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Isotypic analysis of specific antibody response in serum, saliva, gastric and rectal homogenates of Helicobacter pylori-infected patients
285
FEMS Immunology and Medical Microbiology 10 (1995) 285-288 0 1995 Federation of European Microbiological Societies 0928-8244/95/$09.50 Published by Elsevier
FEMSIM 00460
Isotypic analysis of specific antibody response in serum, saliva, gastric and rectal homogenates of Helicobacter pylori-infected patients F. Luzza,
M. Imeneo,
M. Maletta,
Dipartimento di Medicina Spkrzentale,
G. Monteleone,
P. Doldo,
L. Biancone
and F. Pallone
*
Universitci di Reggio Calabria, I/is T. Catnpanella, 88100 Catanzaro, Italy
(Received 23 August 1994; accepted 10 October 1994)
Abstract: The relationship
b,etween systemic and local humoral immune response to Helicobacter pylon’ is poorly understood. To further address this issue we measured, using ELISA, H. &or&specific IgG and IgA antibodies in serum, saliva, gastric and rectal homogenates of H. pylon’-infected patients. A total of 107 patients who underwent upper GI endoscopy and/or sigmoidoscopy were studied. The isotypic pattern of H. pylon’-specific antibodies appeared to differ at the serum, salivary, gastric and rectal mucosa level. Serum H. pyhri IgG titers were higher than those of the serum-specific IgA. On the contrary, in saliva samples H. pylon’ IgA titers were higher than specific IgG titers. In gastric homogenates, specific IgG and IgA titers were similar. H. pylon’-specific IgG were detectable in recta1 homogenates but no or very low H. pylori-specific IgA were found in the same material. Furthermore, no difference was found in H. pylon’ IgG and IgA in serum, saliva and gastric homogenates between duodenal ulcer and non-ulcer dyspepsia patients. Data of the present study indicate that, in H. pylori-infected patients, the specific immune response is as follows: (1) it involves the secretory immune system; (2) it is paralleled by the specific salivary IgA; (3) it does not differentiate duodenal ulcer from non-ulcer dyspepsia patients; and (4) it does not take place in the large bowel. Key words: Helicobacter pylori; ELISA, IgG antibody; IgA antibody
Introduction Helicobacter pylon’ infection is the most common chronic bacterial :infection in man. The host’s response to the bacterium is considered as being a key factor in understanding the pathogenesis and the natural history of H. pylon’ infection [1,2]. Evidence is accumulating that H. pylon’ induces a marked humoral specific immune re-
* Corresponding author. + 39.961.775373. SSDI 092%8244(94)00069-7
Tel:
+ 39.961.770144;
Fax:
sponse detectable in the serum [3-51, in the salivary secretion 161and in the gastric mucosa [7,8]. However, the relationship between systemic and local humoral immune response and the pathogenic role, if any, are poorly understood. Furthermore, the recent isolation of H. pylon’ in faeces [9] raises the question of whether the colonic mucosa also harbour H. pylon’ and a specific local antibody response is elicited. The aim of our study was to define the isotypic pattern of the serum, salivary, gastric and rectal H. pylon’-specific antibody responses and to explore their interrelationships.
