289
Requests for reprints should
be addressed
to
J.G.K.
REFERENCES 1. Barr, D. P., Russ, E. M., Eder, H. V. Am. J. Med. 1951, 11, 480 2. Wiklund, O., Gustafson, A., Wilhelmsen, L. Artery, 1975, 1, 399. 3. Rössner, S., Kjellin, K. G., Mettinger, K. L., Sidén, A., Söderström, C. E.
Lancet, 1978, i, 577.
G., Gustafson, A., Kral, J., Scherstén, T., Sjöström, L. Artery, 1976,2,200. 5. Kral, J. G., Bondjers, G., Gustafson, A. Eur. J. clin. Invest, 1978, 8, 119. 6. Payne, J. H., de Wind, L. T. Am. J. Surg. 1969, 118, 141. 7. Bondjers, G., Björkerud, S. Analyt. Biochem. 1971, 42, 363. 8. Kissane, J. M., Robins, E. J. biol. Chem. 1958, 233, 184. 9. Bondjers, G., Björkerud, S. Artery, 1974, i, 3. 10. Walton, K. W. Proceedings IVth International Conference on Atherosclerosis. Aug. 24, 1976, Tokyo, Japan. 11. Green, P. H. R., Tall, A. R., Glickman, R. M. J. clin. Invest. 1978, 61, 528. 4. Bondjers,
Fig.
1—Noma
affecting
the
nose
and upper lip with inflamma-
tory tedeoia of the adjacent tissue.
NOMA NEONATORUM: ITS ÆTIOPATHOGENESIS S. P. GHOSAL A. K.
P. C. SEN GUPTA
MUKHERJEE
Departments of Pædiatrics and Pathology, Institute Health, Calcutta 700 017, India MOMOTA CHOUDHURY
of Child
gested that it was a definite nosological entity and named it "noma neonatorum". In a preliminary investigation of a small number of cases, we isolated Pseudomonas œruginosa from post-mortem blood-cultures.1 The results of detailed bacteriological and histopathological studies of 35 cases of noma neonatorum are discussed in this paper.
NEILLE DUTTA
Patients
A. K. SARKAR Vivekananda Institute Sciences, Calcutta 700 026, India
Department of Pædiatrics,
Summary
of Medical
Noma neonatorum,
a gangrenous prothe nose, lips, mouth, anal affecting and eyelids, affects and the scrotum region, occasionally neonates, especially low-birth-weight and premature ill babies, and is usually fatal 1-3 days after onset. In 35 cases of noma neonatorum Pseudomonas æruginosa was isolated from blood-culture (86·3%), gangrenous areas (96·0%), rectal swabs (58·3%), and cerebrospinal fluid (60·0%). Blood-vessels in the deep cutis or subcutis were affected and the gangrenous process extended superficially. Noma in older children and adults is caused by fusospirochætosis but noma neonatorum appears to be due to P. æruginosa septicæmia.
cess
Introduction BETWEEN 1970 and 1975 we saw 48 low-birth-weight, usually premature, newborn babies who, during the first two weeks of life had gangrene of the nose, lips, mouth, anal region, and occasionally the eyelids and some other areas. Almost all the babies died 1-3 days after gangrene began. Because all the babies appeared to have the same condition, which was quite distinct from other gangrenous processes known in this age group, we sug-
35 neonates with noma neonatorum and 10 controls from the same nursery were studied. The patients were aged 6-23 days (meanis.D. 9.2±5.1) and their birth-weight was 1-0-2-7 kg (mean+s.D. 1.75±0.38). 27 patients had noma in the mouth and oral cavity (tongue, 7; lips, 7; gum, 7; palate, 6) and in others noma was found in the nose and alae nasi (11), anogenital region (perianal, 6; scrotum, 1), and eyelids (3). More than one site was involved in 12 cases (fig. 1).
