A critical appraisal of impact of compounding factors in limb salvage decision making in combat extremity vascular trauma.

OBJECTIVE: For more than half a century, surgeons who managed vascular injuries were guided by a 6-hour maximum ischaemic time dogma in their decision to proceed with vascular reconstruction or not. Contemporary large animal survival model experiments aimed at redefining the critical ischaemic time threshold concluded this to be less than 5 hours. Our clinical experience from recent combat vascular trauma contradicts this dogma with limb salvage following vascular reconstruction with an average ischaemic time of 6 hours. METHODS: During an 8-month period of the Sri Lankan Civil War, all patients with penetrating extremity vascular injuries were prospectively recorded by a single surgeon and retrospectively analysed. A total of 76 arterial injuries was analysed for demography, injury anatomy and physiology, treatment and outcomes. Subsequent statistical analysis was performed to evaluate the impact of independent variables to include; injury anatomy, concomitant venous, skeletal trauma, shock at presentation and time delay from injury to reconstruction. RESULTS: In this study, the 76 extremity arterial injuries had a median ischaemic time of 290 (IQR 225-375) min. Segmental arterial injury (p=0.02), skeletal trauma (p=0.05) and fasciotomy (p=0.03) were found to have a stronger correlation to subsequent amputation than ischaemic time. CONCLUSIONS: Multiple factors affect limb viability following compromised distal circulation and our data show a trend towards various subsets of limbs that are more vulnerable due to inherent or acquired paucity of collateral circulation. Early identification and prioritisation of these limbs could achieve functional limb salvage if recognised. Further prospective research should look into the clinical, biochemical and morphological markers to facilitate selection and prioritisation of limb revascularisation.

Effectiveness of TaqMan probes for detection of fish eggs and larvae in the stomach contents of a teleost predator.

Experiments were conducted on the ability of TaqMan molecular probes to detect plaice Pleuronectes platessa DNA from eggs, and cod Gadus morhua DNA from eggs and larvae following ingestion by a teleost predator, whiting Merlangius merlangus. Estimated half-life detection rate (T50) for eggs was 31 h, and 26 h for larvae, with some positive detections occurring even after visual inspection indicated complete gut clearance. Because TaqMan probes are taxon specific, the results presented demonstrate that this technique can provide a means of rapid and unambiguous detection of predation by teleosts on fish eggs and larvae.

Propagation of giant migrating contractions between the small intestine, cecum and colon during radiation.

BACKGROUND: Radiation increases the frequency of small intestinal and colonic giant migrating contractions (GMCs). These contractions contribute to the diarrhea and cramping after radiation therapy and are coordinated with one another across the ileocolonic (IC) junction. METHODS: We investigated the coordination of contractile activity between the small intestine, cecum and colon in five canines following circumferential myotomy on the ileum at the IC junction and compared it to intact animals. Studies were performed before and during a radiation schedule. KEY RESULTS: Myotomy increased the frequency of small intestinal GMCs prior to irradiation. In intact animals, the duration and amplitude of cecal GMCs decreased when multiple contractions occurred in rapid succession. This is in contrast to small intestinal and colonic GMCs and suggests a different mechanism of propagation for GMCs within the cecum. Ileal myotomy dramatically decreased the frequency of propagating radiation-induced colonic GMCs. The total number of colonic GMCs was not altered. Colonic contractile activity was disrupted in intact animals during irradiation. However, after ileal myotomy, irradiation did not affect the pattern of colonic contractile states. Diarrhea in irradiated animals with myotomy started earlier than intact animals. This may be related to the frequency of small intestinal GMCs. CONCLUSIONS & INFERENCES: Our findings suggest importance of the enteric neural connections at the IC region to contractile disorders of both the small and large intestine. The anatomic relationship between the canine IC junction is similar to the human ileo-appendiceal-colonic region and surgical manipulations of this area may likewise affect human contractile activity.

