Cyberbiosecurity Challenges of Pathogen Genome Databases.

Pathogen detection, identification, and tracking is shifting from non-molecular methods, DNA fingerprinting methods, and single gene methods to methods relying on whole genomes. Viral Ebola and influenza genome data are being used for real-time tracking, while food-borne bacterial pathogen outbreaks and hospital outbreaks are investigated using whole genomes in the UK, Canada, the USA and the other countries. Also, plant pathogen genomes are starting to be used to investigate plant disease epidemics such as the wheat blast outbreak in Bangladesh. While these genome-based approaches provide never-seen advantages over all previous approaches with regard to public health and biosecurity, they also come with new vulnerabilities and risks with regard to cybersecurity. The more we rely on genome databases, the more likely these databases will become targets for cyber-attacks to interfere with public health and biosecurity systems by compromising their integrity, taking them hostage, or manipulating the data they contain. Also, while there is the potential to collect pathogen genomic data from infected individuals or agricultural and food products during disease outbreaks to improve disease modeling and forecast, how to protect the privacy of individuals, growers, and retailers is another major cyberbiosecurity challenge. As data become linkable to other data sources, individuals and groups become identifiable and potential malicious activities targeting those identified become feasible. Here, we define a number of potential cybersecurity weaknesses in today's pathogen genome databases to raise awareness, and we provide potential solutions to strengthen cyberbiosecurity during the development of the next generation of pathogen genome databases.

Exceptional Multiple Foreign-Body Ingestion by a Patient with Schizophrenia.

Swallowing foreign body in adult is uncommon. This mostly occurs accidentally or in psychologically unsound patient. A 32-years-old male patient with abdominal pain admitted in surgery department of Mymensingh Medical College Hospital, Mymensingh, Bangladesh with a history of swallowing various objects. After endoscopic confirmation and psychological evaluation he underwent laparotomy and 29 different objects were removed from his stomach by Gastrotomy. He was psychiatrically evaluated after recovery from operation and was found to be suffering from Schizophrenia with cannabis use. The aim of reporting this case can raise awareness at the patients complains should be taken seriously to prevent morbidity and even mortality.

Duration of benefit following completion of pulmonary rehabilitation in interstitial lung disease-an observational study.

BACKGROUND: It remains unclear for how long the benefits of pulmonary rehabilitation (PR) last in interstitial lung disease (ILD). An increasing number of ILD patients complete PR and it is vital they be offered the most beneficial approaches. METHODS: This is a retrospective, observational study of a cohort with ILD who had completed PR. Incremental shuttle walk (ISWT) and chronic respiratory disease questionnaire (CRDQ) were compared before PR, at course completion, and 6/12 months follow-up. Focus group discussions with ILD participants who had completed PR and their carers established qualitative views on existing and potential future PR provision. RESULTS: 79 participants with ILD were identified at course completion, with 39 followed to 12 months. 11 participants died during follow-up. Initial benefits from PR were not sustained at 6 months (ISWT change 0.0m (95% CI-23.2 to 23.2 m), CRDQ change 2.5 (95% CI-2.4 to 7.4)) and 12 months (ISWT change-0.7 m (95% CI-37.3 to 35.9 m), CRDQ change 4.0 (95% CI-2.2 to 10.2)). Continued home exercise gave longer lasting benefit in exercise capacity. Focus group discussions highlighted the value attached to PR and suggested areas for improvement. CONCLUSIONS: Standard PR gives initial benefits in participants with ILD who complete the course, however these are not sustained. Tailored approaches to this group would be appreciated by this group and should be explored.

Small Bowel Perforation during Suprapubic Trocar Cystotomy: A Case Report.

A 35 years old man presented with retention of urine secondary to meatal stenosis with bulbar urethral stricture. He had a distended, palpable, tender urinary bladder. Urethral catheterization and dilatation was tried but failed. A trocar cystostomy was performed under local anaesthesia, which led to the injury to the small bowel when least expected. This is a rare but well recognized complication of small bowel injury following blind trocar suprapubic cystostomy when it was least expected and as such had a significant bearing on its management. We discuss its subsequent management and possible mechanism underlying this unexpected and unfortunate complication in the given circumstances.

Admission levels and early changes in serum interleukin-10 are predictive of poor outcome in acute liver failure and decompensated cirrhosis.

