The face of postural tachycardia syndrome - insights from a large cross-sectional online community-based survey.

BACKGROUND: Patients with postural tachycardia syndrome (POTS) experience chronic symptoms of orthostatic intolerance. There are minimal data detailing the demographics, clinical features and clinical course of this condition. This online, community-based survey highlights patients' experience with POTS. It consists of the largest sample of POTS patients reported to date. OBJECTIVES: To describe the demographics, past medical history, medications, treatments and diagnostic journey for patients living with POTS. METHODS: Postural tachycardia syndrome patients completed an online, community-based, cross-sectional survey. Participants were excluded if they had not received a diagnosis of POTS from a physician. The questions focused on the patient experience and journey, rather than physiological responses. RESULTS: The final analysis included 4835 participants. POTS predominantly affects white (93%) females (94%) of childbearing age, with approximately half developing symptoms in adolescence (mode 14 years). POTS is a chronic multisystem disorder involving a broad array of symptoms, with many patients diagnosed with comorbidities in addition to POTS. POTS patients often experience lengthy delays [median (interquartile range) 24 (6-72) months] and misdiagnosis, but the diagnostic delay is improving. POTS patients can present with a myriad of symptoms most commonly including lightheadedness (99%), tachycardia (97%), presyncope (94%), headache (94%) and difficulty concentrating (94%). CONCLUSIONS: These data provide important insights into the background, clinical features and diagnostic journey of patients suffering from POTS. These data should serve as an essential step for moving forward with future studies aimed at early and accurate diagnoses of these patients leading to appropriate treatments for their symptoms.

Targeted expression of the anti-apoptotic gene CrmA to NOD pancreatic islets protects from autoimmune diabetes.

The activation of apoptosis is a critical mechanism by which pancreatic beta cells are destroyed in type 1 diabetes (T1DM). Strategies aimed at interfering with the apoptotic pathways could therefore be of potential therapeutic value. To this end, we generated NOD transgenic mice with targeted expression of the anti-apoptotic gene Cytokine response modifier A (CrmA) to pancreatic beta cells using the rat insulin promoter and the reverse tetracycline transactivator to express CrmA in a temporally controlled manner. Two lines of transgenic mice were studied whose expression of CrmA occurred only after feeding doxycycline food. Islet expression of CrmA partially protected pancreatic beta cells from the cytokine-mediated cytotoxicity in vitro and reduced modestly the spontaneous development of diabetes in NOD mice in vivo. In addition, beta cells from NOD CrmA mice were significantly protected from the destruction by diabetogenic T cells after adoptive transfer. More strikingly, NODCrmA mice were significantly resistant to the diabetogenic activity of a potent insulin-specific CD8 T-cell clone. Since these adoptive transfer models mainly represent the effector phase rather than the initiation phase of autoimmune diabetes, our data suggest that the latter is more sensitive to CrmA protection. We conclude that anti-apoptotic genes such as CrmA might be potential candidates to enhance islet graft survival in T1DM.

Cost-effective analysis of candidate genes using htSNPs: a staged approach.

We have previously shown that the selection of haplotype tag single nucleotide polymorphisms (htSNPs) and their statistical analysis in a multi-locus transmission/disequilibrium test (TDT) results in a more cost-effective genotyping strategy in disease association studies of genes by minimising redundancy due to linkage disequilibrium between SNPs. Further savings can be achieved by the use of a two-stage genotyping strategy. This approach is illustrated here in conjunction with the multi-locus TDT in determining whether common alleles of the immune regulatory genes RANK and its ligand TRANCE (RANKL) are associated with type 1 diabetes (T1D). A saving of approximately 75% of potential genotyping reactions could be made with minimal loss of power. There was little evidence from our analysis for association between the TRANCE and RANK genes and T1D in the populations tested.

A dual role for TNF-alpha in type 1 diabetes: islet-specific expression abrogates the ongoing autoimmune process when induced late but not early during pathogenesis.