Materials and Methods Heart-blood was cultured soon after death in 31 cases. In 13 blood was taken ante mortem from the femoral vein. Aspirates from the junction of normal and gangrenous tissues (25 cases), rectal swabs (12 cases), and cerebrospinal fluid (c.s.F.) (5 cases) were obtained at necropsy. All materials for bacteriological examination were cultured on blood-agar and MacConkey’s medium. Standard methods were used to isolate and identify organisms.2 As controls, blood-cultures were obtained ante mortem and at necropsy from 10 babies of similar gestation period and birth-weight from the same nursery who had been very ill but who had not had gangrene. For histopathological examination, a small specimen which included gangrenous tissue and adjacent unaffected tissue was taken soon after death in 14 cases. These were fixed in 10% formol saline and given routine histological examination: paraffin sections were stained with haematoxylin and eosin (H and E), Gram’s stain, the periodic-acid/Schiff (PAS) reaction, and Pterocarpus santalinus stain (for elastic tissue3). cases
BACTERIOLOGICAL EXAMINATION
290 in
specimen. Necrosed strongly P.A.s.-positive in all
seen or
one
moderately specimens. Elastic-
areas were
the
tissue stain showed that the thrombosed arterioles and the tissues surrounding them lacked elastic fibres. The lesion seemed to originate in and around the blood-vessels in the subcutis and the muscle layer, and then to extend more superficially to the different layers of the dermis (fig. 2). The epidermis was necrosed when the papillary layer of the dermis was involved. In a few cases there was an acute inflammatory reaction probably due to the associated bacterial infection. Discussion
Fig. 2—Affected tissue of noma (early stage). A blood-vessel in the subcutis is thrombosed with perivascular necrosis and dense cellular infiltration extending superficially to the dermis. (H. and E x 84). Results
Bacteriological Examination (see table) P. œruginosa was isolated from 38 of 44 specimens (86-3%) from affected babies. There was pure growth from 34 specimens (77.2%), of which 25 were from postand 9 from ante-mortem blood-cultures. Both and post-mortem blood-culture was positive for P. ceruginosa in 9 cases. In 2 ante-mortem and 2 post-mortem cultures, P. œruginosa was found with Escherichia coli (2) and Klebsiella (2). In one case the ante-mortem blood-culture was positive for P. œruginosa 3 days before noma appeared despite treatment for pseudomonas septicaemia. P. ceruginosa was isolated from aspirates of tissue adjoining the gangrenous area in 24 out of 25 cases (96%); growth was pure in 11 cases and in the remaining 13 cases also included one or more of the following: E. coli, Klebsiella œrogenes, Staphylococcus pyogenes, mortem
ante-mortem
’
Proteus rettgeri. In 7 of 12 cases, P. œruginosa was grown from a rectal swab along with E. coli and/or K. cerogenes and in one case it was grown with Salmonella alachua. In 5, only E. coli and K. œrogenes were isolated. Of the c.s.F. specimens cultured, 3 gave pure growths of P. œruginosa and 2 gave no growth. Of the 86 specimens cultured from cases of noma, 72 (83.77%) grew P. œruginosa (as pure growth in 48, 55.8%, and with other organisms in 24, 27.9%). Blood-culture was sterile in the 8 babies of the control group; in 1 blood-culture gave S. alachua (ante-mortem culture) and in the other blood-culture gave P. mirabilis
(post-mortem culture). Histopathological examination On H and
E
stain, the necrosed
stained light blue with
area was
diffusely
hmmatoxylii, was fibrillar, and anuclear in specimens from most cases. Necrosed collagen, stained deeply with eosin and lacking nuclei, was
The isolation of P. œruginosa from blood (86.3% cases) and from the site of noma (96% cases) suggests that the organism. is important in the xtiology of noma neonatorum. Invasion of gangrenous lesions by secondary organisms, seen in 13 (52%) of cases, and was probably dependent on what organisms were abundant at the oronasal and anorectal sites of infection. The influence of blood-borne infection (septicaemia) on the gangrenous process was also evident from the histological changes. In ecthyma gangrenosum, a condition caused by P. œruginosa, the skin shows similar histological changes.4 These are thought to be produced by the exotoxin and enzymes of the organism.56 The loss of elastic tissue in affected areas was very probably due to the effect of the elastase of P. ceruginosa. Pseudomonas species are dangerous and frequently encountered in hospital infection, particularly of neonates and children.7-11 