The contribution of the fusiform gyrus and superior temporal sulcus in processing facial attractiveness: neuropsychological and neuroimaging evidence.

Current cognitive models suggest that the processing of dynamic facial attributes, including social signals such as gaze direction and facial expression, involves the superior temporal sulcus, whereas the processing of invariant facial structure such as the individuals' identity involves the fusiform face area. Where facial attractiveness, a social signal that may emerge from invariant facial structure, is processed within this dual-route model of face perception is uncertain. Here, we present two studies. First, we investigated the explicit judgments of facial attractiveness and attractiveness-motivated behavior in patients with acquired prosopagnosia, a deficit in familiar face recognition usually associated with damage to medial occipitotemporal cortex. We found that both abilities were impaired in these patients, with some weak residual ability for attractiveness judgments found only in those patients with unilateral right occipitotemporal or bilateral anterior temporal lesions. Importantly, deficits in attractiveness perception correlated with the severity of the face recognition deficit. Second, we performed a functional magnetic resonance imaging study in healthy subjects that included an implicit and explicit processing of facial attractiveness. We found increased neural activity when explicitly judging facial attractiveness within a number of cortical regions including the fusiform face area, but not the superior temporal sulcus, indicating a potential contribution of the fusiform face area to this judgment. Thus, converging neuropsychological and neuroimaging evidence points to a critical role of the inferior occipitotemporal cortex in the processing of facial attractiveness.

Physical exam and occult post-traumatic vascular lesions: implications for the evaluation and management of arterial injuries in modern warfare in the endovascular era.

Based on complexity of extremity wounds sustained in recent combat, arteriography had been used routinely in evaluations for delayed or occult arterial injuries. This report aims to quantitatively analyze the sensitivity and specificity of physical exam (PE) in predicting the presence of these injuries. United States service members sustaining extremity trauma in the Global War on Terrorism were evacuated to our medical center and evaluated by a senior vascular surgeon. Those with an abnormal PE, at risk based on wounding patterns, or previously treated for vascular injury underwent arteriography. Data from each patient were prospectively entered into a Vascular Injury registry. Comprehensive information about the injuries, interventions, arteriogram results, and any periprocedural complications were analyzed. Twenty-five endovascular or open surgical interventions were performed in forty-six of 99 patients that had lesions on arteriography. Seventy-three patients had a normal PE, of which 36 had lesions that prompted 6 subsequent interventions. Twenty-two of 26 patients with an abnormal PE had lesions that prompted 19 interventions. For PE, sensitivity was 38%, specificity was 90%, and positive predictive value (PPV) and negative predictive value (NPV) were 85% and 51%, respectively. In proximity injuries, PPV improved to 100%, but was only 15% sensitive with a NPV of 60%. In conclusion normal PE did not reliably predict post-traumatic arterial lesions in these military extremity injuries. These lesions are amenable to endovascular therapies, and should be considered in cases of complex trauma involving high amounts of energy, penetrating mechanisms, or wounding patterns in proximity to named vessels.

Case of adolescent with Paget-Schroetter syndrome and underlying thrombophilia due to an elevated lipoprotein (A).

Upper extremity deep vein thrombosis (UEDVT) is a rare disorder in children and is most often associated with a central venous catheter (CVC), cancer, or an underlying thrombophilia. In adults, repetitive or strenuous upper extremity activity has been linked to mechanical compression of the thoracic outlet resulting in subclavian vein thrombosis, also known as Paget-Schroetter Syndrome (PSS). We present the case of a 14 year old female who was found to have an UEDVT consistent with PSS. On subsequent thrombophila screening, she was noted to have a significantly elevated lipoprotein (a) level of 83 (normal: <30 mg/dL). She underwent 3 months of anticoagulation therapy prior to her first rib resection and in follow-up was noted to have complete patency of her subclavian vein. This case illustrates the necessity for thrombophilia screening in the pediatric patient with UEDVT despite evidence of anatomic abnormality or mechanical venous compression.

