- Email: [email protected]
PARTICLES ASSOCIATED WITH MICROVILLOUS BORDER OF INTESTINAL MUCOSA
931 intention of questioning the underlying principle or the validity of the technique. We merely wish to place on record that we disapprove of the practice of publishing results before was no
publishing the validation
of the’method used
to
obtain them.
University Department
of Physiology,
J. S. DAVISON
Dundee DD1 4HN.
Faculty of Medicine, University of Calgary, Alberta, Canada.
D. M. HAY
DOES NITROUS OXIDE HARM THE DENTIST ?
SIR,-Ever since Bruce and his co-workerssuggested link between the pollution of operating-theatres with anxsthetic gases and causes of death in anxsthetists there has been concern over the problem. Halothane has been the main object of this concern but nitrous oxide is not innocuous. Lassen and his colleagueshave reported severe bone-marrow depression after prolonged nitrousoxide anaesthesia, and the gas has also been shown to be teratogenic under experimental conditions. 3-5 This latter effect may be associated with an increased spontaneousabortion rate in female doctors and nurses.6 Pollution of the dental surgery with anxsthetic gases is also common. Levels of halothane have been found to be high,’ and a were highest in the region of the dental surgeon’s face." there is an in the use of e nitrous oxide Today upsurge as a means of sedation in dentistry. It appears safe for the patient who inhales it for a relatively short time. But is it safe for the dentist and his staff who may inhale it over a long period ?? As part of our investigation of the problem we have exposed albino rats to a 1 % level of nitrous oxide for six hours per day, five days per week. This pollution level is equivalent to that reported in a dental surgery by Millard and Corbett,9 and the exposure-time fairly closely mimics a typical dentist’s day when using nitrous-oxide/oxygen sedation (relative analgesia). After nine weeks’ exposure no difference was found between the hsmatological appearances of the peripheral blood in experimental and control animals. However, after five weeks’ exposure to nitrous oxide there was a marked increase in the numbers of mast cells, particularly in the bone-marrow where they averaged 10 per oil-immersion field. The exact role of the mast cells in this experimental situation is not yet clear, but their presence does suggest that the bone-marrow is under stress. 10 Further experiments are under way and full details will be published
a
PARTICLES ASSOCIATED WITH MICROVILLOUS BORDER OF INTESTINAL MUCOSA
SIR,-Electron-microscope observations often form part of studies on viral and other infections of the intestine, in both man and animals. Micrographs of the apical border of normal intestinal epithelial cells commonly depict a regular array of intact microvilli of equal length, while pathological change in an epithelial cell often features the loss of many microvilli, the remainder being of irregular distribution and length. An electron-microscope study of human jejunal biopsies by Dr Tomkins and others (July 5, p. 36) showed particles resembling oncornavirus, arenavirus, and mycoplasma. One of their micrographs (their fig. 3), in addition to demonstrating mycoplasma-like particles, showed particles with a circular profile aligned in the spaces between microvilli. We have observed similar particles in intestinal samples from pigs. They resemble viral particles in size and appearance and, in addition to their intrinsic interest, their differentiation from known infectious agents needs to be taken into consideration. The
specimens
were
fixed in
glutaraldehyde, post-fixed
tetroxide, and embedded in ’Araldite’; ultrathin
in osmium
stained with uranyl acetate and lead citrate and examined in a Philips EM 300 electron microscope. In each specimen the particles were seen associated with a minority of cells. Fig. 1 shows particles associated with an epithelial cell with rather dense cytoplasm. The microvilli appear to be pinched and beaded in such a way that the particles may be derived from the beads. Higher magnification (fig, 2) shows that the particles have a unit membrane and diameter of 60-75 nm. The particles were seen in the duodenal, Iejunal, and ileal portions of the small intestine of a 9-day-old gnotobiotic piglet and m the jejunal and ileal portions of the intestine of 3 siblings similarlv reared but experimentally infected with neonatal-calf-diarrhoea reovirus-like agent (rotavirus). The particles were not seen in a further umnfected sibling or in 3 further rotavirus-tnfected siblings. The particles were also seen in a mucosal sample obtained from a 1-week-old gnotobiotic pig and maintained for 30 minutes in organ culture in the presence of Escherichia. coli. They were not seen in a sample from the same pig maintained without E. coli. Neither were they seen in samples, maintained in organ culture with or without E. coli, from 3 other pigs. A search made on a further uninoculated gnotobiotic pig, on 2 gnotobiotic pigs inoculated with transmissible gastroenteritis virus, and on 4 pigs inoculated with a rotavirus and an enterovirus did not demonstrate the particles. sections were
These results show that the particles can be found in gnotobiotic pigs, whether or not these pigs have been experimentally infected. They could be differentiated from the virus particles
elsewhere. Dental Research Unit, South African Medical Research Council and University of the Witwatersrand, Jan Smuts Avenue,
Johannesburg 2001, South Africa.