P. œruginosa can cause bactersmia and septicæmia4,10—17 and can give rise to gastroenteritis, pneumonitis, and meningitis in neonates.7,8, to, tSkin involvement may lead to patchy areas of necrosis and ulceration in some cases.4,11,12,16 Curtin et al. 12 found ecthyma gangrenosum in only 1 of 18 premature babies with pseudomonas septicaemia. Localised, dark red, tender areas of cellulitis can appear in the perianal region of children with cancer who have pseudomonas septicæmia.16 In noma neonatorum, P. œruginosa septicæmia affects the mucous membrane and the mucocutaneous junction, mainly in the oronasal and perianal regions. The appearance of noma in older children and adults with other diseases is thought to be due to lowered resistance to infection.18,19 The neonates in this study were of low birth-weight, mostly premature, and very ill; noma thus probably developed because their immunological function was impaired. In older children and adults, cancrum oris seems to be caused by fusospirochætosis18 19 but noma neonatorum seems to be due to P. œruginosa septicoemia and thus must be added to the list of dangerous clinical conditions caused by - this organism in newborns. We thank the Secretary, Vivekananda Institute of Medical Sciences, the Director, Institute of Child Health, Calcutta, and Prof. S. K. Roy for permission to publish this paper. We also thank Dr K. K. Pramanick and Dr D. Chatterjee for allowing us to include the cases under their care, the resident staff for help in collecting materials, and Dr T. K. Banerjee for the photograph.
Requests for reprints to S.P.G., Department of Pediatrics, Institute of Child Health, Calcutta 700 017, India.
291
Preliminary
Communication
ENDOGENOUS OPIOID PEPTIDES
IN NEURONS OF THE HUMAN BRAIN
mation has been obtained in laboratory animals there are indications that opiate-like substances are present in the human brain.13 The present report describes the direct microscopical visualisation of .neurons in the human brain containing an opiate-like peptide, as revealed by
immunocytochemistry.
A. C. CUELLO MATERIALS AND METHODS
Medical Research Council Neurochemical Pharmacology Unit,
Department of Pharmacology, Medical School, Hills Road, Cambridge CB2 2QD Immunoreactive leucine-encephalin has been found in nerve terminals and cell bodies of neurons in the human brain. The most intense reaction was found in the globus pallidus, followed by the spinal nucleus of the trigeminal nerve. These observations suggest that in man endogenous opioid substances may play an important part in neurotransmission in areas of the central nervous system related to pain and locomotor activity.
Summary
INTRODUCTION
RECEPTORS to morphine have been well characterised in a variety of peripheral organs.1,2 The presence in the mammalian brain of receptors which respond specifically to opiates has also been proposed.3,4 These observations led to the speculation that mammalian brains produce an opioid substance as the natural ligand for such receptors. Hughes et al. demonstrated the existence of two brain pentapeptides, leucine-encephalin and methionine-encephalin, with opiate-like pharmacological actions which were blocked by morphine antagonists. Other opiate-like substances have been also described in the mammalian brain, and they, like Met-encephalin, correspond to 7different portions of the larger peptide p-lipotrophin. 6, Of all endogenous opiate-like substances, the encephalins seem to be the most abundant in the brain. They have been found by radioimmunoassay and immunocytochemistry,8-12 to be unevenly distributed in the central nervous system of laboratory animals. They are particularly concentrated in areas related to pain perception, but they are also found in the extrapyramidal system. Although the bulk of this infor-
obtained up to 32 h after death, and diffixed in cold 4% paraformaldehyde in 0.11 mol/I phosphate buffer (pH 7-0). Brains from Wistar adult male rats were also fixed by immersion or by intracardiac perfusion with the same fixative. The tissue samples were fixed for 2-4 h and kept for up to 5 days in the same phosphate buffer containing 5% sucrose. Sections 10 .m thick were obtained at - 20°C in a cryostat and stained with the indirect immunofluorescent technique.14 A rabbit leucine-encephalin antiserum prepared by Dr Richard Miller (Chicago University) was used in this study. This antiserum did not cross-react with p-Iipotrophin or the adrenocorticotrophic hormone. The cross-reactivity with methionine-encephalin was 1% and with &bgr;-endorphin
Requests for reprints should
be addressed
to
J.G.K.