TaqMan DNA technology confirms likely overestimation of cod (Gadus morhua L.) egg abundance in the Irish Sea: implications for the assessment of the cod stock and mapping of spawning areas using egg-based methods.

Recent substantial declines in northeastern Atlantic cod stocks necessitate improved biological knowledge and the development of techniques to complement standard stock assessment methods (which largely depend on accurate commercial catch data). In 2003, an ichthyoplankton survey was undertaken in the Irish Sea and subsamples of 'cod-like' eggs were analysed using a TaqMan multiplex, PCR (polymerase chain reaction) assay (with specific probes for cod, haddock and whiting). The TaqMan method was readily applied to the large number of samples (n = 2770) generated during the survey and when combined with a manual DNA extraction protocol had a low failure rate of 6%. Of the early stage 'cod-like' eggs (1.2-1.75 mm diameter) positively identified: 34% were cod, 8% haddock and 58% whiting. As previous stock estimates based on egg surveys for Irish Sea cod assumed that the majority of 'cod-like' eggs were from cod, the TaqMan results confirm that there was probably substantial contamination by eggs of whiting and haddock that would have inflated estimates of the stock biomass.

Comparison of fibroglandular tissue distributions for microwave tomographic breast images with complementary MR T2 weighted images.

We have recently demonstrated good correlation between the recovered permittivity from microwave imaging (MIS) and the recovered water content from near infrared imaging (NIR) for a common set of normal patients undergoing associated breast examinations. We have subsequently conducted a small sample of comparison breast examinations between microwave imaging and MR to assess possible correlation between the location and extent of the fibroglandular as seen on MR images with increased permittivity zones of the microwave images. From various physiological and MR breast studies, it has been shown that the fibroglandular regions are generally comprised of significantly higher levels of water than the more dominant adipose tissue. The initial results of this study are quite encouraging and demonstrate obvious correlations between the permittivity and MR-recovered fibroglandular regions for a set of patients with widely varying tissue type variations. In addition, they illustrate the value of extracting diagnostic information from multiple modalities especially where the amount of direct in vivo property measurements is limited or nonexistent.

Growth factors can influence cell growth and survival through effects on glucose metabolism.

Cells from multicellular organisms are dependent upon exogenous signals for survival, growth, and proliferation. The relationship among these three processes was examined using an interleukin-3 (IL-3)-dependent cell line. No fixed dose of IL-3 determined the threshold below which cells underwent apoptosis. Instead, increasing growth factor concentrations resulted in progressive shortening of the G(1) phase of the cell cycle and more rapid proliferative expansion. Increased growth factor concentrations also resulted in proportional increases in glycolytic rates. Paradoxically, cells growing in high concentrations of growth factor had an increased susceptibility to cell death upon growth factor withdrawal. This susceptibility correlated with the magnitude of the change in the glycolytic rate following growth factor withdrawal. To investigate whether changes in the availability of glycolytic products influence mitochondrion-initiated apoptosis, we artificially limited glycolysis by manipulating the glucose levels in the medium. Like growth factor withdrawal, glucose limitation resulted in Bax translocation, a decrease in mitochondrial membrane potential, and cytochrome c redistribution to the cytosol. In contrast, increasing cell autonomous glucose uptake by overexpression of Glut1 significantly delayed apoptosis following growth factor withdrawal. These data suggest that a primary function of growth factors is to regulate glucose uptake and metabolism and thus maintain mitochondrial homeostasis and enable anabolic pathways required for cell growth. Consistent with this hypothesis, expression of the three genes involved in glucose uptake and glycolytic commitment, those for Glut1, hexokinase 2, and phosphofructokinase 1, was found to rapidly decline to nearly undetectable levels following growth factor withdrawal.

Common femoral artery ligation and local debridement: a safe treatment for infected femoral artery pseudoaneurysms.