P. CLEATON-JONES J. C. AUSTIN D. BANKS E. VIEIRA.
National Research Institute for
Occupational Diseases, South African Medical Research
Council, Civic Centre,
Johannesburg 2001, South Africa.
E. KAGAN.
Bruce, D. L., Eide, K. A., Linde, H. W., Eckinhoff, J. E. Anesthesiology, 1968, 29, 565. 2. Lassen, H. C. A., Henrickson, E., Neukirch, F., Kirstensen, H. S. Lancet, 1956, i, 527. 3. Rector, G. H. M., Eastwood, D. N. Anesthesiology, 1964, 25, 109. 4 Smith, B. E., Gaub, M. L., Moya, F. Anesth. Analg. 1965, 44, 726. 5. Fink, B. R., Shepard, T. H., Blandau, R. J. Nature, 1967, 214, 146. 6 Vaisman, A. I. Éksp. Khir. Anest. 1967, 3, 44. 7. Strunin, L., Strunin, J. M., Mallios, C. C. Br. med. J. 1973, iv, 459. 8. Mallios, C. C., Strunin, J. M., Strunin, L. Br. dent. J. 1974, 136, 1.
449.
Millard, R. I., Corbett, T. H. J. Oral Surg. 1974, 32, 593. 10. Selye, H. The Mast Cells; p. 403, Washington, 1965. 9.
Fig. 1-Apical surface of mucosal cell. Particles with a circular profile associated with microvilli having a beaded appearance.
are
932 COMPLEMENT VALUES IN THREE GROUPS
lower than that in the controls (85 mg/100 ml) we found 18 M.S. patients with C3 level below 65 mg/100 ml (lowest value in our control group). The frequency of low C3 in the M.S. group (29 5%) is significantly higher than that in the normal control group (0%) and in the o.N.D. group (96%;
significantly
U=1545). Yet
x2=15-45; p<0-01). Hypocomplementaemia was confirmed by decreased levels of CH 50 (below 40 units CH 50) in 60% of patients
Fig. 2-High-power
view of microvilli,
showing particles with enveloping
membranes.
in this low-C3 group. Serial studies in 13 hypocomplementaemic patients showed persistently low C3 in 8 cases during several months and variable levels of C3 (low or normal) in 5 patients, not clearly related to attacks. Investigation of C4 and properdin factor B (B) in hypocomplementaemic M.S. showed a marked decrease of B level in 59% (11 cases), while except for 2 cases C4 was normal. These results indicate that about 30% of all M.s. patients have either a ersistent or a fluctuating hypocomplementaemia. This phenomenon has hitherto been overlooked because the mean values recorded masked the prevalence of these low values and serial studies were not ,
also seen in the specimens from infected pigs. They found in all pigs examined, and this suggests that their distribution is limited. Their occurrence and appearance suggests that the particles are more likely to arise from an undocumented change in the microvilli than to represent any known infectious agent.