BACKGROUND & AIM: Immunoparesis contributes to prognosis in acute liver failure (ALF) and decompensated cirrhosis, a phenomenon thought to be mediated by the anti-inflammatory cytokine interleukin (IL)-10. We investigated the prognostic value of admission IL-10 levels and their evolution during the early phase of treatment in intensive care, in comparison to the pro-inflammatory cytokines IL-6 and tumour necrosis factor (TNF)-alpha. METHODS: We measured these cytokines within 48 h of admission in 51 ALF and 39 decompensated cirrhosis patients admitted to intensive care, and obtained follow-up measurement a median of 2 days later in 35 patients. RESULTS: Levels of all cytokines were higher in those with a poor outcome. IL-10 performed as well as TNF-alpha and IL-6 in the whole cohort (area under receiver operator curve 0.73 vs 0.66 and 0.72). However IL-10 outperfomed pro-inflammatory cytokines in the subgroups with ALF (0.80 vs 0.63 and 0.70) and acetaminophen-induced ALF (0.92 vs 0.67 and 0.81). Levels of all cytokines rose significantly in non-surviving patients (n=15); IL-10 by a factor of 2, TNF-alpha by 2.6 and IL-6 by 1.13. No significant changes were seen in the surviving patients. In ALF, IL-10 was an independent predictor of outcome in multivariate analysis. CONCLUSION: The magnitude of the compensatory anti-inflammatory response at admission, and its development during the early phase of treatment, predicts outcome as well as the pro-inflammatory response in acute hepatic syndromes and supports a vital role for this immunological phenomenon in the outcome of these patients.

Determination of methadone in urine using ion trap GC/MS in positive ion chemical ionization mode.

Methadone is a potent analgesic and sedative. It is widely used in the treatment of heroin addiction and is often encountered in forensic specimens. In electron impact (EI) gas chromatography/mass spectrometry (GC/MS) mode, methadone produces predominantly a m/z 72 ion, which is not sufficiently characteristic for identification. Determination of the molecular ion, which can be achieved using chemical ionization (CI) provides diagnostic information and better identification of the drug. This paper describes the development of a positive ion CI GC/MS procedure, using a liquid reagent gas and ion trap instrumentation, for the determination of methadone and its metabolites in urine. CI generally produces a single molecular ion spectrum, but optimization of the reagent gas parameters increases the fragmentation of the molecule, allowing determination of ion ratios if required. The procedure is sensitive, diagnostic and is currently in routine use in our laboratory.

High affinity binding between lipoprotein lipase and lipoproteins involves multiple ionic and hydrophobic interactions, does not require enzyme activity, and is modulated by glycosaminoglycans.

Lipoprotein lipase (LPL) physically associates with lipoproteins and hydrolyzes triglycerides. To characterize the binding of LPL to lipoproteins, we studied the binding of low density lipoproteins (LDL), apolipoprotein (apo) B17, and various apoB-FLAG (DYKDDDDK octapeptide) chimeras to purified LPL. LDL bound to LPL with high affinity (K(d) values of 10(-12) m) similar to that observed for the binding of LDL to its receptors and 1D1, a monoclonal antibody to LDL, and was greater than its affinity for microsomal triglyceride transfer protein. LDL-LPL binding was sensitive to both salt and detergents, indicating the involvement of both hydrophobic and hydrophilic interactions. In contrast, the N-terminal 17% of apoB interacted with LPL mainly via ionic interactions. Binding of various apoB fusion peptides suggested that LPL bound to apoB at multiple sites within apoB17. Tetrahydrolipstatin, a potent enzyme activity inhibitor, had no effect on apoB-LPL binding, indicating that the enzyme activity was not required for apoB binding. LDL-LPL binding was inhibited by monoclonal antibodies that recognize amino acids 380-410 in the C-terminal region of LPL, a region also shown to interact with heparin and LDL receptor-related protein. The LDL-LPL binding was also inhibited by glycosaminoglycans (GAGs); heparin inhibited the interactions by approximately 50% and removal of trace amounts of heparin from LPL preparations increased LDL binding. Thus, we conclude that the high affinity binding between LPL and lipoproteins involves multiple ionic and hydrophobic interactions, does not require enzyme activity and is modulated by GAGs. It is proposed that LPL contains a surface exposed positively charged amino acid cluster that may be important for various physiological interactions of LPL with different biologically important molecules. Moreover, we postulate that by binding to this cluster, GAGs modulate the association between LDL and LPL and the in vivo metabolism of LPL.