PURPOSE: The management of infected femoral artery pseudoaneurysms (IFAPs) is difficult and controversial. Use of synthetic or autologous conduit during arterial revascularization in these cases is complicated by the presence of sepsis and unavailability of autologous venous conduit. We present the results of common femoral artery (CFA) ligation and local drainage with debridement for the treatment of IFAP. METHODS: A retrospective chart review of six consecutive patients from 1995 to 1999 who presented with IFAP from intravenous drug abuse was performed. Inpatient records, anesthesia records, and outpatient clinic charts were reviewed. All patients were men with right-sided lesions. All six patients abused heroin, and five (83%) abused heroin and cocaine. All six patients had a duplex ultrasound scan, and five (83%) patients had a digital subtraction angiogram to confirm the clinical diagnosis. Proximal vascular control was achieved retroperitoneally through an oblique suprainguinal incision. After vascular isolation and test clamping of the distal external iliac artery (EIA), the pedal pulses were examined with continuous wave Doppler scan. If a Doppler signal was present, this was followed with CFA ligation and local drainage and debridement of the IFAP. RESULTS: Pain at injection site and fever with chills were present in five (83%) and three (50%) patients, respectively. A pulsatile groin mass and thigh or leg edema were present in five (83%) patients. Three patients (50%) had a palpable pedal pulse, and all six had a Doppler signal over a pedal artery at presentation. The mean white cell count was 15.6 thousand per cubic millimeter (range, 9.2-19.3). All patients had a Doppler signal over a pedal artery after distal EIA/CFA test occlusion and ligation. None of the patients required an amputation, and all six patients regained their preoperative ambulatory status. The mean follow-up period was 25 months. Two patients had mild claudication (5-6 blocks) at 18 months and 2 years after surgery. All six patients received drug rehabilitation, but they admitted to drug abuse after surgery and rehabilitation. CONCLUSION: CFA ligation and local debridement are safe treatment modalities for IFAP, if there is an intraoperative Doppler signal over a pedal artery during test occlusion of the distal EIA/CFA. CFA ligation avoids the use and therefore the risk of synthetic conduit infection, because there is a high incidence of postoperative drug injection despite aggressive drug rehabilitation.

A prospective series of out-patient endoscopic retrograde cholangiopancreatography.

OBJECTIVE: Out-patient endoscopic retrograde cholangiopancreatography (ERCP) is widely practised but the safety of this approach has only been addressed in a limited number of prospective series mainly from specialist North American centres. Our objective was to determine prospectively the safety and admission rates of out-patient ERCPs. STUDY DESIGN AND PARTICIPANTS: Patients were selected for out-patient ERCP if in relatively good health, without major risk factors for complications following ERCP and with adequate social support. Our series consisted of 136 consecutive cases of which 82 were therapeutic. SETTING AND OUTCOME MEASURES: A district general hospital in the UK, which also performs ERCPs for neighbouring health districts. Out-patient ERCP patients were followed up at 30 days using standard criteria for defining complications. RESULTS: Procedures were 60 biliary sphincterotomy, 10 stone removal, nine stenting procedures, two dilatations and one pancreatic intervention. Complications were pancreatitis in seven patients (six moderate severity, one mild), cholangitis in three patients, haemorrhage in one patient. Nine patients required admission for complications, two from the endoscopy unit and seven from home; their average in-patient stay was 6 days. Seventeen patients were admitted for observation or for further management. There was one death unrelated to ERCP. Overall, 110 of 136 patients did not require inpatient care following out-patient ERCP. CONCLUSIONS: Our complication rates were similar to those of other series. Out-patient ERCP for selected cases, with adequate post-discharge arrangements for advice and readmission, appears safe and would reduce healthcare costs.

Avoiding the cost of males in obligately asexual Daphnia pulex (Leydig).

