Keratinocytes derived from psoriatic plaques are resistant to apoptosis compared with normal skin.

Previously we observed that hyperplastic epidermal keratinocytes characteristic of psoriasis had abundant amounts of the cell survival protein Bcl-xL; however, whether this overexpression correlated with enhanced survival was unclear because the majority of epidermal cells possess nuclei that are positively labeled by an assay typically regarded as indicative of cells undergoing apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining). To clarify this apparent discrepancy, we explored the propensity of keratinocytes derived from psoriatic plaques to undergo apoptosis and also determined the reliability of TUNEL staining as an indicator of apoptosis in keratinocytes in vitro and in vivo. First, a keratinocyte cell line, HaCat, was examined before and after being suspended in semisolid medium (methylcellulose) using flow cytometry to detect TUNEL-positive cells, and the percentage of positive cells was correlated to the presence or absence of double-stranded DNA fragmentation using pulsed field gel electrophoresis. After 18 hours in methylcellulose suspension, apoptosis was detected in HaCat cells when at least 5% of the cell population was undergoing programmed cell death. Second, we examined 23 clinical specimens of skin (13 from psoriatic patients and 10 from healthy control subjects) and observed that no double-stranded DNA fragmentation was present in any of the freshly isolated keratinocytes from either normal or psoriatic patients. Keratinocytes from 9 of 12 normal skin samples underwent double-stranded DNA fragmentation after being in methylcellulose for 18 to 24 hours, which contrasts with keratinocytes from lesions of psoriasis where only 1 of 13 of the skin samples had these changes. Third, two-color immunofluorescence staining of psoriatic plaques revealed that numerous TUNEL-positive keratinocytes were also positive for proliferating cell nuclear antigen and Ki-67 antigens and that by flow cytometry TUNEL-positive keratinocytes obtained from psoriatic plaques possessed a DNA content profile indicative of proliferating and not dying cells. These results demonstrate that keratinocytes within psoriatic plaques do not have double-stranded DNA breaks, that they have a prolonged capacity to resist induction of apoptosis compared with normal-skin-derived keratinocytes or cultured HaCat cells, and that caution is necessary for proper interpretation related to detection of 3'-OH DNA ends (ie, TUNEL positivity) in skin, as it can be associated with DNA synthesis as well as cell death.     

p53-Regulated Apoptosis Is Differentiation Dependent in Ultraviolet B-Irradiated Mouse Keratinocytes

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (−/−) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53−/− mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m² UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53−/− cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53−/− cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and is therefore of questionable significance in protection against skin cancer induction.     

Sensitivity of SARS‐CoV‐2 to different temperatures

This study was designed to investigate the sensitivity of SARS‐CoV‐2 to different temperatures, to provide basic data and a scientific basis for the control of COVID‐19 epidemic. The virus was dispersed in 1 mL basal DMEM medium at a final concentration of 10^3.2 TCID₅₀/mL and then incubated at 4, 22, 30, 35, 37, 38, 39 and 40°C for up to 5 days. The infectivity of residual virus was titrated using the Vero E6 cell line. The results showed that the virus remained viable for 5 days at 4°C, and for 1 day only at 22 and 30°C. We found that the infectivity of the virus was completely lost after less than 12 hours at 37, 38 and 39°C, while at 40°C, the inactivation time of the virus was rapidly reduced to 6 hours. We show that SARS‐CoV‐2 is sensitive to heat, is more stable at lower temperatures than higher temperature, remains viable for longer at lower temperatures, and loses viability rapidly at higher temperatures.     

Ultrastructural and confocal laser scanning microscopic examination of TUNEL-positive cells

TdT-mediated dUTP-biotin nick end labelling (TUNEL) has been widely used for detecting cells with DNA fragmentation or apoptotic cells. However, since the concept of apoptosis is based on cellular ultrastructure, it is important to identify the morphological features of TUNEL-positive cells. In this study, we performed TUNEL and electron microscopic observation on serial semithin and ultrathin sections of pancreas from bilaterally adrenalectomized rats with caerulein-induced pancreatitis. TUNEL-positive cells were identified with two different ultrastructural patterns. One was characteristic of apoptosis, with condensed nuclei, intact mitochondria, and zymogen granules. The other pattern was one of marked cellular degeneration, possibly representing the end stage of cell death. Cells which did not demonstrate these ultrastructural patterns were not labelled by the TUNEL method. The three-dimensional structure of TUNEL-positive cells was also investigated by confocal laser scanning microscopy (CLSM), which showed the apoptotic nuclei exhibited various three-dimensional structures. These results confirm the utility of the TUNEL method in detecting apoptosis; application of the technique reported in this study will contribute to the further characterization of individual TUNEL-positive cells. © 1997 John Wiley & Sons, Ltd.     