Immunopathogenesis of hepatitis B virus recurrence after liver transplantation.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) recurrence after orthotopic liver transplantation is associated with inflammatory graft changes, despite immunosuppression and donor/recipient HLA mismatch. We investigated whether immune mechanisms are involved in the pathogenesis of hepatitis B after liver transplantation. METHODS: The virus-specific T helper (Th) cell response, activation of Th1/Th2 subpopulations, donor/recipient HLA, and expression of tumor necrosis factor (TNF)-alpha/TNF receptors were determined in 28 patients who underwent transplantation for HBV-related cirrhosis (17 with HBV recurrence and 11 without recurrence) in comparison to 30 nontransplant patients with chronic hepatitis B. RESULTS: Orthotopic liver transplantation recipients with HBV recurrence showed significant hepatitis B core antigen-specific T-cell proliferation, comparable to nontransplant patients, which was not present in transplant recipients without recurrence. In addition, hepatic and serum interleukin (IL)-2, interferon-gamma, and TNF-alpha were enhanced, without changes in IL-4 and IL-10. Phenotypically, hepatic infiltrates in allografts with HBV recurrence were comprised of CD4+ lymphocytes and macrophages with a correlation between interferon-gamma- and TNF-alpha-producing cells and the degree of necroinflammatory activity. There was a marked up-regulation of both TNF-alpha receptors, significantly greater than in nontransplant patients. CONCLUSIONS: These findings suggest that despite immunosuppression, HLA class I-independent immune mechanisms have a significant pathogenic role in liver damage associated with HBV recurrence after liver transplantation.

Cytokine overproduction in healthy first degree relatives of patients with IDDM.

Healthy family members of patients with insulin-dependent diabetes mellitus (IDDM) are known to share a number of immunological abnormalities with their affected relatives. Since monocyte and type 1 T-cell-derived cytokines contribute to the pathogenesis of IDDM, we studied the production of these cytokines in the healthy first degree relatives of 29 children with IDDM. We report that circulating tumour necrosis factor-alpha (TNF-alpha) and soluble interleukin-2 (sIL-2) receptor were present in increased amounts in non-diabetic family members at levels similar to those found in the diabetic children (duration of disease 3 months-5 years). Furthermore, marked hypersecretion of IL-1alpha and TNF-alpha by mitogen-stimulated peripheral blood mononuclear cells was found in both diabetic and healthy family members. Abnormalities of cytokine production in healthy relatives did not correlate with the presence of islet cell antibodies or with HLA DR type. These data indicate that healthy family members of patients with IDDM exhibit overproduction of a number of cytokines that have been implicated in diabetogenesis.

Lymphocyte vaccination protects prediabetic non-obese diabetic mice from developing diabetes mellitus.

In the therapeutic manoeuvre termed "lymphocyte vaccination", activated lymphocytes capable of transferring an autoimmune disease are instead attenuated and given in vaccine form. We have previously shown that such a therapy administered to non-obese diabetic (NOD) mice at 6 weeks of age prevents diabetes mellitus. To assess whether this therapy has potential clinical relevance, in the present study lymphocyte vaccination was applied in NOD mice in 3 weekly doses commencing in the immediate prediabetic period (age 12 weeks), when insulitis is advanced and diabetes incipient. Of 30 NOD mice receiving active vaccine (composed of attenuated lymphocytes from diabetic NOD mice) 13 (43.3%) remained non-diabetic to the age of 30 weeks, in comparison with 2 of 30 (6.7%; p < 0.01) mice receiving a control vaccine (composed of attenuated lymphocytes from non-diabetic NOD/B10 mice) and 5 of 26 (19.2%; p < 0.01) mice receiving saline carrier alone. Moreover, in an additional group of 10 NOD mice receiving active vaccine weekly between 12 and 30 weeks, 8 remained diabetes free at the end of the treatment. The most notable effect of the vaccine was that the delay in diabetes onset was accompanied by a reduction in insulitis and in some cases a complete absence of infiltrating lymphocytes at 30 weeks of age. Immunocytochemistry indicated that when present, islet infiltrating lymphocytes in non-diabetic mice that received active vaccine showed significantly reduced staining for interferon-gamma, compared with the infiltrate seen in diabetic mice receiving the control vaccine or saline. This study demonstrates that the rapid progression to diabetes typically seen in 12-week-old NOD mice can be delayed by lymphocyte vaccination, supporting the possibility that a vaccine composed of attenuated autologous peripheral blood lymphocytes could be effective in high risk first degree relatives of patients with insulin dependent diabetes mellitus.