286
Materials
and Methods
Subjects and sampling
The first group of patients consisted of 79 patients (42 male, 27 female; mean age: 48 f 12) who underwent upper GI endoscopy. Seventy-four were found to be H. pylori-positive by the urease quick test (CP-test) confirmed by histology (Giemsa staining). Thirty-one had duodenal ulcer (DU) and 43 complained of non-ulcer dyspepsia (NUD). The remaining 5 subjects were H. pylorinegative healthy controls. The second group of patients consisted of 28 patients (17 male, 11 female; mean age: 46 f 12) undergoing sigmoidoscopy for functional bowel disorders. Among these, 22 also underwent upper GI endoscopy as a part of a diagnostic work up and were found to be H. pylori-positive by CP-test and Giemsa staining. In all patients, samples of serum and unstimulated saliva were collected. In the 79 patients of the first group and in the 22 gastroscoped from the second group, 4 antral biopsies were taken from each patient. Four additional rectal biopsies were taken from each patient of the second group. Serum and saliva samples were spun, coded and stored at - 20” until tested. Antral and rectal biopsy samples were separately homogenated on ice using a tissue homogenizer (Ystral, GmbH, Dottingen, Germany). The homogenates were spun (600 x g for 20 min at 4”C), coded and stored at -80°C until tested. H. pylon’-specific IgG and IgA were measured in each sample of serum, saliva and mucosal (gastric and/or rectal) homogenate by direct ELISA. H. pylori-specific IgG and IgA measurement A sonicate of a whole H. pylon’ strain was used
as a source of antigen. The antigen preparation was diluted with coating buffer, added to each well and incubated for 2 h at 37°C. The plates were washed with washing buffer and binding sites were blocked by adding 2% serum albumin in washing buffer and incubated for 18 h at 4°C. The working dilution was 1:lOO for serum and 1:4 for saliva. Homogenates were normalized (10 pg ml-‘) by measuring the total protein concentration (Bio-Rad Protein Assay). Diluted serum,
saliva and homogenates were separately added to each well in duplicate and incubated for 90 min at 37°C. The plates were washed again and either anti-human IgG or IgA peroxidase conjugate (Sigma Immunochemicals) was added and incubated for 1 h at 37°C. The plates were washed again and substrate was added and left for 10 min and 35 min (for serum IgG and IgA, respectively), 35 min (for salivary and mucosal IgG and IgA) in a dark room. Stopping solution was finally added and the optical density of the wells was read at 405 nm using a microelisa plate reader (Labsystern Multiskan MCC/340). All samples were processed in the same ELISA assay. Serum H. pylori positivity was defined by using a pool of 100 negative control sera as a reference for IgG determinations (cut-off point of 2 standard deviations above the mean of the standard negative control sera) which gave a sensitivity and a specificity of 94% and 95%, respectively (data not shown). Results were expressed as mean optical density + S.D. Student’s t-test was used for data analysis.
Results
As expected, in the first group all 74 patients had a positive serology as indicated by the serum
/I OD
I
1n W 1 0 L--l IgA
(
II
I,5
1
a5
/
/
OV Serum
Fig. 1. Isotypic pylon’-infected
Saliva
/
G.Homogenates
pattern of specific antibody response in 74 H. patients (OD = optical density, mean f SD.).
287
Table 1 H. pylori-specific IgG and IgA in patients negative for H. pylon’ on histology
IgG IgA
Serum
Saliva
G. Homogenate
0.549 f. 0.143 0.298&0.165
0.222 + 0.163 0.458 + 0.372
0.098 f 0.080 0.161 kO.177
OD/ I,5
1
H. pyhi IgG (1.192 f 0.308) that were significantly (P < 0.001) higher than serum H. pylori IgA (0.592 f 0.398). In contrast, in saliva samples, H. pylon’ IgG concentration (0.825 f 0.682) was lower than that of H. pylori IgA (1.014 f 0.721). In gastric homogenates, the mean concentration of H. pylon’ IgA (0.605 f 0.467) was similar to that of H. pylori IgG (0.610 k 0.623) (Fig. 1). In the 5 negative healthy controls, specific IgG were significantly lower in all examined areas, whilst H. pylon’-specific IgA appear to differ only in terms of mucosal concentration (Table 1). No difference was found in H. pylori IgG and IgA in serum, saliva and gastric homogenates between DU and NUD patients (Fig. 2). In the second group, 26 of the 28 patients were found to be H. pylon! seropositive. In the rectal homogenates, H. pylon’ IgG were detectable in all patients, with a mean concentration of about
1
OD 1.5
-
0.5
OV
/
Sl?WtTl
/ saliva
gastric
/ horn.
/ rectal
horn.
Fig. 3. Specific IgG and IgA in serum, saliva, gastric and rectal homogenates of 22 H. pylon’-infected patients (OD = optical density, mean f S.D.).
one third of that detected in gastric homogenates (0.239 k 0.214). Specific H. pylon’ IgA were undetectable in the rectal homogenates of 12 patients and present in trace amounts in the remaining 16. In contrast, the gastric homogenates showed similar titers for H. pylori IgG (0.763 f 0.702) and H. pylori IgA (0.647 + 0.486). In the 22 patients with both gastric and rectal homogenates, both H. pylon’ IgG and IgA were significantly higher in gastric samples than in rectal samples (0.763 f 0.702 vs. 0.230 + 0.230 for IgG, P < 0.001 and 0.647 + 0.486 vs. 0.060 + 0.059 for IgA, P < 0.0001 respectively). Serum and salivary figures confirmed the first group patients’ results (Fig. 3).