We report here that islet-specific expression of TNF-alpha can play a dual role in autoimmune diabetes, depending on its precise timing in relation to the ongoing autoimmune process. In a transgenic model (rat insulin promoter-lymphocytic choriomeningitis virus) of virally induced diabetes, TNF-alpha enhanced disease incidence when induced through an islet-specific tetracycline-dependent promoter system early during pathogenesis. Blockade of TNF-alpha during this phase prevented diabetes completely, suggesting its pathogenetic importance early in disease development. In contrast, TNF-alpha expression abrogated the autoimmune process when induced late, which was associated with a reduction of autoreactive CD8 lymphocytes in islets and their lytic activities. Thus, the fine-tuned kinetics of an autoreactive process undergo distinct stages that respond in a differential way to the presence of TNF-alpha. This observation has importance for understanding the complex role of inflammatory cytokines in autoimmunity.

The temporal importance of TNFalpha expression in the development of diabetes.

The inflammatory cytokine tumor necrosis factor alpha (TNFalpha) has been linked to the development of several autoimmune diseases. By adapting the tetracycline-regulated gene transcription system, we generated a murine model where islet-specific expression of TNFalpha could be repressed/derepressed within 48 hr following introduction/removal of tetracycline in the drinking water. Here we describe the temporal importance of TNFalpha in diabetes development in mice expressing islet-specific B7-1 and TNFalpha. We show that the duration of TNFalpha-mediated inflammation, not the putative maturity of the immune system at the time of TNFalpha expression, determines diabetes progression. Further, we have described an interval between 21 and 25 days following initiation of TNFalpha expression where the fate of islet-reactive T cells is decided.

Neonatal tumor necrosis factor alpha promotes diabetes in nonobese diabetic mice by CD154-independent antigen presentation to CD8(+) T cells.

Neonatal islet-specific expression of tumor necrosis factor (TNF)-alpha in nonobese diabetic mice promotes diabetes by provoking islet-infiltrating antigen-presenting cells to present islet peptides to autoreactive T cells. Here we show that TNF-alpha promotes autoaggression of both effector CD4(+) and CD8(+) T cells. Whereas CD8(+) T cells are critical for diabetes progression, CD4(+) T cells play a lesser role. TNF-alpha-mediated diabetes development was not dependent on CD154-CD40 signals or activated CD4(+) T cells. Instead, it appears that TNF-alpha can promote cross-presentation of islet antigen to CD8(+) T cells using a unique CD40-CD154-independent pathway. These data provide new insights into the mechanisms by which inflammatory stimuli can bypass CD154-CD40 immune regulatory signals and cause activation of autoreactive T cells.

Tumor necrosis factor-alpha and the progression of diabetes in non-obese diabetic mice.

In the past decade, a wealth of information has accumulated through studies in non-obese diabetic (NOD) mice regarding the molecular and cellular events that participate in the progression to diabetes in insulin-dependent diabetes mellitus (IDDM). One molecule that has received considerable attention is the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). TNF-alpha has been demonstrated to have a positive or negative effect on the progression to diabetes in NOD mice, although the mechanism by which TNF-alpha exerts these differential outcomes is unknown. Here we describe a new NOD model for analyzing the role of TNF-alpha in IDDM, TNF-alpha-NOD mice. TNF-alpha-NOD mice express TNF-alpha solely in their islets from neonatal life onwards, and develop accelerated progression to diabetes. This rapid progression to diabetes is related to earlier and more aggressive infiltration of the islets with immune cells and an enhancement in the presentation of islet antigen in situ in the islets by islet-infiltrating antigen-presenting cells to T cells. Although adoptive transfer studies demonstrated that TNF-alpha can enhance presentation of islet antigen to both effector CD4+ and CD8+ T cells, further investigations in TNF-alpha-NOD mice deficient in either CD4+ or CD8+ T cells demonstrated that diabetes progression is dependent on CD8+ T cells, with CD4+ T cells playing a lesser role. The data accumulating from TNF-alpha-NOD mice, described in this review, indicates novel pathways by which inflammatory stimuli can precipitate autoimmunity, and suggests newer approaches in the design of therapeutic treatments that prevent beta-cell destruction in IDDM.

The initiation of autoimmune diabetes.