which
were
were not
grateful to Dr S. Bradbury, of the department of human anatomy, Oxford University, for helpful discussion. We also wish to thank Mr R. J. Sellwood for provision of specimens and Mr H. S. Anger for skilled technical assistance. We
are
Institute for Research
on
Animal Diseases
R. L. CHANDLER R. G. BIRD A. P. BLAND
Compton, Newbury, Berks., and London School of Hygiene and Tropical Medicine.
performed.’.’z Low properdin-factor-B
values with normal C4 values C3 activation suggest mainly through the alternate in pathway hypocomplementsemic M.s. This situation seems to be analogous to the chronic hypocomplementsmic glomerulonephritis described some years ago.6-10 a
Laboratoire de Neuro-
Immunologie, Hôpital Neurologique, 59 Boulevard Pinet 69003
MULTIPLE SCLEROSIS WITH HYPOCOMPLEMENTÆMIA
Lyon; et Clinique de Nephrologie, Hôpital Edouard Herriot, Place d’Arsonval, 69003 Lyon, France.
P. TROUILLAS G. AIMARD F. BERTHOUX M. DEVIC
SIR,-Studies of the complement system in the serum of multiple-sclerosis (M.S.) patients have given conflicting results. Kuwert et al. reported low mean complement component (Cl, C3, C4) activities in acute cases " whilst Link2 found normal C3 and C4 concentrations in both chronic and acute cases. In 61 M.S. patients who were selected according to the criteria of Broman et awl. at random stages of evolution and who had not received steroid therapy for 3 months, we have measured the complement components C3 (PiC), C4, and properdin factor B (B) by radial immunodiffusion
MOLLARET’S MENINGITIS
"
techniques4 (Behringwerke plates). Total complement activity (CH 50) was measured by the method of Mayer.5 Results were compared with those in a control group (52 normals) and in 27 patients with neurological diseases (O.N.D.) other than M.S. The mean values of C3 in M.S. (80 mg/100 ml) are not
SIR,-In response to the letter of Dr Gledhill and his colleagues (Aug. 30, p. 415), I should like to point out that some
meningitis (also known as benign meningitis) recover spontaneously, as happened with patients. The first case was reported in 1943" (before the publication of Mollaret’s paper") and the other in 1950." Both patients are still in good health, having had no
patients
of the disease. deny that Mollaret’s meningitis may be a periodic disease or that colchicine is effective in treatment. Dr Gledhill and his colleagues mentioned the use of colchicine in the treatment of familial Mediterranean fever. I have also treated two patients with this disease with colchicine (I mg per day for six recurrence
I do
not
West, C. D., MacAdams, A. J., MacConville, J. M., Davis, N. C., Holland, D. J. Pediat. 1965, 67, 1089. 7. Gotoff, S. D., Fellers, F. X., Vawter, G. E., Janeway, C. A., Rosen, F. S. New Engl. J. Med. 1965, 273, 524. 8. Ogg, C. S., Cameron, J. S., White, R. H. R. Lancet, 1968, ii, 78. 9. Michael, A. F., Herdman, R. C., Fish, A. J., Pickering, R. J., Vernier, R. L. Transplant. Proc. 1969, 1, 925. 10. MacLean, R. H., Michael, A. F. J. clin. Invest. 1973, 52, 634. 11. Calvo Melendro, J. Revta clin. esp. 1943, 10, 125. 12. Mollaret, M. P. Bull Mém Soc. méd. Hôp. 1944, no. 11 & 12, p. 121 13. Calvo Melendro, J. Presse med. 1950, 55, 952. 6.
Kuwert, E., Noll, K., Firnhaber, W. Z. Immun. Forsch. exp. Ther. 1968, 135, 462. 2. Link, H. Acta neurol. scand. 1972, 48, 521. 3. Broman, T., Bergmann, L., Fog, T., Gilland, T., Hyllested, K., Lindberg-Broman, A. M., Pedersen, E., Presthus, J. ibid. 1965, 41, suppl. 13, p. 543. 4. Mancini, G., Carbonara, A. O., Heremans, J. F. Immunochemistry, 1965, 2, 235. 5. Mayer, M. M. in Experimental Immunochemistry (edited by E. A. Kabat and M. M. Mayer); p. 133. Springfield, I11. 1961. 1.
with Mollaret’s
recurrent two of my
publishing the validation
of the’method used
to
obtain them.