Discussion
OI_L_L-L N”D SERUM
DU
NUD SALIVA
D”
NW DU G. HOMOGENATES
Fig. 2. Isotypic pattern of specific antibody response in 43 NUD and in 31 DU H. &on’-infected patients (OD = optical density, mean f S.D.).
It has become clear that H. pylon’ plays an etiological role in chronic gastritis, peptic ulcer disease, and possibly in gastric cancer. Nevertheless, information on the pathogenesis and natural history of H. pylori infection is limited. Studies of the specific circulating antibody response to H. pylori focussed primarily on the diagnostic rather than the pathogenetic role of such a response [3-51. Only recently has it been appreciated that the gastric mucosal immune response may repre-
288
sent one main pathogenetic mechanism in H pylon’-associated diseases [2,8]. In this study we further investigated the systemic and local humoral immune responses to H. pylon’ and their interrelationships. The isotypic pattern of H. pylon’-specific antibodies appeared to differ at serum, salivary, gastric and rectal mucosa level. Serum H. pylon’ IgG titers were higher than those of the serum-specific IgA. On the contrary, in saliva samples H. pylori IgA titers were higher than specific IgG titers. In gastric homogenates, specific IgG and IgA titers were similar. The marked H. pylon’ IgA response at the salivary and gastric mucosa level indicate that the specific humoral immune response to H. pylori involves the secretory immune system. Furthermore, differential analysis of the isotypic H. pylon’-specific antibody pattern in DU and NUD patients failed to produce evidence of any difference in IgG/IgA ratio in the three examined compartments of the two groups of patients. H. pylon’-specific IgG were detectable also in rectal homogenates of our patients, but in contrast to gastric mucosa, none or very low H. pylori-specific IgA were found in the same material. Data also suggest that the H. pylon’-specific local humoral immune response does not seem to occur at the rectal level but that H. pylon’ antibodies are likely to be partly of systemic origin. Consequently, our data do not support the concept that colonic mucosa harbour H. pyfori.
References Blaser, M.J. (1992) Hypotheses on the pathogenesis and natural history of Helicobacierpylorj-induced inflammation. Gastroenterology 102, 720-727. Hatz, R.A., Brooks, W.P., Kramling, H. and Enders, G. (1992) Stomach immunology and Helicobacter pylon’ infection. Curr. Opin. Gastroenterol. 8, 993-lOill. Newell, D.G. and Stacey, A.R. (19891 The serology of Campylobacter pylon’ infection. In: Campylobacter pylon’ and Gastroduodenal Disease (Rathbone, B.J. and Heatley, R.V., Eds.1, pp. 190-196. Blackwell Scientific Publications, London. Evans, D.J., Evans, D.G., Graham D.Y. and Klein, P.D. (1989) A sensitive and specific serologic test for detection of Campylobacter pylon’ infection. Gastroenterology 96, 10041008. Kosunen, T.U., Seppala, K., Sama, S. and Sipponen, P. (1992) Diagnostic value of decreasing IgG, IgA and IgM antibody titres after eradication of Helicobacter pylon’. Lancet 339, 893-895. Pate], P., Mendall, M.A., Khulusi, S. Molineaux, N. and Northfield, T.C. (1993) Detection of H. pylon’ infection by saliva. Gut 34(l), S36-T139. Rathbone, B.J., Wyatt, J.I., Worsley, B.W., Shires, S.E., Trejdosiewicz, L.K., Heatley, R.V. and Losowsky, MS. (1986) Systemic and local antibody responses to gastric Campylobacter pyloridis in non-ulcer dyspepsia. Gut 27, 642-647. 8 Crabtree, LE., Taylor J.D., Wyatt, J.I., Heatley, R.V., Shallcross, T.M., Tompkins, D.S. and Rathbone, B.J. (1991) Mucosal IgA recognition of Helicobacter pylon’ 120 kDa protein, peptic ulceration, and gastric pathology. Lancet 338, 332-335. 9 Thomas, J.E., Gibson, G.R., Darboe,M.K., Dale, A. and Weaver, L.T. (1992) Isolation of Helicobacter pylori from human faeces. Lancet 340, 1194-1195.
