. A. is supported by a grant from the Medical Research Council. Requests for reprints should be addressed to J. D. A. REFERENCES 1. London, W. T., DiFiglia, M., Sutnick, A. I., Blumberg, B. S. New Engl. J. Med. 1969, 281, 571. 2. Hawe, B. J., Goldsmith, H. J., Jones, P. O. Br. med. J. 1971, i, 540. 3. Almeida, J. D. Postgrad. med. J. 1971, 47, 484. 4. Krugman, S., Giles, J. P., Hammond, J. J. Am. med. Ass. 1967, 200, 365.
" The introduction of a BILL into our legislature for amending ’ thepoor laws ’ of England, has given rise to a discussion ... The principle which has been advocated in Parliament by the supporters of this measure, is neither more nor less than this,-that the present generation being the owners of the soil, have it in their power to decree... that a portion of the succeeding generation shall be deprived of the means of subsistence. Whether such a horrible principle as this can be worked out into detail, events, possibly of a sanguinary character, will determine... the labouring population should demand a revival of that statute which in olden times required that every landowner should till a third of his estate, or in failure thereof forfeit that third to the public ... It should be remembered that the strength of the labourer is his property, and that whengoaded to desperation, the combined multitude of labourers may feel disposed to inquire, why their wealth is the only property in the state which is denied the protection of the law."-Lancet, 1834, ii, 666 ,
M.R.C. Rheumatism Research Unit, Canadian Red Cross Memorial Hospital, Taplow, Maidenhead, Berkshire
By a membrane staining technique, fluorescein-conjugated antisera to im-
munoglobulins were reacted with purified human blood lymphocyte suspensions. A proportion of peripheral-blood and lymph-node lymphocytes were shown to have immunoglobulins on their surfaces. The clinical observations suggest that they belong to the bonemarrow-derived " B "-cell population. Patients with active rheumatoid arthritis show an increased proportion in their peripheral blood, and the lymphocytes from three patients with chronic lymphocytic leukæmia had both IgG and IgM on their surfaces. INTRODUCTION
RECENT evidence suggests the existence of a distinct subpopulation of lymphocytes bearing immunoglobulins on their surfaces. 1-7 Most of this evidence stems from animal experiments which identify such immunoglobulin-bearing cells as " marrowed-derived" bursaequivalent " " B " cells. Whether they are antigenreactive cells, plasma-cell precursors, or circulating memory cells remains an open question. We describe here the presence and numbers of lymphocytes bearing immunoglobulins on their surfaces in human blood and lymphoid tissue. MATERIALS AND METHODS
Peripheral-blood Lymphocytes Laboratory staff, newborn cord-blood, and fetal blood were the source of normal human lymphocytes. Lymphocytes from patients with definite active rheumatoid arthritis, none of whom were on steroids or cytotoxic therapy, were also studied, and cells from 3 patients with chronic lymphocytic leukaemia, 3 with multiple myeloma, and 1 with acquired primary hypogammaglobulinsemia were
also included.
Tissue-culture Fluid Medium 199 (BDH Chemicals Ltd., Poole, England) with 5% fetal calf-serum was used as washing and diluting fluid throughout the experiments.
Lymphocyte Separation 25 ml. peripheral blood was collected by venepuncture into heparinised plastic tubes. The buffy-coat" leucocyte-rich cell layer was obtained by light centrifugation, layered carefully on to 10 ml. of a mixture of’Ficoll’ (Pharmacia, Sweden) 6% and triosyl 13-3% (’ 75 Glaxo Laboratories, Greenford, England), and centrifuged at 2000 g. for 30 minutes at laboratory temperature. The resultant lymphocyte-rich layer above the interface was aspirated and washed three times in 199. The cells thus obtained consisted of 95% lymphocytes and 5% monocytes with an occasional polymorph. In some cases, contaminating red cells were lysed by exposure to 0-83% ammonium "
chloride for 10 minutes washing twice in 199.
at room
This
temperature followed by the
procedure facilitated