Elevated serum levels of macrophage-derived cytokines precede and accompany the onset of IDDM.

To determine whether cytokines could have a role in the development of insulin-dependent diabetes mellitus (IDDM), we measured serum levels of cytokines derived from T helper 1 (interleukin-2 and interferon-gamma), T helper 2 (interleukin-4 and interleukin-10) lymphocytes and macrophages (tumour necrosis factor-alpha, interleukin-1 alpha and interleukin-1 beta) in patients before and after the onset of IDDM. Recently diagnosed IDDM patients had significantly higher levels of interleukin-2, interferon-gamma, tumour necrosis factor-alpha and interleukin-1 alpha than patients with either long-standing IDDM, non-insulin-dependent diabetes (NIDDM), Graves' disease, or control subjects (p < 0.05 for all). Compared with control subjects, patients with long-standing IDDM and those with NIDDM had higher interleukin-2 and tumour necrosis factor-alpha levels (p < 0.01 for all). Interleukin-4 and interleukin-10 were detectable in sera of patients with Graves' disease only, while interleukin-1 beta was not detectable in the serum of any control or test subject. To investigate whether high cytokine levels precede the onset of IDDM, we studied 28 non-diabetic identical co-twins of patients with IDDM, followed-up prospectively for up to 6 years after the diagnosis of the index. Levels of tumour necrosis factor-alpha and interleukin-1 alpha were elevated above the normal range more frequently in the eight twins who developed diabetes than in those 20 who did not (p < 0.005). Analysis of T helper 1 and T helper 2 profiles of the twin groups did not reveal a clear difference between prediabetic twins and twins remaining non-diabetic. These results support the notion that T helper 1 lymphocytes may play a role in the development of IDDM. This is associated with release of macrophage-derived cytokines, which is also a feature of the prediabetic period. The lack of evidence of a dominant T helper 1 profile of cytokine release before diabetes onset suggests that additional events, activating this arm of the cellular immune response, are required in the immediate prediabetic period.

Hepatic expression of tumour necrosis factor-alpha in chronic hepatitis B virus infection.

AIM: To determine the hepatic expression of tumour necrosis factor-alpha (TNF alpha) in patients with chronic hepatitis B virus (HBV) infection. METHODS: Frozen liver biopsy sections from 19 patients with chronic HBV infection were studied, 12 of whom were HBeAg positive and 10 serum HBV DNA positive. Hepatic expression of TNF alpha was determined using immunohistochemistry. RESULTS: Only infiltrating mononuclear cells showed immunoreactive staining for TNF alpha (median 2, range 0-3; n = 19) which appeared as diffuse positive staining material in the cytoplasm. Patients with active liver disease, assessed histologically and biochemically, had a higher level of expression, both in the number of TNF alpha positive cells and the proportion of TNF alpha positive infiltrating mononuclear cells. There was no correlation between the expression of TNF alpha and serological parameters of viral infection (HBeAg and HBV DNA status and HBV DNA concentrations). CONCLUSION: Hepatic expression of TNF alpha is increased in chronic HBV infection and is related to the activity of liver disease and not to the level of HBV replication.

Cellular expression of tumour necrosis factor-alpha and interferon-gamma in the liver biopsies of children with chronic liver disease.