FEMS Immunology and Medical Microbiology 10 (1995) 285-288 0 1995 Federation of European Microbiological Societies 0928-8244/95/$09.50 Published by Elsevier
FEMSIM 00460
Isotypic analysis of specific antibody response in serum, saliva, gastric and rectal homogenates of Helicobacter pylori-infected patients F. Luzza,
M. Imeneo,
M. Maletta,
Dipartimento di Medicina Spkrzentale,
G. Monteleone,
P. Doldo,
L. Biancone
and F. Pallone
*
Universitci di Reggio Calabria, I/is T. Catnpanella, 88100 Catanzaro, Italy
(Received 23 August 1994; accepted 10 October 1994)
Abstract: The relationship
b,etween systemic and local humoral immune response to Helicobacter pylon’ is poorly understood. To further address this issue we measured, using ELISA, H. &or&specific IgG and IgA antibodies in serum, saliva, gastric and rectal homogenates of H. pylon’-infected patients. A total of 107 patients who underwent upper GI endoscopy and/or sigmoidoscopy were studied. The isotypic pattern of H. pylon’-specific antibodies appeared to differ at the serum, salivary, gastric and rectal mucosa level. Serum H. pyhri IgG titers were higher than those of the serum-specific IgA. On the contrary, in saliva samples H. pylon’ IgA titers were higher than specific IgG titers. In gastric homogenates, specific IgG and IgA titers were similar. H. pylon’-specific IgG were detectable in recta1 homogenates but no or very low H. pylori-specific IgA were found in the same material. Furthermore, no difference was found in H. pylon’ IgG and IgA in serum, saliva and gastric homogenates between duodenal ulcer and non-ulcer dyspepsia patients. Data of the present study indicate that, in H. pylori-infected patients, the specific immune response is as follows: (1) it involves the secretory immune system; (2) it is paralleled by the specific salivary IgA; (3) it does not differentiate duodenal ulcer from non-ulcer dyspepsia patients; and (4) it does not take place in the large bowel. Key words: Helicobacter pylori; ELISA, IgG antibody; IgA antibody
Introduction Helicobacter pylon’ infection is the most common chronic bacterial :infection in man. The host’s response to the bacterium is considered as being a key factor in understanding the pathogenesis and the natural history of H. pylon’ infection [1,2]. Evidence is accumulating that H. pylon’ induces a marked humoral specific immune re-
* Corresponding author. + 39.961.775373. SSDI 092%8244(94)00069-7
Tel:
+ 39.961.770144;
Fax:
sponse detectable in the serum [3-51, in the salivary secretion 161and in the gastric mucosa [7,8]. However, the relationship between systemic and local humoral immune response and the pathogenic role, if any, are poorly understood. Furthermore, the recent isolation of H. pylon’ in faeces [9] raises the question of whether the colonic mucosa also harbour H. pylon’ and a specific local antibody response is elicited. The aim of our study was to define the isotypic pattern of the serum, salivary, gastric and rectal H. pylon’-specific antibody responses and to explore their interrelationships.