REFERENCES 1. Barr, D. P., Russ, E. M., Eder, H. V. Am. J. Med. 1951, 11, 480 2. Wiklund, O., Gustafson, A., Wilhelmsen, L. Artery, 1975, 1, 399. 3. Rössner, S., Kjellin, K. G., Mettinger, K. L., Sidén, A., Söderström, C. E.
Lancet, 1978, i, 577.
G., Gustafson, A., Kral, J., Scherstén, T., Sjöström, L. Artery, 1976,2,200. 5. Kral, J. G., Bondjers, G., Gustafson, A. Eur. J. clin. Invest, 1978, 8, 119. 6. Payne, J. H., de Wind, L. T. Am. J. Surg. 1969, 118, 141. 7. Bondjers, G., Björkerud, S. Analyt. Biochem. 1971, 42, 363. 8. Kissane, J. M., Robins, E. J. biol. Chem. 1958, 233, 184. 9. Bondjers, G., Björkerud, S. Artery, 1974, i, 3. 10. Walton, K. W. Proceedings IVth International Conference on Atherosclerosis. Aug. 24, 1976, Tokyo, Japan. 11. Green, P. H. R., Tall, A. R., Glickman, R. M. J. clin. Invest. 1978, 61, 528. 4. Bondjers,
Fig.
1—Noma
affecting
the
nose
and upper lip with inflamma-
tory tedeoia of the adjacent tissue.
NOMA NEONATORUM: ITS ÆTIOPATHOGENESIS S. P. GHOSAL A. K.
P. C. SEN GUPTA
MUKHERJEE
Departments of Pædiatrics and Pathology, Institute Health, Calcutta 700 017, India MOMOTA CHOUDHURY
of Child
gested that it was a definite nosological entity and named it "noma neonatorum". In a preliminary investigation of a small number of cases, we isolated Pseudomonas œruginosa from post-mortem blood-cultures.1 The results of detailed bacteriological and histopathological studies of 35 cases of noma neonatorum are discussed in this paper.
NEILLE DUTTA
Patients
A. K. SARKAR Vivekananda Institute Sciences, Calcutta 700 026, India
Department of Pædiatrics,
Summary
of Medical
Noma neonatorum,
a gangrenous prothe nose, lips, mouth, anal affecting and eyelids, affects and the scrotum region, occasionally neonates, especially low-birth-weight and premature ill babies, and is usually fatal 1-3 days after onset. In 35 cases of noma neonatorum Pseudomonas æruginosa was isolated from blood-culture (86·3%), gangrenous areas (96·0%), rectal swabs (58·3%), and cerebrospinal fluid (60·0%). Blood-vessels in the deep cutis or subcutis were affected and the gangrenous process extended superficially. Noma in older children and adults is caused by fusospirochætosis but noma neonatorum appears to be due to P. æruginosa septicæmia.
cess
Introduction BETWEEN 1970 and 1975 we saw 48 low-birth-weight, usually premature, newborn babies who, during the first two weeks of life had gangrene of the nose, lips, mouth, anal region, and occasionally the eyelids and some other areas. Almost all the babies died 1-3 days after gangrene began. Because all the babies appeared to have the same condition, which was quite distinct from other gangrenous processes known in this age group, we sug-
35 neonates with noma neonatorum and 10 controls from the same nursery were studied. The patients were aged 6-23 days (meanis.D. 9.2±5.1) and their birth-weight was 1-0-2-7 kg (mean+s.D. 1.75±0.38). 27 patients had noma in the mouth and oral cavity (tongue, 7; lips, 7; gum, 7; palate, 6) and in others noma was found in the nose and alae nasi (11), anogenital region (perianal, 6; scrotum, 1), and eyelids (3). More than one site was involved in 12 cases (fig. 1).