The liver biopsies of patients with autoimmune liver diseases have a dense portal tract mononuclear cell infiltrate. To investigate whether these cells produce tumour necrosis factor-alpha and interferon-gamma, cytokines which could be involved in the autoimmune attack through a direct cytopathic effect and/or through induction/enhancement of major histocompatibility complex antigen expression, we immunohistochemically stained cryostat liver sections from 21 children with autoimmune liver disease and from 15 children with metabolic liver disorders and histological evidence of portal tract inflammation as controls. Tumour necrosis factor-alpha and interferon-gamma producing cells were detected simultaneously within the inflammatory cell infiltrate in the liver biopsies of 18 patients with autoimmune liver disease, but only one patient with a metabolic disorder was positive for tumour necrosis factor-alpha. There was a significant correlation between frequency of tumour necrosis factor-alpha and interferon-gamma producing cells, intensity of inflammatory cell infiltrate (p < 0.03 and p < 0.05, respectively) and transaminase levels (p < 0.008 and p < 0.03, respectively). These results suggest that tumour necrosis factor-alpha and interferon-gamma play a pathogenic role in autoimmune liver cell damage.

CD5+ B-cells at the onset of type I diabetes and in the prediabetic period.

OBJECTIVE: B-cells expressing CD5 are associated with the production of autoantibodies and are present at increased levels in several autoimmune diseases. The aim of this study was to investigate the relationship of these cells to the development of type I diabetes and the presence of organ and non-organ-specific autoantibodies. RESEARCH DESIGN AND METHODS: We measured percentage levels of CD5+ B-cells in patients with recent-onset (n = 34) and long-standing (n = 21) type I diabetes and in a cohort of 18 identical twins of patients with type I diabetes studied prospectively, 8 of whom became diabetic (prediabetic twins) during the study; the rest remained nondiabetic after at least 7 years and are now unlikely to develop the disease. Forty-seven healthy individuals were studied as control subjects. RESULTS: Percentage levels of total B-cells (CD20+) and the proportion expressing CD5 were increased in patients with recent-onset (P < 0.001 for both) but not long-standing type I diabetes compared with control subjects. Percentage levels of CD20+ B-cells were increased in prediabetic twins throughout the prediabetic period (P < 0.05), and there was an increased proportion of CD5-expressing B-cells that failed to reach statistical significance (P = 0.08). Percentage levels of CD20+ B-cells and the proportion expressing CD5 were normal throughout the study in twins remaining nondiabetic. No relationship between percentage levels of CD5+ B-cells and islet cell antibody, thyroid autoantibodies, or non-organ-specific autoantibodies was found. CONCLUSIONS: These results show an increase in B-cell percentage levels at the diagnosis of type I diabetes, which is because of an expansion of the CD5+ subset. These changes are also evident in twins throughout the prediabetic period, which suggests that they are related to the processes that lead to diabetes.

Interleukin-1 alpha, soluble interleukin-2 receptor, and IgG concentrations in cystic fibrosis treated with prednisolone.

The cytokines interleukin-1 and interleukin-2 participate in the inflammatory response, and may contribute to hypergammaglobulinaemia G and the development of lung injury in cystic fibrosis. Anti-inflammatory treatment with corticosteroids may attenuate this response. The effect of a 12 week course of oral prednisolone on spirometry and serum concentrations of interleukin-1 alpha (IL-1 alpha), soluble interleukin-2 receptor (sIL-2R), and IgG was investigated in 24 children with cystic fibrosis. Prednisolone was administered, in a double blind and placebo controlled manner, at an initial dose of 2 mg/kg daily for 14 days and tapered to 1 mg/kg on alternate days for 10 weeks. The treated group (n = 12) experienced an increase in forced expiratory volume in one second and forced vital capacity at 14 days, however, these changes were smaller at 12 weeks. In the treated group, change in pulmonary function was associated with decreased serum IgG and cytokine concentrations. Prednisolone suppresses serum concentrations of these cytokines, which may participate in the inflammatory response, the excessive synthesis of IgG, and airflow obstruction observed in cystic fibrosis patients.

Increased expression of T-cell markers of immunological memory associated with protection from type I diabetes. A study of identical twins.