University Department
of Physiology,
J. S. DAVISON
Dundee DD1 4HN.
Faculty of Medicine, University of Calgary, Alberta, Canada.
D. M. HAY
DOES NITROUS OXIDE HARM THE DENTIST ?
SIR,-Ever since Bruce and his co-workerssuggested link between the pollution of operating-theatres with anxsthetic gases and causes of death in anxsthetists there has been concern over the problem. Halothane has been the main object of this concern but nitrous oxide is not innocuous. Lassen and his colleagueshave reported severe bone-marrow depression after prolonged nitrousoxide anaesthesia, and the gas has also been shown to be teratogenic under experimental conditions. 3-5 This latter effect may be associated with an increased spontaneousabortion rate in female doctors and nurses.6 Pollution of the dental surgery with anxsthetic gases is also common. Levels of halothane have been found to be high,’ and a were highest in the region of the dental surgeon’s face." there is an in the use of e nitrous oxide Today upsurge as a means of sedation in dentistry. It appears safe for the patient who inhales it for a relatively short time. But is it safe for the dentist and his staff who may inhale it over a long period ?? As part of our investigation of the problem we have exposed albino rats to a 1 % level of nitrous oxide for six hours per day, five days per week. This pollution level is equivalent to that reported in a dental surgery by Millard and Corbett,9 and the exposure-time fairly closely mimics a typical dentist’s day when using nitrous-oxide/oxygen sedation (relative analgesia). After nine weeks’ exposure no difference was found between the hsmatological appearances of the peripheral blood in experimental and control animals. However, after five weeks’ exposure to nitrous oxide there was a marked increase in the numbers of mast cells, particularly in the bone-marrow where they averaged 10 per oil-immersion field. The exact role of the mast cells in this experimental situation is not yet clear, but their presence does suggest that the bone-marrow is under stress. 10 Further experiments are under way and full details will be published
a
PARTICLES ASSOCIATED WITH MICROVILLOUS BORDER OF INTESTINAL MUCOSA
SIR,-Electron-microscope observations often form part of studies on viral and other infections of the intestine, in both man and animals. Micrographs of the apical border of normal intestinal epithelial cells commonly depict a regular array of intact microvilli of equal length, while pathological change in an epithelial cell often features the loss of many microvilli, the remainder being of irregular distribution and length. An electron-microscope study of human jejunal biopsies by Dr Tomkins and others (July 5, p. 36) showed particles resembling oncornavirus, arenavirus, and mycoplasma. One of their micrographs (their fig. 3), in addition to demonstrating mycoplasma-like particles, showed particles with a circular profile aligned in the spaces between microvilli. We have observed similar particles in intestinal samples from pigs. They resemble viral particles in size and appearance and, in addition to their intrinsic interest, their differentiation from known infectious agents needs to be taken into consideration. The
specimens
were
fixed in
glutaraldehyde, post-fixed
tetroxide, and embedded in ’Araldite’; ultrathin
in osmium
stained with uranyl acetate and lead citrate and examined in a Philips EM 300 electron microscope. In each specimen the particles were seen associated with a minority of cells. Fig. 1 shows particles associated with an epithelial cell with rather dense cytoplasm. The microvilli appear to be pinched and beaded in such a way that the particles may be derived from the beads. Higher magnification (fig, 2) shows that the particles have a unit membrane and diameter of 60-75 nm. The particles were seen in the duodenal, Iejunal, and ileal portions of the small intestine of a 9-day-old gnotobiotic piglet and m the jejunal and ileal portions of the intestine of 3 siblings similarlv reared but experimentally infected with neonatal-calf-diarrhoea reovirus-like agent (rotavirus). The particles were not seen in a further umnfected sibling or in 3 further rotavirus-tnfected siblings. The particles were also seen in a mucosal sample obtained from a 1-week-old gnotobiotic pig and maintained for 30 minutes in organ culture in the presence of Escherichia. coli. They were not seen in a sample from the same pig maintained without E. coli. Neither were they seen in samples, maintained in organ culture with or without E. coli, from 3 other pigs. A search made on a further uninoculated gnotobiotic pig, on 2 gnotobiotic pigs inoculated with transmissible gastroenteritis virus, and on 4 pigs inoculated with a rotavirus and an enterovirus did not demonstrate the particles. sections were
These results show that the particles can be found in gnotobiotic pigs, whether or not these pigs have been experimentally infected. They could be differentiated from the virus particles
elsewhere. Dental Research Unit, South African Medical Research Council and University of the Witwatersrand, Jan Smuts Avenue,
Johannesburg 2001, South Africa.