286
Materials
and Methods
Subjects and sampling
The first group of patients consisted of 79 patients (42 male, 27 female; mean age: 48 f 12) who underwent upper GI endoscopy. Seventy-four were found to be H. pylori-positive by the urease quick test (CP-test) confirmed by histology (Giemsa staining). Thirty-one had duodenal ulcer (DU) and 43 complained of non-ulcer dyspepsia (NUD). The remaining 5 subjects were H. pylorinegative healthy controls. The second group of patients consisted of 28 patients (17 male, 11 female; mean age: 46 f 12) undergoing sigmoidoscopy for functional bowel disorders. Among these, 22 also underwent upper GI endoscopy as a part of a diagnostic work up and were found to be H. pylori-positive by CP-test and Giemsa staining. In all patients, samples of serum and unstimulated saliva were collected. In the 79 patients of the first group and in the 22 gastroscoped from the second group, 4 antral biopsies were taken from each patient. Four additional rectal biopsies were taken from each patient of the second group. Serum and saliva samples were spun, coded and stored at - 20” until tested. Antral and rectal biopsy samples were separately homogenated on ice using a tissue homogenizer (Ystral, GmbH, Dottingen, Germany). The homogenates were spun (600 x g for 20 min at 4”C), coded and stored at -80°C until tested. H. pylon’-specific IgG and IgA were measured in each sample of serum, saliva and mucosal (gastric and/or rectal) homogenate by direct ELISA. H. pylori-specific IgG and IgA measurement A sonicate of a whole H. pylon’ strain was used
as a source of antigen. The antigen preparation was diluted with coating buffer, added to each well and incubated for 2 h at 37°C. The plates were washed with washing buffer and binding sites were blocked by adding 2% serum albumin in washing buffer and incubated for 18 h at 4°C. The working dilution was 1:lOO for serum and 1:4 for saliva. Homogenates were normalized (10 pg ml-‘) by measuring the total protein concentration (Bio-Rad Protein Assay). Diluted serum,
saliva and homogenates were separately added to each well in duplicate and incubated for 90 min at 37°C. The plates were washed again and either anti-human IgG or IgA peroxidase conjugate (Sigma Immunochemicals) was added and incubated for 1 h at 37°C. The plates were washed again and substrate was added and left for 10 min and 35 min (for serum IgG and IgA, respectively), 35 min (for salivary and mucosal IgG and IgA) in a dark room. Stopping solution was finally added and the optical density of the wells was read at 405 nm using a microelisa plate reader (Labsystern Multiskan MCC/340). All samples were processed in the same ELISA assay. Serum H. pylori positivity was defined by using a pool of 100 negative control sera as a reference for IgG determinations (cut-off point of 2 standard deviations above the mean of the standard negative control sera) which gave a sensitivity and a specificity of 94% and 95%, respectively (data not shown). Results were expressed as mean optical density + S.D. Student’s t-test was used for data analysis.
Results
As expected, in the first group all 74 patients had a positive serology as indicated by the serum
/I OD
I
1n W 1 0 L--l IgA
(
II
I,5
1
a5
/
/
OV Serum
Fig. 1. Isotypic pylon’-infected
Saliva
/
G.Homogenates
pattern of specific antibody response in 74 H. patients (OD = optical density, mean f SD.).
287
Table 1 H. pylori-specific IgG and IgA in patients negative for H. pylon’ on histology
IgG IgA
Serum
Saliva
G. Homogenate
0.549 f. 0.143 0.298&0.165
0.222 + 0.163 0.458 + 0.372
0.098 f 0.080 0.161 kO.177
OD/ I,5
1
H. pyhi IgG (1.192 f 0.308) that were significantly (P < 0.001) higher than serum H. pylori IgA (0.592 f 0.398). In contrast, in saliva samples, H. pylon’ IgG concentration (0.825 f 0.682) was lower than that of H. pylori IgA (1.014 f 0.721). In gastric homogenates, the mean concentration of H. pylon’ IgA (0.605 f 0.467) was similar to that of H. pylori IgG (0.610 k 0.623) (Fig. 1). In the 5 negative healthy controls, specific IgG were significantly lower in all examined areas, whilst H. pylon’-specific IgA appear to differ only in terms of mucosal concentration (Table 1). No difference was found in H. pylori IgG and IgA in serum, saliva and gastric homogenates between DU and NUD patients (Fig. 2). In the second group, 26 of the 28 patients were found to be H. pylon! seropositive. In the rectal homogenates, H. pylon’ IgG were detectable in all patients, with a mean concentration of about
1
OD 1.5
-
0.5
OV
/
Sl?WtTl
/ saliva
gastric
/ horn.
/ rectal
horn.
Fig. 3. Specific IgG and IgA in serum, saliva, gastric and rectal homogenates of 22 H. pylon’-infected patients (OD = optical density, mean f S.D.).
one third of that detected in gastric homogenates (0.239 k 0.214). Specific H. pylon’ IgA were undetectable in the rectal homogenates of 12 patients and present in trace amounts in the remaining 16. In contrast, the gastric homogenates showed similar titers for H. pylori IgG (0.763 f 0.702) and H. pylori IgA (0.647 + 0.486). In the 22 patients with both gastric and rectal homogenates, both H. pylon’ IgG and IgA were significantly higher in gastric samples than in rectal samples (0.763 f 0.702 vs. 0.230 + 0.230 for IgG, P < 0.001 and 0.647 + 0.486 vs. 0.060 + 0.059 for IgA, P < 0.0001 respectively). Serum and salivary figures confirmed the first group patients’ results (Fig. 3).