Asexual organisms are thought to gain an advantage by avoiding the cost of producing males. In the cladoceran Daphnia pulex (Leydig), male production is determined by the environment and is independent of the origin of the asexual obligate parthenogens from the sexual cyclical parthenogens. If there is a cost to producing males, successful obligate parthenogens should have reduced or eliminated male production. Field and laboratory observations showed that obligate parthenogens have much-reduced male production compared to cyclical parthenogens. Although the reduction or elimination of males in the obligate parthenogens suggests that the cost of males is avoided, the coexistence of sexual and asexual forms of D. pulex may be partially explained by cyclical parthenogens compensating for the cost of males by having greater fecundity. In addition, the absence of a mating constraint for the obligate parthenogens may favour an increased allocation to asexual diapausing eggs earlier in the season compared to the cyclical parthenogens which require mating with males to produce sexual diapausing eggs. No difference in the production of diapausing eggs was observed, probably because males were abundant in populations of cyclical parthenogens and do not appear to limit the production of sexual diapausing eggs. D. pulex is a useful system for determining the ecological consequences of abandoning sexual reproduction and explaining the coexistence of sexual and asexual forms of a species.

Two genetic loci regulate T cell-dependent islet inflammation and drive autoimmune diabetes pathogenesis.

Insulin-dependent diabetes mellitus (IDDM) is a polygenic disease caused by progressive autoimmune infiltration (insulitis) of the pancreatic islets of Langerhan, culminating in the destruction of insulin-producing beta cells. Genome scans of families with diabetes suggest that multiple loci make incremental contributions to disease susceptibility. However, only the IDDM1 locus is well characterized, at a molecular and functional level, as alleleic variants of the major histocompatibility complex (MHC) class II HLA-DQB1, DRB1, and DPB1 genes that mediate antigen presentation to T cells. In the nonobese diabetic (NOD) mouse model, the Idd1 locus was shown to be the orthologous MHC gene I-Ab. Inheritance of susceptibility alleles at IDDM1/Idd1 is insufficient for disease development in humans and NOD mice. However, the identities and functions of the remaining diabetes loci (Idd2-Idd19 in NOD mice) are largely undefined. A crucial limitation in previous genetic linkage studies of this disease has been reliance on a single complex phenotype-diabetes that displays low penetrance and is of limited utility for high-resolution genetic mapping. Using the NOD model, we have identified an early step in diabetes pathogenesis that behaves as a highly penetrant trait. We report that NOD-derived alleles at both the Idd5 and Idd13 loci regulate a T lymphocyte-dependent progression from a benign to a destructive stage of insulitis. Human chromosomal regions orthologous to the Idd5 and -13 intervals are also linked to diabetes risk, suggesting that conserved genes encoded at these loci are central regulators of disease pathogenesis. These data are the first to reveal a role for individual non-MHC Idd loci in a specific, critical step in diabetes pathogenesis-T cell recruitment to islet lesions driving destructive inflammation. Importantly, identification of intermediate phenotypes in complex disease pathogenesis provides the tools required to progress toward gene identification at these loci.

Analysis of ketamine responses in the pulmonary vascular bed of the cat.

OBJECTIVE: To test the hypothesis that pulmonary vasodilator responses of ketamine are dependent on activation of L-type calcium channels, independent of synthesis of nitric oxide from L-arginine, activation of adenosine triphosphate-sensitive potassium channels, and the release of cyclooxygenase products. DESIGN: Prospective study. SETTING: Research laboratory. SUBJECTS: Isolated lobar lung preparation, mongrel cats. INTERVENTIONS: In separate experiments, the effects of nicardipine; N omega-I-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase; glibenclamide, an adenosine triphosphate-sensitive potassium channel antagonist; and meclofenamate, a cyclooxygenase blocker, were investigated in the pulmonary vascular bed of the cat. The effects of these agents were evaluated on the pulmonary arterial responses of ketamine, acetylcholine, and isoproterenol during elevated tone conditions induced by the thromboxane A2 mimic, U46619 (Upjohn, Kalamazoo, MI). MEASUREMENTS: Lobar arterial perfusion pressure, systemic pressure, and left atrial pressure were continuously monitored, electronically averaged, and permanently recorded. MAIN RESULTS: Under elevated tone conditions in the isolated left lower lobe vascular bed of the cat, N omega-I-nitro-L-arginine methyl ester, glibenclamide, and meclofenamate had no significant effect on the vasodilator responses to ketamine. Nicardipine, in a dose that reduced significantly vasopressor effects to BAY K 8644, a calcium-channel opener, attenuated significantly vasodilator responses to ketamine, whereas the L-type calcium-channel blocker had no significant effects on responses to acetylcholine and to isoproterenol. CONCLUSIONS: These data show that ketamine has significant vasodilator activity in the pulmonary vascular bed of the cat. The present data also suggest that responses to ketamine during elevated tone conditions may in part be mediated by the activation of L-type calcium channels.