P. CLEATON-JONES J. C. AUSTIN D. BANKS E. VIEIRA.
National Research Institute for
Occupational Diseases, South African Medical Research
Council, Civic Centre,
Johannesburg 2001, South Africa.
E. KAGAN.
Bruce, D. L., Eide, K. A., Linde, H. W., Eckinhoff, J. E. Anesthesiology, 1968, 29, 565. 2. Lassen, H. C. A., Henrickson, E., Neukirch, F., Kirstensen, H. S. Lancet, 1956, i, 527. 3. Rector, G. H. M., Eastwood, D. N. Anesthesiology, 1964, 25, 109. 4 Smith, B. E., Gaub, M. L., Moya, F. Anesth. Analg. 1965, 44, 726. 5. Fink, B. R., Shepard, T. H., Blandau, R. J. Nature, 1967, 214, 146. 6 Vaisman, A. I. Éksp. Khir. Anest. 1967, 3, 44. 7. Strunin, L., Strunin, J. M., Mallios, C. C. Br. med. J. 1973, iv, 459. 8. Mallios, C. C., Strunin, J. M., Strunin, L. Br. dent. J. 1974, 136, 1.
449.
Millard, R. I., Corbett, T. H. J. Oral Surg. 1974, 32, 593. 10. Selye, H. The Mast Cells; p. 403, Washington, 1965. 9.
Fig. 1-Apical surface of mucosal cell. Particles with a circular profile associated with microvilli having a beaded appearance.
are
932 COMPLEMENT VALUES IN THREE GROUPS
lower than that in the controls (85 mg/100 ml) we found 18 M.S. patients with C3 level below 65 mg/100 ml (lowest value in our control group). The frequency of low C3 in the M.S. group (29 5%) is significantly higher than that in the normal control group (0%) and in the o.N.D. group (96%;
significantly
U=1545). Yet
x2=15-45; p<0-01). Hypocomplementaemia was confirmed by decreased levels of CH 50 (below 40 units CH 50) in 60% of patients
Fig. 2-High-power
view of microvilli,
showing particles with enveloping
membranes.
in this low-C3 group. Serial studies in 13 hypocomplementaemic patients showed persistently low C3 in 8 cases during several months and variable levels of C3 (low or normal) in 5 patients, not clearly related to attacks. Investigation of C4 and properdin factor B (B) in hypocomplementaemic M.S. showed a marked decrease of B level in 59% (11 cases), while except for 2 cases C4 was normal. These results indicate that about 30% of all M.s. patients have either a ersistent or a fluctuating hypocomplementaemia. This phenomenon has hitherto been overlooked because the mean values recorded masked the prevalence of these low values and serial studies were not ,
also seen in the specimens from infected pigs. They found in all pigs examined, and this suggests that their distribution is limited. Their occurrence and appearance suggests that the particles are more likely to arise from an undocumented change in the microvilli than to represent any known infectious agent.
which
were
were not
grateful to Dr S. Bradbury, of the department of human anatomy, Oxford University, for helpful discussion. We also wish to thank Mr R. J. Sellwood for provision of specimens and Mr H. S. Anger for skilled technical assistance. We
are
Institute for Research
on
Animal Diseases
R. L. CHANDLER R. G. BIRD A. P. BLAND
Compton, Newbury, Berks., and London School of Hygiene and Tropical Medicine.