Materials and Methods Heart-blood was cultured soon after death in 31 cases. In 13 blood was taken ante mortem from the femoral vein. Aspirates from the junction of normal and gangrenous tissues (25 cases), rectal swabs (12 cases), and cerebrospinal fluid (c.s.F.) (5 cases) were obtained at necropsy. All materials for bacteriological examination were cultured on blood-agar and MacConkey’s medium. Standard methods were used to isolate and identify organisms.2 As controls, blood-cultures were obtained ante mortem and at necropsy from 10 babies of similar gestation period and birth-weight from the same nursery who had been very ill but who had not had gangrene. For histopathological examination, a small specimen which included gangrenous tissue and adjacent unaffected tissue was taken soon after death in 14 cases. These were fixed in 10% formol saline and given routine histological examination: paraffin sections were stained with haematoxylin and eosin (H and E), Gram’s stain, the periodic-acid/Schiff (PAS) reaction, and Pterocarpus santalinus stain (for elastic tissue3). cases
BACTERIOLOGICAL EXAMINATION
290 in
specimen. Necrosed strongly P.A.s.-positive in all
seen or
one
moderately specimens. Elastic-
areas were
the
tissue stain showed that the thrombosed arterioles and the tissues surrounding them lacked elastic fibres. The lesion seemed to originate in and around the blood-vessels in the subcutis and the muscle layer, and then to extend more superficially to the different layers of the dermis (fig. 2). The epidermis was necrosed when the papillary layer of the dermis was involved. In a few cases there was an acute inflammatory reaction probably due to the associated bacterial infection. Discussion
Fig. 2—Affected tissue of noma (early stage). A blood-vessel in the subcutis is thrombosed with perivascular necrosis and dense cellular infiltration extending superficially to the dermis. (H. and E x 84). Results
Bacteriological Examination (see table) P. œruginosa was isolated from 38 of 44 specimens (86-3%) from affected babies. There was pure growth from 34 specimens (77.2%), of which 25 were from postand 9 from ante-mortem blood-cultures. Both and post-mortem blood-culture was positive for P. ceruginosa in 9 cases. In 2 ante-mortem and 2 post-mortem cultures, P. œruginosa was found with Escherichia coli (2) and Klebsiella (2). In one case the ante-mortem blood-culture was positive for P. œruginosa 3 days before noma appeared despite treatment for pseudomonas septicaemia. P. ceruginosa was isolated from aspirates of tissue adjoining the gangrenous area in 24 out of 25 cases (96%); growth was pure in 11 cases and in the remaining 13 cases also included one or more of the following: E. coli, Klebsiella œrogenes, Staphylococcus pyogenes, mortem
ante-mortem
’
Proteus rettgeri. In 7 of 12 cases, P. œruginosa was grown from a rectal swab along with E. coli and/or K. cerogenes and in one case it was grown with Salmonella alachua. In 5, only E. coli and K. œrogenes were isolated. Of the c.s.F. specimens cultured, 3 gave pure growths of P. œruginosa and 2 gave no growth. Of the 86 specimens cultured from cases of noma, 72 (83.77%) grew P. œruginosa (as pure growth in 48, 55.8%, and with other organisms in 24, 27.9%). Blood-culture was sterile in the 8 babies of the control group; in 1 blood-culture gave S. alachua (ante-mortem culture) and in the other blood-culture gave P. mirabilis
(post-mortem culture). Histopathological examination On H and
E
stain, the necrosed
stained light blue with
area was
diffusely
hmmatoxylii, was fibrillar, and anuclear in specimens from most cases. Necrosed collagen, stained deeply with eosin and lacking nuclei, was
The isolation of P. œruginosa from blood (86.3% cases) and from the site of noma (96% cases) suggests that the organism. is important in the xtiology of noma neonatorum. Invasion of gangrenous lesions by secondary organisms, seen in 13 (52%) of cases, and was probably dependent on what organisms were abundant at the oronasal and anorectal sites of infection. The influence of blood-borne infection (septicaemia) on the gangrenous process was also evident from the histological changes. In ecthyma gangrenosum, a condition caused by P. œruginosa, the skin shows similar histological changes.4 These are thought to be produced by the exotoxin and enzymes of the organism.56 The loss of elastic tissue in affected areas was very probably due to the effect of the elastase of P. ceruginosa. Pseudomonas species are dangerous and frequently encountered in hospital infection, particularly of