It is speculated that presentation of self-peptides to autoreactive T cells normally results in T cell tolerance. In autoimmune conditions, breakdown in the tolerization process results in activation of self-reactive T cells and an immune attack on host tissues. Our understanding of the immune cells and signaling pathways that contribute to this breakdown in T cell tolerization mechanisms is beginning to be deciphered. In particular, the elucidation of the mechanisms that contribute to the release of host antigen, the identification of the antigen-presenting cells that present the host peptides to self-reactive T cells and the role of members of the tumor necrosis factor receptor/ligand families that contribute to inappropriate activation of self-reactive T cells is advancing. The accumulating data from these studies will hopefully provide new ideas for combating autoimmunity.

Local expression of TNFalpha in neonatal NOD mice promotes diabetes by enhancing presentation of islet antigens.

The relationship of inflammation to autoimmunity has been long observed, but the underlying mechanisms are unclear. Here, we demonstrate that islet-specific expression of TNFalpha in neonatal nonobese diabetic mice accelerated diabetes. In neonatal transgenic mice, disease was preceded by apoptosis of some beta cells, upregulation of MHC class I molecules on residual islet cells, and influx and activation of both antigen-presenting cells bearing MHC-islet peptide complexes and T cells. Infiltrating dendritic cells/macrophages, but not B cells, from neonatal islets activated islet-specific T cells in vitro. Thus, inflammation can trigger autoimmunity by recruiting and activating dendritic cells/macrophages to present self-antigens to autoreactive T cells.

Insulin resistance syndrome in Australian aboriginal people.

1. Like many indigenous populations, Australian Aboriginal people have developed high rates of obesity, non-insulin-dependent diabetes mellitus (NIDDM) and cardiovascular and renal disease following the transition from a traditional to an 'urbanized' lifestyle. These conditions tend to cluster as part of the insulin resistance syndrome. 2. The prevalence of overweight people and obesity in Australian Aboriginal populations ranges from 0% in communities with a traditionally orientated lifestyle to well over 50% in the worst affected communities. There is a predominantly central pattern of fat deposition in both men and women, which is associated with greater insulin resistance and cardiovascular risk than is peripheral fat deposition. 3. Data from four previously published, population-based surveys in Aboriginal communities were combined to give a cohort of 1079 subjects of 15 years and older. Several conditions of the insulin resistance syndrome had a strong, positive association with increasing body mass index (BMI): NIDDM (both cross-sectionally and longitudinally), hypertension, dyslipidaemia and albuminuria. Remaining lean (BMI < 20 kg/m2) protected even older Aboriginal people from these conditions to a large extent. 4. Community based programmes to increase physical activity and improve dietary quality are likely to be the major means by which conditions associated with insulin resistance can be prevented in Aboriginal populations.

Construction, purification and immunogenicity of antigen-antibody-LTB complexes.

An oligonucleotide, encoding a short epitope peptide tag, termed Pk, was inserted at the 3'-end of the gene coding B-subunit of Escherichia coli heat-labile enterotoxin (LTB). The presence of the Pk epitope on LTB-Pk was used to construct novel macromolecular assemblies comprising LTB-Pk, an anti-Pk mAb, (mAb SV5-P-k) and Pk-linked recombinant SIV proteins. The 1:1:1 stoichiometry of such complexes was ensured by binding LTB-Pk to one arm of mAb SV5-P-k and an SIV-Pk antigen to the other arm of the antibody. Such SIV-mAb-LTB macromolecular complexes bound to GM1-ganglioside in vitro, and when immunized systemically into mice were highly immunogenic, inducing both humoral and cell-mediated responses to the recombinant SIV antigens.

Expression and purification of nonglycosylated SIV proteins, and their use in induction and detection of SIV-specific immune responses.

