Integrative Diabetes and Cardiovascular Diseases β-cell Related Amyloidosis Localized to the Islets of Langerhans , Type II Diabetes Mellitus and Liponecrotic Pancreatitis in Rheumatoid Arthritis : A Postmortem Clinicopathologic Statistical Study of 234 Autopsy Patients

The aim of this study was to determine the prevalence of systemic AA amyloidosis (AAa), islet amyloidosis (IA) and liponecrotic pancreatitis (LnP) including acute liponecrotic (aLnP), acute relapsing liponecrotic (aRelLnP), and chronic liponecrotic pancreatitis (chrLnP) in rheumatoid arthritis (RA), and to analyse the possible relationship between them. Patients and methods: At the National Institute of Rheumatology 11558 patients died between 1969 and 1998; among them 234 with RA, and all of them were autopsied. RA was confirmed clinically according to the criteria of the American College of Rheumatology (ACR). The diagnosis of DM was based on clinical data. Tissue samples of pancreas were available for histologic evaluation in 164 of 234 patients. AAa, IA and LnP were diagnosed histologically. Demographics of different patient cohorts were compared with the Student (Welch) t-probe. The relationships between AAa and IA, furthermore between IA and DM or LnP (including aLnP, aRelLnP, chrLnP) were analyzed by Pearson’s chi-squared (c2) test. Results: AAa complicated RA in 42 (25.61%) of 164 patients. IA localized to the islets of Langerhans was observed in 16 (9.76%) of 164 pancreases. Clinically diagnosed DM was associated with RA in 31 (18.90%) of 164 patients. Pancreatitis with multiple liponecrotic foci (LnP) was found in 19 (11.58%) of 164 patients; aLnP existed in 9 (47.37%), aRelLnP in 4 (21.05%), and liponecrotic foci in combination with chronic fibrotic pancreatitis (chrLnP) in 6 (31.58%) of these 19 patients. Discussion and conclusions: There was no significant difference between female and male RA patients associated with AAa, IA, DM and LnP. The age, sex and onset of disease did not influence basically the prevalence of AAa, IA, DM and LnP except male patients with IA, whose mean age at death was significantly higher than the general RA population. IA (fibrillar amyloid IAPP deposits -AIAPP) is related to the activity of b-cells and may presumably be a faulty product of b-cells (normal islets of Langerhans do not contain IA deposits). The progressive deposition of IAPP prohormon fragments inhibits the function of b-cells because of their toxic effect and/or blocking mechanically the blood supply of b-cells and they “die in their own product”. The significant correlation between IA and DM refers to a close connection between them, but not necessarily a direct cause and effect relationship; it may be an indirect result of damaged (apoptotic) b-cells. The early stage of IA is characterized by minimal IAPP deposits involving only a few islets, which represents a clinically latent DM, and the advanced stage of IA is characterized by massive IAPP Research Article ISSN: 2637-4587

The aim of this study was to determine the prevalence of AAa, IA and liponecrotic pancreatitis (LnP) including acute liponecrotic (aLnP), acute relapsing liponecrotic (aRelLnP), and chronic liponecrotic pancreatitis (chrLnP) in RA, and to analyse the possible relationship between them.

Patients and Methods
At the National Institute of Rheumatology 11558 patients died between 1969 and 1998; among them 234 with RA, and all of them were autopsied.RA was confirmed clinically according to the criteria of the American College of Rheumatology (ACR) [42].The diagnosis of DM was based on clinical data.
AAa and IA (amyloid A and IAPP deposition) was diagnosed histologically according to Romhányi [43] by a modified (more sensitive) Congo red staining [44].AAa and IA deposits were identified in serial sections by immunohistochemical and histochemical methods [45,46].Tissue samples of pancreas were available for deposits involving most of the islets, which correspond to clinically manifest DM.Based on the positive and significant correlation between IA and clinically not diagnosed DM, IA may be a good indicator of potential DM in the latent stage of disease.Therefore we recommend that all biopsy material and surgical specimens of pancreas to be tested for IA or IAPP deposition.

Glossary of definitions
Prevalence of AAa was specified histologically based on the presence of amyloid A in blood vessels of different calibers or in different tissue structures of five organs (heart, lung, liver, kidney and pancreas) in each patient [50].Prevalence of IA concerns the presence of islet amyloid polypeptide (IAPP) prohormone fragment deposition localized to the islets of Langerhans [2].

Histologic patterns of pancreatitis:
Liponecrotic pancreatitis (LnP) -multiple acinar liponecrotic foci, with or without inflammatory reaction, with or without hemorrhage.

Acute liponecrotic pancreatitis (aLnP):
Acinar liponecrotic foci usually in the same stage and similar size of necrosis, with or without inflammatory reaction, with or without hemorrhage.