neonates and children.7-11 P. œruginosa can cause bactersmia and septicæmia4,10—17 and can give rise to gastroenteritis, pneumonitis, and meningitis in neonates.7,8, to, tSkin involvement may lead to patchy areas of necrosis and ulceration in some cases.4,11,12,16 Curtin et al. 12 found ecthyma gangrenosum in only 1 of 18 premature babies with pseudomonas septicaemia. Localised, dark red, tender areas of cellulitis can appear in the perianal region of children with cancer who have pseudomonas septicæmia.16 In noma neonatorum, P. œruginosa septicæmia affects the mucous membrane and the mucocutaneous junction, mainly in the oronasal and perianal regions. The appearance of noma in older children and adults with other diseases is thought to be due to lowered resistance to infection.18,19 The neonates in this study were of low birth-weight, mostly premature, and very ill; noma thus probably developed because their immunological function was impaired. In older children and adults, cancrum oris seems to be caused by fusospirochætosis18 19 but noma neonatorum seems to be due to P. œruginosa septicoemia and thus must be added to the list of dangerous clinical conditions caused by - this organism in newborns. We thank the Secretary, Vivekananda Institute of Medical Sciences, the Director, Institute of Child Health, Calcutta, and Prof. S. K. Roy for permission to publish this paper. We also thank Dr K. K. Pramanick and Dr D. Chatterjee for allowing us to include the cases under their care, the resident staff for help in collecting materials, and Dr T. K. Banerjee for the photograph.
Requests for reprints to S.P.G., Department of Pediatrics, Institute of Child Health, Calcutta 700 017, India.
291
Preliminary
Communication
ENDOGENOUS OPIOID PEPTIDES
IN NEURONS OF THE HUMAN BRAIN
mation has been obtained in laboratory animals there are indications that opiate-like substances are present in the human brain.13 The present report describes the direct microscopical visualisation of .neurons in the human brain containing an opiate-like peptide, as revealed by
immunocytochemistry.
A. C. CUELLO MATERIALS AND METHODS
Medical Research Council Neurochemical Pharmacology Unit,
Department of Pharmacology, Medical School, Hills Road, Cambridge CB2 2QD Immunoreactive leucine-encephalin has been found in nerve terminals and cell bodies of neurons in the human brain. The most intense reaction was found in the globus pallidus, followed by the spinal nucleus of the trigeminal nerve. These observations suggest that in man endogenous opioid substances may play an important part in neurotransmission in areas of the central nervous system related to pain and locomotor activity.
Summary
INTRODUCTION
RECEPTORS to morphine have been well characterised in a variety of peripheral organs.1,2 The presence in the mammalian brain of receptors which respond specifically to opiates has also been proposed.3,4 These observations led to the speculation that mammalian brains produce an opioid substance as the natural ligand for such receptors. Hughes et al. demonstrated the existence of two brain pentapeptides, leucine-encephalin and methionine-encephalin, with opiate-like pharmacological actions which were blocked by morphine antagonists. Other opiate-like substances have been also described in the mammalian brain, and they, like Met-encephalin, correspond to 7different portions of the larger peptide p-lipotrophin. 6, Of all endogenous opiate-like substances, the encephalins seem to be the most abundant in the brain. They have been found by radioimmunoassay and immunocytochemistry,8-12 to be unevenly distributed in the central nervous system of laboratory animals. They are particularly concentrated in areas related to pain perception, but they are also found in the extrapyramidal system. Although the bulk of this infor-
obtained up to 32 h after death, and diffixed in cold 4% paraformaldehyde in 0.11 mol/I phosphate buffer (pH 7-0). Brains from Wistar adult male rats were also fixed by immersion or by intracardiac perfusion with the same fixative. The tissue samples were fixed for 2-4 h and kept for up to 5 days in the same phosphate buffer containing 5% sucrose. Sections 10 .m thick were obtained at - 20°C in a cryostat and stained with the indirect immunofluorescent technique.14 A rabbit leucine-encephalin antiserum prepared by Dr Richard Miller (Chicago University) was used in this study. This antiserum did not cross-react with p-Iipotrophin or the adrenocorticotrophic hormone. The cross-reactivity with methionine-encephalin was 1% and with &bgr;-endorphin