performed.’.’z Low properdin-factor-B
values with normal C4 values C3 activation suggest mainly through the alternate in pathway hypocomplementsemic M.s. This situation seems to be analogous to the chronic hypocomplementsmic glomerulonephritis described some years ago.6-10 a
Laboratoire de Neuro-
Immunologie, Hôpital Neurologique, 59 Boulevard Pinet 69003
MULTIPLE SCLEROSIS WITH HYPOCOMPLEMENTÆMIA
Lyon; et Clinique de Nephrologie, Hôpital Edouard Herriot, Place d’Arsonval, 69003 Lyon, France.
P. TROUILLAS G. AIMARD F. BERTHOUX M. DEVIC
SIR,-Studies of the complement system in the serum of multiple-sclerosis (M.S.) patients have given conflicting results. Kuwert et al. reported low mean complement component (Cl, C3, C4) activities in acute cases " whilst Link2 found normal C3 and C4 concentrations in both chronic and acute cases. In 61 M.S. patients who were selected according to the criteria of Broman et awl. at random stages of evolution and who had not received steroid therapy for 3 months, we have measured the complement components C3 (PiC), C4, and properdin factor B (B) by radial immunodiffusion
MOLLARET’S MENINGITIS
"
techniques4 (Behringwerke plates). Total complement activity (CH 50) was measured by the method of Mayer.5 Results were compared with those in a control group (52 normals) and in 27 patients with neurological diseases (O.N.D.) other than M.S. The mean values of C3 in M.S. (80 mg/100 ml) are not
SIR,-In response to the letter of Dr Gledhill and his colleagues (Aug. 30, p. 415), I should like to point out that some
meningitis (also known as benign meningitis) recover spontaneously, as happened with patients. The first case was reported in 1943" (before the publication of Mollaret’s paper") and the other in 1950." Both patients are still in good health, having had no
patients
of the disease. deny that Mollaret’s meningitis may be a periodic disease or that colchicine is effective in treatment. Dr Gledhill and his colleagues mentioned the use of colchicine in the treatment of familial Mediterranean fever. I have also treated two patients with this disease with colchicine (I mg per day for six recurrence
I do
not
West, C. D., MacAdams, A. J., MacConville, J. M., Davis, N. C., Holland, D. J. Pediat. 1965, 67, 1089. 7. Gotoff, S. D., Fellers, F. X., Vawter, G. E., Janeway, C. A., Rosen, F. S. New Engl. J. Med. 1965, 273, 524. 8. Ogg, C. S., Cameron, J. S., White, R. H. R. Lancet, 1968, ii, 78. 9. Michael, A. F., Herdman, R. C., Fish, A. J., Pickering, R. J., Vernier, R. L. Transplant. Proc. 1969, 1, 925. 10. MacLean, R. H., Michael, A. F. J. clin. Invest. 1973, 52, 634. 11. Calvo Melendro, J. Revta clin. esp. 1943, 10, 125. 12. Mollaret, M. P. Bull Mém Soc. méd. Hôp. 1944, no. 11 & 12, p. 121 13. Calvo Melendro, J. Presse med. 1950, 55, 952. 6.
Kuwert, E., Noll, K., Firnhaber, W. Z. Immun. Forsch. exp. Ther. 1968, 135, 462. 2. Link, H. Acta neurol. scand. 1972, 48, 521. 3. Broman, T., Bergmann, L., Fog, T., Gilland, T., Hyllested, K., Lindberg-Broman, A. M., Pedersen, E., Presthus, J. ibid. 1965, 41, suppl. 13, p. 543. 4. Mancini, G., Carbonara, A. O., Heremans, J. F. Immunochemistry, 1965, 2, 235. 5. Mayer, M. M. in Experimental Immunochemistry (edited by E. A. Kabat and M. M. Mayer); p. 133. Springfield, I11. 1961. 1.
with Mollaret’s
recurrent two of my