Low-Grade Enteroviral Infection in Newly Diagnosed T1DM
Low-Grade Enteroviral Infection in Newly Diagnosed T1DM
This study of fresh pancreatic tissue collected close to the time of diagnosis of type 1 diabetes suggests that a low-grade enteroviral infection is sustained within the islets of Langerhans. Thus, enterovirus RNA was amplified successfully by RT-PCR from four of the six patients in two independent laboratories. The presence of enterovirus sequences was also confirmed by sequencing the PCR products. Moreover, the enterovirus capsid protein VP1 was detected immunohistochemically in islet cells of all six patients. The expression of VP1 is known to be most intense during the acute phase of an enterovirus infection, whereas it is reduced during persistent infection. This is because enteroviral persistence is characterized by naturally occurring deletions at the 5' terminus of the genome, which reduce the replication rate of the virus. It was recently shown that these terminally deleted viruses also can persist after inoculation in murine pancreas in the absence of cytopathic virus weeks after the acute infectious period. Hence, the present results are consistent with the possibility that a low-grade enterovirus infection was established and maintained in the islet cells of the patients with type 1 diabetes. The biopsy samples were taken from the pancreatic tail, not discarding that the infection could be affecting other parts first before slowly disseminating to the rest of the tissue. The lack of virus-induced cytopathic effects in islets and exocrine cell clusters during 3–5 days of culture indicates that the virus is not powerfully cytolytic, implying that the virus might be rendered replication deficient during the development of a persistent infection.
It is well understood that the amplification achieved by PCR allows for the detection of even very small quantities of target RNA; thus, a significant finding is that four of the type 1 diabetic patients were positive for viral RNA by RT-PCR in two different laboratories. The detection of positive signals required as many as 40 cycles of amplification or the use of a nested RT-PCR method, indicating that only very small amounts of viral RNA were present. Sequencing confirmed that amplified sequences originated from enteroviruses.
The high sensitivity of PCR makes it susceptible for false-positive results due to viruses that may contaminate the samples during the analysis. Several actions were taken to avoid such contaminations and enable their detection if they occur. First, the two virus laboratories got the same results even though they carried out all RNA extraction and RT-PCR steps independently and used different primers and PCR protocols. Second, the amplified enterovirus sequences differed from each other, suggesting that they originated from different enterovirus strains and thus excluding a common contaminating virus. Third, exocrine cells isolated from the same pancreata in the same time and place as well as virus-negative internal control samples included in each test run were all PCR negative. In addition, all pancreas samples were PCR negative for all other tested viruses. Finally, pancreata from nondiabetic controls were PCR negative as determined using exactly the same PCR procedures and in the same laboratory as the samples from diabetic patients.
The antibody used to detect VP1 is known to recognize this protein from multiple enteroviruses in formalin-fixed samples. However, it has also been shown that under some conditions, the antibody may label certain human proteins. We were careful to use the antibody under conditions optimized to avoid such interactions without compromise of virus-specific binding. Thus, we are confident that the immunolabeling achieved in human pancreas sections is likely to represent the presence of viral protein.
In addition to virus-specific markers, the expression of class I HLA molecules was upregulated in the islets of all type 1 diabetic patients. This fits with previous observations showing that pancreatic islets of type 1 diabetic patients hyperexpress class I HLA molecules and interferon α. This might indicate ongoing virus-induced interferon secretion in the islets. We have previously shown that enterovirus infection in human pancreatic islets leads to such class I HLA hyperexpression in vitro partly mediated by secretion of type 1 interferons.
This study did not include pancreatic biopsy samples from healthy living individuals. Although not ideal, the nondiabetic organ donors from nPOD are clinically well defined and age matched to the patient cases. The mean age of the subjects from whom islets were isolated for the culture studies was higher than the patient cases, but we would emphasize that they served mainly as methodological controls in PCR analyses, being negative for all tested viruses in PCR.
In conclusion, DiViD is the first study of living, newly diagnosed type 1 diabetic patients to demonstrate the presence of enterovirus in pancreatic islets using multiple techniques across several independent laboratories, including the detection of enterovirus-specific sequences in the islets. The results do not prove causality between enterovirus infection and type 1 diabetes, but they support the view that a low-grade enteroviral infection is present in the islets of Langerhans at diagnosis of type 1 diabetes. These findings should encourage studies in which antiviral medication and/or vaccines against enteroviruses could be tested to reduce disease progression and prevent type 1 diabetes.