Effect of probe design on accuracy and reliability of pulse oximetry in pediatric patients.

STUDY OBJECTIVE: To determine if the traditional band-wrap disposable pulse oximeter probe is more accurate or reliable for oxygen monitoring in children than the reusable clip-type probe. DESIGN: Prospective, observational clinical study. SETTING: Operating room of a large university hospital. PATIENTS: 18 children 11 years of age or younger, who were scheduled for general anesthesia with placement of an intraarterial catheter. INTERVENTIONS: Pulse oximetry values were obtained using both band and clip probes of three pulse oximeters (Nellcor, Hayward CA; Novametrix, Wallingford, CT; Ohmeda, Boulder, CO) and compared with simultaneous hemoximetry values. Dropout rate (percent of down time) also was recorded for each probe-machine combination. MEASUREMENTS AND MAIN RESULTS: Data were analyzed using bias and precision and t-test. p < 0.05 is considered significant. Bias (mean SpO2-SaO2) is less than 2% for all probe-machine combinations. The range of error observed between SpO2 and SaO2 for all data points was greatest using the Nellcor band (27.8%) and least using the Ohmeda band (11.4%). In cyanotic children, the greatest bias and precision were observed with Nellcor band (N = 11, -5.12 +/- 9.74) and the best agreement with Novametrix band (N = 17, 0.08 +/- 4.21). The difference in bias for any test units above or below the median weight of 13.75 kg did not vary by more than 1%. Dropout rate was minimal for all units in nonbypass situations. After cardiopulmonary bypass, no data dropout was observed with Ohmeda band; observed down time with the other units varied between 34% and 55%. CONCLUSIONS: The type of probe selected has little effect on accuracy of pulse oximetry in children. After cardiopulmonary bypass, using the Ohmeda band combination may improve the likelihood of obtaining consistent readings and decreasing down time.

Independent genetic regulation of T-cell and antigen-presenting cell participation in autoimmune islet inflammation.

Genetic susceptibility to type 1 diabetes in the nonobese diabetic (NOD) mouse involves at least 17 Idd loci. Idd1 has been mapped to a class II gene in the major histocompatibility complex (MHC), whereas the products and functions of the remaining Idd loci are unresolved. To investigate how non-MHC Idd genes regulate islet inflammation and IDDM progression, NOD mice were compared with the nonobese diabetes-resistant (NOR) mouse, a related MHC-identical strain that possesses a subset of the NOD-derived alleles at the Idd loci. Using quantitative reverse transcriptase-polymerase chain reaction amplification and immunohistochemistry, we observed that disease resistance in NOR mice is reflected by a protracted block at the earliest stage of insulitis. In NOD islets, early antigen-presenting cell (APC) recruitment to islet lesions was temporally coincident with progressive T-cell infiltration. In striking contrast, islet infiltrates in NOR mice were composed of APCs with minimal contribution from T-cells and T-cell-derived inflammatory cytokines, conferring apparent resistance to invasive insulitis and beta-cell destruction. This is the first evidence that a subset of Idd susceptibility loci independently regulate T-cell and APC participation in insulitis progression. As progress is made toward identification of the Idd gene products, it will be crucial to determine how they regulate diabetogenesis. Our data define distinct cellular stages of IDDM pathogenesis in which the impact of Idd genes can be readily analyzed.