The tunel assay in the diagnosis of graft-versus-host disease: caveats for interpretation

Acute graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality following bone marrow transplantation, and early detection is important to allow effective therapy. Since the presence of apoptotic keratinocytes (dyskeratotic bodies) has been suggested as a useful diagnostic criterion for GVHD, attention has focused on the use of the TUNEL assay to detect apoptosis in clinical specimens. We reviewed clinical specimens upon which TUNEL had been performed for possible artifacts that might interfere with accurate evaluation for GVHD. Several distinct types of artifact were found and could be re-created in experimental systems. Artifacts in TUNEL staining generally resulted from the lack of specificity of this reaction for apoptotic cell death. Artifacts were found resulting from inadequate fixation, over-exposure of the TUNEL reaction, and proximity to the section edge. In addition, a novel artifact, apparently resulting from DNA shearing during the sectioning process, was noted and confirmed using confocal microscopy of experimental specimens. The TUNEL assay must therefore must be interpreted with caution in the clinical setting. In our laboratory, we consider TUNEL-positive cells as apoptotic only when accompanied by apoptotic morphology. Although these criteria clearly miss some cells in the early stages of apoptosis, they provide the highest specificity for apoptotic cell death.     

Radiosensitivity of human ovarian carcinoma and melanoma cells to γ-rays and protons

Introduction

Proton radiation offers physical advantages over conventional radiation. Radiosensitivity of human 59M ovarian cancer and HTB140 melanoma cells was investigated after exposure to γ-rays and protons.

Material and methods

Irradiations were performed in the middle of a 62 MeV therapeutic proton spread out Bragg peak with doses ranging from 2 to 16 Gy. The mean energy of protons was 34.88 ±2.15 MeV, corresponding to the linear energy transfer of 4.7 ±0.2 keV/µm. Irradiations with γ-rays were performed using the same doses. Viability, proliferation and survival were assessed 7 days after both types of irradiation while analyses of cell cycle and apoptosis were performed 48 h after irradiation.

Results

Results showed that γ-rays and protons reduced the number of viable cells for both cell lines, with stronger inactivation achieved after irradiation with protons. Surviving fractions for 59M were 0.91 ±0.01 for γ-rays and 0.81 ±0.01 for protons, while those for HTB140 cells were 0.93 ±0.01 for γ-rays and 0.86 ±0.01 for protons. Relative biological effectiveness of protons, being 2.47 ±0.22 for 59M and 2.08 ±0.36 for HTB140, indicated that protons provoked better cell elimination than γ-rays. After proton irradiation proliferation capacity of the two cell lines was slightly higher as compared to γ-rays. Proliferation was higher for 59M than for HTB140 cells after both types of irradiation. Induction of apoptosis and G2 arrest detected after proton irradiation were more prominent in 59M cells.

Conclusions

The obtained results suggest that protons exert better antitumour effects on ovarian carcinoma and melanoma cells than γ-rays. The dissimilar response of these cells to radiation is related to their different features.     

Biological and physical properties of a human γM-cryoglobulin and its monomer subunit

A patient with Waldenstrom's macroglobulinaemia was found to have a serum cryoprecipitate which consisted entirely of a γMK-macroglobulin. This protein had no detectable antibody activity against a variety of other serum components and fixed only minimal amounts of complement over temperatures ranging from 4°C to 37°C. Precipitation of the cryoglobulin began at about 30°C and was complete at temperatures below 20°C. In contrast to other macroglobulins that have been reported, cryoprecipitability persisted after the protein was dissociated into its monomer subunits. Isolated subunits formed cryoprecipitates as did the hybrid macroglobulin formed from equal parts of such subunits and the subunits of a non-cryoprecipitating macroglobulin.     

Assessing the Validity of Aural Thermometry for Measuring Internal Temperature in Patients With Exertional Heat Stroke