Discussion
This study of fresh pancreatic tissue collected close to the time of diagnosis of type 1 diabetes suggests that a low-grade enteroviral infection is sustained within the islets of Langerhans. Thus, enterovirus RNA was amplified successfully by RT-PCR from four of the six patients in two independent laboratories. The presence of enterovirus sequences was also confirmed by sequencing the PCR products. Moreover, the enterovirus capsid protein VP1 was detected immunohistochemically in islet cells of all six patients. The expression of VP1 is known to be most intense during the acute phase of an enterovirus infection, whereas it is reduced during persistent infection. This is because enteroviral persistence is characterized by naturally occurring deletions at the 5' terminus of the genome, which reduce the replication rate of the virus. It was recently shown that these terminally deleted viruses also can persist after inoculation in murine pancreas in the absence of cytopathic virus weeks after the acute infectious period. Hence, the present results are consistent with the possibility that a low-grade enterovirus infection was established and maintained in the islet cells of the patients with type 1 diabetes. The biopsy samples were taken from the pancreatic tail, not discarding that the infection could be affecting other parts first before slowly disseminating to the rest of the tissue. The lack of virus-induced cytopathic effects in islets and exocrine cell clusters during 3–5 days of culture indicates that the virus is not powerfully cytolytic, implying that the virus might be rendered replication deficient during the development of a persistent infection.
It is well understood that the amplification achieved by PCR allows for the detection of even very small quantities of target RNA; thus, a significant finding is that four of the type 1 diabetic patients were positive for viral RNA by RT-PCR in two different laboratories. The detection of positive signals required as many as 40 cycles of amplification or the use of a nested RT-PCR method, indicating that only very small amounts of viral RNA were present. Sequencing confirmed that amplified sequences originated from enteroviruses.
The high sensitivity of PCR makes it susceptible for false-positive results due to viruses that may contaminate the samples during the analysis. Several actions were taken to avoid such contaminations and enable their detection if they occur. First, the two virus laboratories got the same results even though they carried out all RNA extraction and RT-PCR steps independently and used different primers and PCR protocols. Second, the amplified enterovirus sequences differed from each other, suggesting that they originated from different enterovirus strains and thus excluding a common contaminating virus. Third, exocrine cells isolated from the same pancreata in the same time and place as well as virus-negative internal control samples included in each test run were all PCR negative. In addition, all pancreas samples were PCR negative for all other tested viruses. Finally, pancreata from nondiabetic controls were PCR negative as determined using exactly the same PCR procedures and in the same laboratory as the samples from diabetic patients.
The antibody used to detect VP1 is known to recognize this protein from multiple enteroviruses in formalin-fixed samples. However, it has also been shown that under some conditions, the antibody may label certain human proteins. We were careful to use the antibody under conditions optimized to avoid such interactions without compromise of virus-specific binding. Thus, we are confident that the immunolabeling achieved in human pancreas sections is likely to represent the presence of viral protein.
In addition to virus-specific markers, the expression of class I HLA molecules was upregulated in the islets of all type 1 diabetic patients. This fits with previous observations showing that pancreatic islets of type 1 diabetic patients hyperexpress class I HLA molecules and interferon α. This might indicate ongoing virus-induced interferon secretion in the islets. We have previously shown that enterovirus infection in human pancreatic islets leads to such class I HLA hyperexpression in vitro partly mediated by secretion of type 1 interferons.
This study did not include pancreatic biopsy samples from healthy living individuals. Although not ideal, the nondiabetic organ donors from nPOD are clinically well defined and age matched to the patient cases. The mean age of the subjects from whom islets were isolated for the culture studies was higher than the patient cases, but we would emphasize that they served mainly as methodological controls in PCR analyses, being negative for all tested viruses in PCR.
In conclusion, DiViD is the first study of living, newly diagnosed type 1 diabetic patients to demonstrate the presence of enterovirus in pancreatic islets using multiple techniques across several independent laboratories, including the detection of enterovirus-specific sequences in the islets. The results do not prove causality between enterovirus infection and type 1 diabetes, but they support the view that a low-grade enteroviral infection is present in the islets of Langerhans at diagnosis of type 1 diabetes. These findings should encourage studies in which antiviral medication and/or vaccines against enteroviruses could be tested to reduce disease progression and prevent type 1 diabetes.
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