Discussion
OI_L_L-L N”D SERUM
DU
NUD SALIVA
D”
NW DU G. HOMOGENATES
Fig. 2. Isotypic pattern of specific antibody response in 43 NUD and in 31 DU H. &on’-infected patients (OD = optical density, mean f S.D.).
It has become clear that H. pylon’ plays an etiological role in chronic gastritis, peptic ulcer disease, and possibly in gastric cancer. Nevertheless, information on the pathogenesis and natural history of H. pylori infection is limited. Studies of the specific circulating antibody response to H. pylori focussed primarily on the diagnostic rather than the pathogenetic role of such a response [3-51. Only recently has it been appreciated that the gastric mucosal immune response may repre-
288
sent one main pathogenetic mechanism in H pylon’-associated diseases [2,8]. In this study we further investigated the systemic and local humoral immune responses to H. pylon’ and their interrelationships. The isotypic pattern of H. pylon’-specific antibodies appeared to differ at serum, salivary, gastric and rectal mucosa level. Serum H. pylon’ IgG titers were higher than those of the serum-specific IgA. On the contrary, in saliva samples H. pylori IgA titers were higher than specific IgG titers. In gastric homogenates, specific IgG and IgA titers were similar. The marked H. pylon’ IgA response at the salivary and gastric mucosa level indicate that the specific humoral immune response to H. pylori involves the secretory immune system. Furthermore, differential analysis of the isotypic H. pylon’-specific antibody pattern in DU and NUD patients failed to produce evidence of any difference in IgG/IgA ratio in the three examined compartments of the two groups of patients. H. pylon’-specific IgG were detectable also in rectal homogenates of our patients, but in contrast to gastric mucosa, none or very low H. pylori-specific IgA were found in the same material. Data also suggest that the H. pylon’-specific local humoral immune response does not seem to occur at the rectal level but that H. pylon’ antibodies are likely to be partly of systemic origin. Consequently, our data do not support the concept that colonic mucosa harbour H. pyfori.
References Blaser, M.J. (1992) Hypotheses on the pathogenesis and natural history of Helicobacierpylorj-induced inflammation. Gastroenterology 102, 720-727. Hatz, R.A., Brooks, W.P., Kramling, H. and Enders, G. (1992) Stomach immunology and Helicobacter pylon’ infection. Curr. Opin. Gastroenterol. 8, 993-lOill. Newell, D.G. and Stacey, A.R. (19891 The serology of Campylobacter pylon’ infection. In: Campylobacter pylon’ and Gastroduodenal Disease (Rathbone, B.J. and Heatley, R.V., Eds.1, pp. 190-196. Blackwell Scientific Publications, London. Evans, D.J., Evans, D.G., Graham D.Y. and Klein, P.D. (1989) A sensitive and specific serologic test for detection of Campylobacter pylon’ infection. Gastroenterology 96, 10041008. Kosunen, T.U., Seppala, K., Sama, S. and Sipponen, P. (1992) Diagnostic value of decreasing IgG, IgA and IgM antibody titres after eradication of Helicobacter pylon’. Lancet 339, 893-895. Pate], P., Mendall, M.A., Khulusi, S. Molineaux, N. and Northfield, T.C. (1993) Detection of H. pylon’ infection by saliva. Gut 34(l), S36-T139. Rathbone, B.J., Wyatt, J.I., Worsley, B.W., Shires, S.E., Trejdosiewicz, L.K., Heatley, R.V. and Losowsky, MS. (1986) Systemic and local antibody responses to gastric Campylobacter pyloridis in non-ulcer dyspepsia. Gut 27, 642-647. 8 Crabtree, LE., Taylor J.D., Wyatt, J.I., Heatley, R.V., Shallcross, T.M., Tompkins, D.S. and Rathbone, B.J. (1991) Mucosal IgA recognition of Helicobacter pylon’ 120 kDa protein, peptic ulceration, and gastric pathology. Lancet 338, 332-335. 9 Thomas, J.E., Gibson, G.R., Darboe,M.K., Dale, A. and Weaver, L.T. (1992) Isolation of Helicobacter pylori from human faeces. Lancet 340, 1194-1195.