Chronic pancreatitis (chrP):
Multifocal or diffuse fibrotic interstitial pancreatitis with more or less pronounced glandular atrophy, with or without ductal changes: pluges of inspissated secretion of exocrine glands), ductal dilatation, ductal proliferation and/or epithelial metaplasia.
The prevalence of chrP and eIP were not evaluated in this study.
Demographics, onset and duration of RA complicated by AAa or associated with IA, DM, LnP (including aLnP, aRelLnP or chrLnP) are summarized in table 1.
Comparing the age, sex, onset of RA, and duration of disease there was no significant difference between female and male RA patients (n = 164) with AAa (n = 42), IA (n = 16), DM (n = 31) and LnP (n = 19, except male patients with IA, whose mean age at death was significantly higher (73.57years versus 65.57; p < 0.035) in comparison with average age of the others.
The mean age of RA patients associated with aLnP was significantly higher at the onset of disease (65.06 years versus 51.43; p < 0.00025), and these patients died significantly earlier (4.31 years versus 14.64; p < 0.026) than the average of others.The tendency was similar both in women (64.08 years versus 50.82; p < 0.00002, and 2.92 years versus 15.32; p < 0.089 -NS) and men (68.00 years versus 52.85; p < 0.556 -NS, and 8.50 years versus 13.05; p < 0.089 -NS).
RA started earlier in patients with aRelLnP (48.00 years versus 51.43; p < 0.046), with longer disease duration (19.75 years versus 14.64; p < 0.563 -NS) than in the average of others.The tendency regarding the onset of RA and disease duration was similar both in women (46.00 years versus 50.82; p < 0.521 -NS, and 19.00 versus 15.32 -NS) and in men (50.00 years versus 52.85; p < 0.00006, and 20.50 years versus 13.05; p < 0.852 -NS).
The statistical links ("p" values of significance) between RA patients complicated by AAa or associated with IA, DM, LnP (including aLnP, aRelLnP or chrLnP) are summarized in table 2.
IA was associated with clinically diagnosed DM in 9 (56.25%) of 16 patients.There was a positive and significant correlation between clinically diagnosed IA and DM (association coefficient: 0.7608, χ² = 16.1324,p < 0.00006).IA was present without the clinical diagnosis of DM in 7 (43.75%) of 16 patients.The relationship between IA and clinically not diagnosed DM was also positive and significant (association coefficient: 0.6014, χ² = 7.1407, p < 0.007).
The statistical links ("p" values of significance) between coexistant AAa, IA, DM and LnP (including aLnP, aRelLnP or chrLnP) are summarized in table 3.
The distribution of a-and b-cells in the islets of Langerhans is demonstrated in figures 1 and 2 and the progressive process of IA in figures 3-15.Figures 3-6 show an early stage of deposition, Figure 7

Discussion
IA (fibrillar amyloid IAPP deposits) is regarded as a key factor in the pathogenesis of type 2 diabetes [38,39].IAPP is a normal b-cell component [51,52], synthetized by the b-cells, co secreted and released with insulin [52,53], unique (other than insulin) [35], and in type 2 diabetes is deposited as amyloid fibrils in the islets of Langerhans [51].
Our data indicate that IAPP -related amyloidosis localized to the islets of Langerhans is a progressive and cumulative process, like any form and type of systemic or localized amyloidosis [1].The ratio of islets of Langerhans with amyloid deposits and the amount of deposited prohormone fragments (IAPP) per islet depends on the stage of insular amyloidosis.According to our data IAPP deposition is a localized process connected to the b-cells in agreement with others [35,[53][54][55][56].
Recent studies confirm the reduced number and abnormal function of β-cells [57][58][59] accompanied with increased insulin secretion by the remaining β-cells [58].In our patients the number of a-, b-, g-, and d-cells decreased in proportion to the amount of deposited IAPP, and the number of apoptotic cells increased.IAPP deposits inhibit the glucagon secretion of a-cells [60].For The high value of association coefficient (c = 0.7608) and the positive and significant correlation between IA and DM (χ² = 16.1324,p < 0.00006) refer to a very close connection, but this does not necessarily mean a cause and effect relationship; an associated phenomenon seems more likely.Several etiologic factors (insulin resistance, defective receptors, hyperglycemia, hyperlipidemia, antigens etc.) may play a role in the development of DM (see below).
The etiology of DM is probably polygenetic, influenced by environmental effects: [63,64].For this reason between identical twins the concordance is only 90-95% [65].It is generally accepted that DM is caused by insulin resistance of peripheral receptors in skeletal muscle, liver, adipose tissue.Insulin resistance is also present in the cells of islets of Langerhans.The alpha-cells produce more glucagon, because of insulin resistance and lack of glucose within the cells [66].According to some theories the defect of insulin receptors is determined genetically [67].Some other popular theories include sparing genes [68,69], hyperglycemia, hyperlipidemia [70], islet amyloidosis [35,36,55] or low birth weight [71].
In our opinion IAPP deposition is not the cause of DM, rather an inherent (accompanying) phenomenon or faulty product of the overloaded and exhausted b-cells Apart from this the positive and significant correlation between IA and clinically not diagnosed DM, IA may be a good indicator of potential DM in the latent stage of disease.This correlation may help recognize DM in its early stage.For this reason we recommend that all biopsy material and surgical specimens of pancreas to be tested for IA or IAPP deposition.Our data do not support the theory that IAPP influences the prevalence of LnP, including aLnP, aRelLnP or chrLnP, in contrast with the opinion of Noel et al. [70] or Lai et al. [71], only patients with DM had a higher risk of acute pancreatitis, more precisely of aLnP, based ont he strong relationship between them (association coefficient = 0.58269, χ² = 4.0526 p < 0.044) [72,73].
Systemic or localized types of amyloidosis may exist simulaneously side by side or may be present independently from each other.AAa and IA are independent phenomena which may coexist in RA, based on the negative association coefficient and absence of significant relationship.Severe AAa may involve blood vessels of different calibers of the pancreas and should be regarded as important factors in the pathogenesis of aRelLnP [74].The role of severe AAa in pathogenesis of fatal acute pancreatitis is confirmed by others as well [75][76][77].
There was no significant difference between female and male RA patients associated with AAa, IA, DM and LnP.The age, sex and onset of disease did not influence basically the prevalence of AAa, IA, DM and LnP except male patients with IA, whose mean age at death was significantly higher (73.57years versus 65.57; p < 0.035) than the general RA population.