Two commercially available expression vectors were modified to generate plasmids pGEXcPk and pQ9cPk. Proteins expressed from pGEXcPk and pQ9cPk had a short oligopeptide tag termed Pk at their carboxy termini and either glutathione S-transferase (GST) or a small histidine (His) tag, respectively, at their N termini. GST fusion proteins can be purified on immobilized glutathione and proteins coupled to the His tag selectively bind to Ni(2+)-NTA columns. The Pk tag is recognized by monoclonal antibody (MAb) SV5-P-k, previously produced in our laboratory. Thus proteins expressed from the pGEXcPk and pQ9cPk vectors can be purified in a two-step procedure, first via the N-terminal tag and second via the C-terminal tag. The combination of two affinity purification steps significantly improves the antigen purity and selects for full-size proteins. Moreover, by using the MAbSV5-P-k in the second purification step, Pk-linked antigens can be assembled directly into solid matrix-antibody-antigen (SMAA) complexes for use as vaccines. The genes for nef, endonuclease, p15, p17, p27, protease, Rev, reverse transcriptase (rt), tat, vif, vpr, and vpx of simian immunodeficiency virus (SIV mac 251) were cloned and expressed as both GST-SIV-Pk and His-SIV-Pk proteins. Multivalent SMAA complexes were made that contained His-p17-Pk, His-p27-Pk, His-rt-Pk, His-vpx-Pk, and His-vpr-Pk. Following two immunizations of mice with this mixture, antibodies could be detected to all five SIV antigens. When compared to single-protein immunizations, the immunogenicity of some of the proteins in this cocktail was either enhanced or decreased. Mice were also immunized with His-p17-Pk or His-p17-Pk-antibody complexes in the presence or absence of alum. The antibody-antigen complexes induced two- to four-fold higher antibody levels than antigen alone but did not appear to be more immunogenic in inducing lymphoproliferative responses. Sera from SIV-infected macaques were tested for the presence of antibodies reacting with the recombinant proteins by Western blot analysis. Antibodies to endonuclease, p15, p17, p27, rt, and vif were readily detected, antibodies against protease and vpx were present at much lower levels, but no antibodies were detected to nef, rev, tat, or vpr. Thus, we have developed a comprehensive range of reagents (available on request) that can be used to examine immune responses to SIV in both mice and monkeys.

Tympanometric evaluation of middle ear barotrauma during recreational scuba diving.

We report the first prospective evaluation of middle ear barotrauma in experienced recreational scuba divers. In this pilot study, tympanometric and otoscopic evaluations were performed daily on two experienced scuba divers engaged in multi-day repetitive diving. Middle ear pressures decreased in proportion to diving frequency, demonstrating eustachian tube dysfunction which promptly reversed upon cessation of diving Otoscopic evidence of traumatic injury to the middle ear occurred in proportion to diving frequency, and also readily reversed upon cessation of diving. Tympanic membrane compliance remained normal, often despite pronounced otoscopic abnormalities. Otologic symptoms and impairment of acuity were not observed. Tympanometry appears to be a valuable modality for the verification of middle ear hemorrhage or tympanic membrane rupture. This preliminary data should assist investigators in planning more comprehensive studies of middle ear barotrauma, including clinical trials of treatment and prophylactic interventions for this common condition.

The role of interferon alpha/beta in the induction of intestinal pathology in mice.

We have investigated the role of interferon-alpha/beta (IFN-alpha/beta) and IFN-dependent effector cells in causing enteropathy in mice. The IFN-inducer polyinosinic:polycytydylic acid (poly I:C) augmented the natural killer (NK) cell activation normally seen in neonatal (CBA x BALB/c)F1 mice with graft-versus-host reaction (GVHR) and exacerbated the systemic and intestinal consequences of GVHR. Poly I:C itself produced a similar pattern of intestinal pathology when administered to normal mice. The effects of poly I:C on NK cell activity and intestinal architecture in normal mice could be reproduced by a single injection of purified IFN-alpha/beta and the intestinal lesions caused by IFN-alpha/beta were prevented by in vivo depletion of NK cells with anti-asialo GM1. These results indicate that IFN-alpha/beta may play an important role in immunologically mediated enteropathies by virtue of its ability to activate NK cells.

Adriamycin-induced lipid peroxidation in mitochondria and microsomes.