ContextThe use of aural thermometry as a method for accurately measuring internal temperature has been questioned. No researchers have examined whether aural thermometry can accurately measure internal body temperature in patients with exertional heat stroke (EHS).ObjectiveTo examine the effectiveness of aural thermometry as an alternative to the criterion standard of rectal thermometry in patients with and those without EHS.DesignCross-sectional study.SettingAn 11.3-km road race.Patients or Other ParticipantsA total of 49 patients with EHS (15 men [age = 38 ± 17 years], 11 women [age = 28 ± 10 years]) and 23 individuals without EHS (10 men [age = 62 ± 17 years], 13 women [age = 45 ± 14 years]) who were triaged to the finish-line medical tent for suspected EHS.Main Outcome Measure(s)Rectal and aural temperatures were obtained on arrival at the medical tent for patients with and those without EHS and at 8.3 ± 5.2 minutes into EHS treatment (cold-water immersion) for patients with EHS.ResultsThe mean difference between temperatures measured using rectal and aural thermometers in patients with EHS at medical tent admission was 2.4°C ± 0.96°C (4.3°F ± 1.7°F; mean rectal temperature = 41.1°C ± 0.8°C [106.1°F ± 1.4°F]; mean aural temperature = 38.8°C ± 1.1°C [101.8°F ± 2.0°F]). Rectal and aural temperatures during cold-water immersion in patients with EHS were 40.4°C ± 1.0°C (104.6°F ± 1.8°F) and 38.0°C ± 1.2°C (100.3°F ± 2.2°F), respectively. Rectal and aural temperatures for patients without EHS at medical tent admission were 38.8°C ± 0.87°C (101.9°F ± 1.6°F) and 37.2°C ± 1.0°C (99.1°F ± 1.8°F), respectively.ConclusionsAural thermometry is not an accurate method of diagnosing EHS and should not be used as an alternative to rectal thermometry. Using aural thermometry to diagnosis EHS can result in catastrophic outcomes, such as long-term sequelae or fatality.     

Long-Term Survival of Streptococcus pneumoniae at Room Temperature on Dorset Egg Medium

Forty-five isolates of Streptococcus pneumoniae were inoculated on Dorset egg and supplemented Columbia agar base media, incubated overnight at 37°C, and then kept at room temperature (RT; 21°C) or 4°C. Long-term viability was best at RT for both media, with all isolates remaining viable on Dorset egg medium for 44 days; viability was 90 and 57% on Columbia agar base medium after 7 and 30 days. We recommend the use of Dorset egg medium for the maintenance of pneumococci at RT.     

The role of anakinra in the modulation of intestinal cell apoptosis and inflammatory response during ischemia/reperfusion

Background/aim Even though interleukin-1 receptor antagonist, IL-1Ra, is used in certain inflammatory diseases, its effect on ischemia-reperfusion injury is a current research topic. We aimed to investigate the protective effects of anakinra, an IL-1Ra, on the I/R induced intestinal injury.Materials and methods The rat model of intestinal ischemia-reperfusion was induced. Rats were randomized into 4 groups: (group 1) control group, (group 2) I/R group, (group 3 and 4) treatment groups (50 mg/kg and 100 mg/kg, respectively). Gene expressions of caspase-3, TNF-α, IL-1α, IL-6, and apoptotic cells in tissue samples were evaluated by PCR and TUNEL methods, respectively. Plasma levels of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were studied by the ELISA method and tissue samples were examined histopathologically as well.Results Anakinra inhibited the expression of IL-1α, IL-6, and TNF-α and decreased the SOD, CAT, and MDA caused by ischemia-reperfusion injury in both treatment groups. Caspase-3 expression and TUNEL-positive cell number in treatment groups were also less. Histopathologically, anakinra better preserved the villous structure of the small intestine at a dose of 100 mg/kg than 50 mg/kg. Conclusion Anakinra decreased the intestinal damage caused by ischemia-reperfusion and a dose of 100 mg/kg was found to be histopathologically more effective.     

Ischemia induces endoplasmic reticulum stress and cell apoptosis in human brain

In animal models, endoplasmic reticulum (ER) stress and apoptosis take place around cerebral infarction areas during ischemia, which presumably protect tissues from necroses-induced injury as well as promote cells toward death. We examined whether these pathological changes, especially temporal occurrence, were present in patients who suffered from cerebral ischemia. The studies by immunohistochemistry show that ER chaperone glucose-regulated protein (GRP78) and caspase-9 elevate around infarction areas. The experiments by terminal deoxynucleotidy transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick-end labeling (TUNEL) illustrate that TUNEL-positive cells are higher around infarction tissues than controls. Moreover, GRP78, caspase-9 and TUNEL cells emerge one after another during ischemia. In conclusion, ER stress, apoptosis initiation and DNA fragment develop sequentially in ischemic human brain. ER stress during excessive ischemia stimulates apoptotic cell death beyond activating a defense for nerve cells being away from injury.     

Rapid cytotoxicity of human B lymphocytes induced by VH4-34 (VH4.21) gene-encoded monoclonal antibodies

We have previously described two human cold agglutinin MoAbs 216 and A6(H4C5), that are derived from the VH4-34 (VH4.21) gene that bind specifically to a cell surface ligand on human B lymphocytes. In this study, we report that binding of 216 and A6(H4C5) leads to rapid killing of target B cells. This complement-independent cytotoxicity was measured by three independent assays, cell viability dye uptake on FACS, ³H-thymidine uptake, and the 3(4,5)-dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxicity was specific for CD20⁺ mononuclear cells in human spleen and peripheral blood. The MoAbs were also cytotoxic to human B cell lines Nalm-6, OCI-LY8, Arent and SUP-B8, but not to T cell lines HuT 78 and PEER. As observed by scanning electron microscopy, membrane pores were formed within 15 min of exposure to the MoAbs. Cytotoxic activity was dependent on MoAb concentration and temperature of exposure. Killing was greater at 4°C than 37°C. Sodium azide and EDTA did not block the cytotoxic activity. No DNA fragmentation typical of apoptosis was observed. This rapid cytotoxic activity, independent of physiologic cellular processes and independent of complement, suggests a novel mechanism of cell death via membrane perturbations.     