Conclusions
IA (fibrillar amyloid IAPP deposits) is related to the activity of b-cells and may presumably be a faulty product of b-cells (normal islets of Langerhans do not contain IA deposits).The progressive deposition of IAPP prohormon fragments inhibits the function of b-cells because of their toxic effect and/or blocking mechanically the blood supply of b-cells and they "die in their own product".The significant correlation between IA and DM refers to a close connection between them, but not necessarily a direct cause and effect relationship; it may be an indirect result of demaged (apoptotic) b-cells.The early stage of IA is characterized by minimal IAPP deposits involving only a few islets, which represents a clinically latent DM, and the advanced stage of it is characterized by massive IAPP deposits involving most of the islets, which correspond to clinically manifest DM.
Based on the positive and significant correlation between IA and clinically not diagnosed DM, IA may be a good indicator of potential DM in the latent stage of disease.Therfore we recommend that all biopsy material and surgical specimens of pancreas to be tested for IA or IAPP deposition.
an advanced, Figures 8-10 a late, and Figures 11-15 the terminal stage of amyloid fibril deposition of IAPP.

Figure 1 :
Figure 1: Brown colour of immunohistochemical reaction indicate the normal distribution of a-cells in islets of Langerhans.a-cells are positive for anti-human Glucagon (a) a: hu Glucagon dilution 1:150 polyclonal antibody A0565; DAKO, Glostrup, Denmark, was used to detect the a-cells, Streptavidin-biotin-complex/horseradish peroxidase reaction, x100; (b) Same as (a) x200.

Figure 5 :Figure 6 :
Figure 5: Pancreas, with minimal deposits of AIAPP localized to islets of Langerhans, early stage of IA.(a) Congo red staining, without alcoholic differentiation, covered with gum arabic, x100; (b) Same as Figure (a) x200.

Figure 7 :
Figure 7: Islets of Langerhans without AIAPP deposits and with moderate amount of AIAPP deposits, advanced stage of IA.(a) H-E, x100; (b) Same as (a) PAS, x100.

Figure 7c :
Figure 7c: Congo red staining according to Romhányi, without alcoholic differentiation, covered with gum arabic, viewed under polarized light, x100.

Figure 7f :
Figure 7f: Islet amyloid polypeptide -AIAPP deposits are negative for P-component (note the positive cross reaction of elastic fibres).

Figure 8 a
Figure 8 a,b: Pancreas, with massive deposits of AIAPP loc

Figure 12 :
Figure 12: Pancreas, massive diffuse deposits of AIAPP localized to islets of Langerhans, terminal stage of IA.

Figure 13 :
Figure 13: Pancreas, massive diffuse deposits of AIAPP localized to islets of Langerhans, terminal stage of IA.(a) Congo red staining, without alcoholic differentiation, covered with gum arabic, x100; (b) Same as Figure (a) x200.

Figure 14 :
Figure 14: Pancreas, massive deposits of AIAPP localized to islets of Langerhans, terminal stage of IA.(a) Congo red staining, without alcoholic differentiation, covered with gum arabic and viewed under polarized light, x100; (b) Same as Figure (a) x200.
Arthritis AAa: systemic AA amyloidosis IA: pancreatic IA amyloidosis LnP: Liponecrotic Pancreatitis aLnP: acute Liponecrotic Pancreatitis aRelLnP: acute Relapsing Liponecrotic Pancreatitis chrLnP: chronic Liponecrotic Pancreatitis normal function the harmonic interactions of a-and bcells are essential [61].Unfortunately, the kinetics of b-cell mass reduction and dysfunction as well as the underlying mechanisms remain unclear [62].

Table 3 :
The statistical links ("p" values of significance) of coexistent complications and associated diseases in 164 RA patients.