The effect of the anti-neoplastic agent adriamycin on the peroxidation of lipids from rat liver and heart mitochondria and rat liver microsomes was investigated. The extent of total lipid peroxidation was determined by assaying for malondialdehyde (MDA), while the degradation of unsaturated fatty acids was monitored using gas chromatography. For liver mitochondria and microsomes, the formation of MDA was dependent on the concentrations of adriamycin, Fe3+, and protein, as well as time. In the presence of 50 microM adriamycin and saturating amounts of NADH, 1.5 +/- 0.2 nmol MDA/mg protein/60 min was produced with liver mitochondria. Upon addition of 25 microM Fe3+, the amount of MDA generated was increased to 6.5 +/- 0.1 nmol/mg protein/60 min. Liver microsomes produced amounts which were approximately 2-fold higher under all conditions. No MDA formation could be detected in rat heart mitochondria. The addition of 50 microM chlorpromazine completely inhibited peroxidation, whereas 0.5 to 1.0 mM p-bromophenacyl bromide blocked MDA formation by 50%. Analysis of fatty acids by gas chromatography showed that there was about a 50% decrease in arachidonic and docosahexaenoic acids in liver mitochondria and microsomes, but no change in the fatty acid content of heart mitochondria when incubated with both 50 microM adriamycin and 25 microM Fe3+ for 1 hr. These results suggest that (1) therapeutic concentrations of adriamycin enhance the peroxidation of lipids in liver mitochondria and microsomes through an enzymatic mechanism, especially in the presence of Fe3+; and (2) toxicity of this drug may be related to the degradation of membrane lipids.

Transport and hydrolysis of glucagon in the proximal nephron.

Transport and hydrolysis of glucagon in the rabbit proximal nephron were studied. Iodinated glucagon (0.34 +/- 0.02 pg/nl, mean +/- SE) was microperfused (16.0 +/- 1.1 nl/min) in vitro through proximal straight nephron segments for 30 min. Radiolabeled material, primarily 125I-tyrosine, appeared in the bathing medium in a linear fashion as a function of time (0.406 pg glucagon X mm tubule length-1 X min-1). Hydrolysis of glucagon by proximal tubule homogenates was pH dependent, with a large peak of activity observed at pH 7.0-7.4 and a smaller one at pH 3.0. Analytical cell fractionation studies of proximal tubule cells revealed glucagon-hydrolyzing activity associated with the brush border and cytosol at pH 7.4. Less than 3% of activity was found associated with the contraluminal membrane. Substantial catabolism was observed at lysosomes on lowering the pH to 5.0. Incubation of glucagon directly in the presence of isolated renal cortical microvilli confirmed the presence of a high-capacity glucagon-degrading hydrolase. In addition to glucagon-hydrolyzing activity associated with the proximal nephron, noncortical activity was observed that was not accounted for by proximal tubule hydrolases. The data suggest several mechanisms for renal extraction of glucagon, including hydrolysis by enzymes at the brush border of the proximal tubule, prior to reabsorption of metabolites there. Conversely, enzymes associated with the contraluminal membrane of the proximal nephron probably contribute little to its hydrolysis. Nonproximal extracortical degradation of glucagon may account for its previously observed peritubular hydrolysis.

Efficacy of amdinocillin and lack of nephrotoxicity when combined with a second beta-lactam antibiotic for therapy of serious gram-negative bacillary infections.

Seventy-eight patients with serious gram-negative bacillary infections were assigned at random to receive either amdinocillin or an aminoglycoside. In addition, each patient was also given a broad-spectrum penicillin or cephalosporin antibiotic. The clinical response to treatment was comparable in the two groups. Cures were effected in 35 (92 percent) of 38 patients treated with amdinocillin and a beta-lactam antibiotic, compared with 37 (93 percent) of 40 patients who were treated with an aminoglycoside/beta-lactam combination. For the entire group, only five (7 percent) of the 75 infecting organisms were resistant in vitro to the treatment beta-lactam or amdinocillin combination, and similarly only two (3 percent) organisms were resistant to the treatment aminoglycoside (p = 0.44). Although drug-related toxicity occurred with equal frequency in the two groups, six patients treated with an aminoglycoside experienced nephrotoxicity compared with none of the patients who received amdinocillin (p = 0.034). Thus, amdinocillin plus a broad-spectrum beta-lactam antibiotic may provide suitable empiric therapy for many patients with presumed gram-negative infection and so avoid the risk of aminoglycoside-induced nephrotoxicity.