U.K. Prospective Diabetes Study 22. Effect of age at diagnosis on diabetic tissue damage during the first 6 years of NIDDM.

OBJECTIVE: To assess the effect of age at diagnosis on the initial prevalence and subsequent risk of the progression of diabetic tissue damage in patients with NIDDM. RESEARCH DESIGN AND METHODS: The prevalence of Q-wave myocardial infarction, absent dorsalis pedis pulses, retinopathy, absent ankle jerks, hypertension, and microalbuminuria were determined at baseline and at 3 and 6 years of follow-up in five consecutive 6-year age-cohorts of 3,027 newly diagnosed white patients aged between 36 and 65 years recruited to the U.K. Prospective Diabetes Study. The effect of age at diagnosis on the initial prevalence and the risk of progression of these complications and associated conditions was analyzed using logistic regression and proportional odds methods, respectively. RESULTS: Q-wave myocardial infarction and hypertension were more prevalent in older patients at presentation, but age at diagnosis did not have a significant effect on the increased risk of either after 6 years of NIDDM. Absent dorsalis pedis pulses and ankle jerks were also more prevalent in the older age-groups at presentation, but age at diagnosis was a significant predictor of the increasing prevalence of both during follow-up. The baseline prevalence of retinopathy and microalbuminuria was not related to age. The subsequent risk of retinopathy, but not microalbuminuria, increased significantly with age at diagnosis. CONCLUSIONS: Age at diagnosis has a variable impact on different types of diabetic tissue damage and may thus be an important variable in epidemiological and intervention studies in NIDDM. Regular ophthalmologic surveillance and examination of the feet increase in importance with increasing age since the diagnosis of NIDDM.

IL-4 expression at the onset of islet inflammation predicts nondestructive insulitis in nonobese diabetic mice.

In nonobese diabetic mice, autoimmune diabetes progresses in an age-linked and gender-dependent manner. Insulitis begins in male and female mice at approximately 1 mo of age; however, 70 to 90% of females, but only 10 to 20% of males, become diabetic by 6 mo. Multiple studies propose that proinflammatory Th1 and immunomodulatory Th2 cytokines impact diabetes pathogenesis, but the role of these cytokines in spontaneous diabetes progression is not yet clear. We used quantitative reverse-transcriptase-coupled PCR to analyze expression of cytokines and APC costimulatory molecules in the islets of 20- to 180-day-old male and female nonobese diabetic littermates, and identified three stages in diabetes progression. At 1 to 2 mo of age, islet-infiltrating T cells displayed a Th1 cytokine bias in females, and a Th2 cytokine bias in males. In females, stage II (2-3 mo of age) was characterized by an increase in islet-infiltrating T cells, APC, and Th1 cytokines, whereas male infiltrates did not increase in size, and Th1 cytokine expression continued to decline during this interval. Islet infiltration reached a plateau (stage III) in 3- to 4-mo-old females, months before overt diabetes onset. Our data imply that Th cytokine expression in early insulitis exerts substantial impact on beta cell destruction and overt diabetes. A clinical implication of our results is that young individuals in the early stages of insulitis are ideal candidates for therapeutic intervention to minimize beta cell destruction and morbidity.

Fluorescence-based signal amplification technology.

The ELF alkaline phosphate substrate can be used to fluorescently label a wide variety of biological targets. This substrate yields a bright, photostable yellow-green fluorescent precipitate at the site of enzymatic activity. ELF labeling can be as much as 40 times as bright and hundreds of times as photostable as labeling with conventional fluorophores and yields signals capable of very fine submicroscopic resolution. Signal development is also extremely rapid, making the signal amplification technology well suited for applications such as RNA in situ hybridization.