Disturbances in the balance of CD4+ helper T-lymphocytes expressing the surface molecules CD45RA and CD45R0, which define naive and memory populations, respectively, are present at diagnosis of type I diabetes. In a prospective study over 10 years, these subsets were analyzed in samples obtained from 18 identical twins of patients with type I diabetes, 8 of whom became diabetic (prediabetic twins), whereas the rest remained nondiabetic after at least 8 years follow-up and are now unlikely to develop the disease (diabetes-protected twins). At the beginning of the study, percentage levels of naive (CD45RA+) CD4+ lymphocytes were significantly elevated in prediabetic twins compared with diabetes-protected twins (P < 0.05) and remained so throughout the study (P < 0.01). Percentage levels of naive cells in diabetes-protected twins were significantly reduced compared with control subjects both at the beginning and throughout the study (P < 0.05, P < 0.01, respectively). In contrast, diabetes-protected twins at the beginning of the study had elevated percentage levels of memory (CD45R0+) CD4+ lymphocytes that persisted throughout the study compared with prediabetic twins (P < 0.05 for both). Percentage levels of memory cells in prediabetic twins were significantly reduced compared with control subjects both at the beginning and throughout the study (P < 0.01, P < 0.05, respectively). Increased percentage levels of a population of CD4+ lymphocytes coexpressing CD45RA and CD45R0 were seen in both twin groups compared with control subjects at entry into and during the study (P < 0.05 for all), but persisted only in the prediabetic twins.(ABSTRACT TRUNCATED AT 250 WORDS)

Importance of persistent cellular and humoral immune changes before diabetes develops: prospective study of identical twins.

OBJECTIVES: To determine the pattern of cellular and humoral immune changes associated with insulin dependent diabetes before diabetes develops. DESIGN: Prospective study over 10 years of 25 non-diabetic identical twins of patients with insulin dependent diabetes. The non-diabetic twins were followed up either till they developed diabetes or to the end of the study. SETTING: Teaching hospital. SUBJECTS: 25 non-diabetic identical cotwins of patients with diabetes; 46 controls of the same sex and similar age tested over the same period. Of the 25 twins (total follow up 144 patient years), 10 developed diabetes (prediabetic twins); the remainder were followed up for a mean of 7.7 years. MAIN OUTCOME MEASURES: Results of glucose tolerance tests or fasting blood glucose concentrations at each sample point. Measurements of activated T lymphocytes, expressing the HLA-DR antigen, islet cell antibodies, and insulin autoantibodies in samples. RESULTS: All 10 prediabetic twins had both cellular and humoral changes initially and in most samples before diabetes was diagnosed (activated T lymphocytes in 39/40, islet cell antibodies in 45/47, and insulin autoantibodies to islet cells and insulin were detected infrequently (in 8/54, 6/69, and 0/69 samples, respectively). The combination of cellular and humoral (islet cell antibodies or insulin autoantibodies) immune changes were detected in all 10 of the prediabetic twins but in only one of the 15 non-diabetic twins (P < 0.001). The positive predictive value in this cohort of increased percentages of activated T cells and the presence of antibodies to islet cells or insulin on two consecutive occasions was 100%. CONCLUSION: Most of the twins had cellular or humoral immune changes at some stage. A combination of cellular and humoral immune changes and their tendency to persist is highly predictive of insulin dependent diabetes and distinguishes twins who develop diabetes from those who do not.

Relationship between the tumour-associated antigen 90K and cytokines in the circulation of persons infected with human immunodeficiency virus.

A tumour-associated antigen known as 90K has been found in high concentrations in the serum of patients infected with human immunodeficiency virus (HIV) even in the absence of neoplastic complications. In order to investigate the relationship between the production of 90K and soluble inflammatory mediators, we studied serum concentrations of the antigen, tumour necrosis factor-alpha (TNF-alpha), interleukin-I-alpha (IL-I-alpha), interferon-gamma (IFN-gamma), IFN-alpha, neopterin and beta 2-microglobulin (beta 2-m) in patients with non-neoplastic HIV infection at various stages of disease and in control persons. The antigen was detected in all those studied but its concentration was higher in HIV-infected patients compared with controls (P < 0.001), increasing progressively with advancing stages of disease. There was a negative correlation between concentrations of 90K and IL-I-alpha in patients in U.S.A. Centers for Disease Control groups II and III (P < 0.02) and also between that of 90K and both TNF-alpha (P < 0.01) and IL-I-alpha (P < 0.05) in control persons. The results indicate that 90K is not merely a tumour-associated antigen and that its production may be part of immune and inflammatory responses in the absence of neoplasia. The correlation between the concentrations of 90K and of some cytokines in asymptomatic patients and healthy persons suggests that 90K may be part of a network of immune and inflammatory reactants.