Rhein induces apoptosis of human gastric cancer SGC-7901 cells via an intrinsic mitochondrial pathway

Rhein is a primary anthraquinone found in the roots of a traditional Chinese herb, rhubarb, and has been shown to have some anticancer effects. The aim of the present study was to investigate the effect of rhein on the apoptosis of the human gastric cancer line SGC-7901 and to identify the mechanism involved. SGC-7901 cells were cultured and treated with rhein (0, 50, 100, 150, and 200 µM) for 24, 48, or 72 h. Relative cell viability assessed by the MTT assay after treatment was 100, 99, 85, 79, 63% for 24 h; 100, 98, 80, 51, 37% for 48 h, and 100, 97, 60, 36, 15% for 72 h, respectively. Cell apoptosis was detected with TUNEL staining and quantified with flow cytometry using annexin FITC-PI staining at 48 h after 100, 200 and 300 µm rhein. The percentage of apoptotic cells was 7.3, 21.9, 43.5%, respectively. We also measured the mRNA levels of caspase-3 and -9 using real-time PCR. Treatment with 100 µM rhein for 48 h significantly increased mRNA expression of caspase-3 and -9. The levels of apoptosis-related proteins including Bcl-2, Bax, Bcl-xL, and pro-caspase-3 were evaluated in rhein-treated cells. Rhein increased the Bax:Bcl-2 ratio but decreased the protein levels of Bcl-xL and pro-caspase-3. Moreover, rhein significantly increased the expression of cytochrome c and apoptotic protease activating factor 1, two critical components involved in mitochondrial pathway-mediated apoptosis. We conclude that rhein inhibits SGC-7901 proliferation by inducing apoptosis and this antitumor effect of rhein is mediated in part by an intrinsic mitochondrial pathway.     

Characterization of two different agglutinators in the latex fixation test, occurring in normal human sera

Using a sensitive modification of the latex fixation test it is possible to detect a small agglutinating effect in about 60 per cent of normal human sera, after these have been heated for 30 minutes at 56°. This was shown to be caused by an IgM globulin with the properties of a rheumatoid factor. The factor is able to react with human IgG globulin and may represent an antibody to the IgG part of circulating antigen—antibody complexes. The heat treatment probably inactivates an inhibitor of the latex fixation reaction.

In addition all normal human sera give an agglutination reaction with IgG coated latex at incubation temperatures of 37° or lower. It was shown that these reactions are caused by a thermolabile, non-reducible component with a sedimentation constant of about 10. This component is probably identical with the complement component C'_1q. The agglutinating activity was found in the α₂—β₁ region after electrophoresis of untreated serum, but in the slow γ region after treatment of the serum with EDTA. This kind of agglutination may cause false positive reactions in latex tests which are carried out at 37° or less.

     

Natural antibody-induced intracellular signalling and growth control in C3H 10T½ fibroblast variants

Cell-surface binding by natural antibody (NAb) places it well for controlling cell function directly through signalling. Flow cytometry revealed an instability of syngeneic NAb binding to C3H 10T½ fibroblast variants at 37°, which could be partially reduced by H7, an inhibitor of the pivotal signalling serine/threonine kinase, protein kinase C (PKC). Cells coated with purified NAb at 4° followed by a rise in temperature to 37° showed an increase in membrane expression of introduced rat PKC-β1 and endogenous PKC-α, in the PKC-β1-overexpressing PKC-4 and v-H-ras-producing I3T2.1, respectively. Tyrosine phosphorylation of membrane-associated 60 000 MW protein including the tyrosine kinase src was markedly reduced. In addition, both the precoated NAb and numerous membrane molecules ranging from 20 000 to 220 000 MW were released into the supernatant, including the receptor-like protein tyrosine phosphatase α (RPTP-α). Furthermore, purified NAb reduced the growth of I3T2.1 cells in culture assessed as a decrease in total cell numbers and an increase in the proportion of cells in the G0/G1 phase of the cell cycle. Together, these data argue that the interaction of NAb with cell surface structures initiated a series of intracellular signalling events leading to the release of membrane molecules and over time the suppression of cell proliferation. This process could provide a biological mechanism for direct NAb control of activated cells in both physiological and pathological conditions.     

An analysis of apoptosis in lymphoid organs and lupus disease in murine systemic lupus erythematosus (SLE)

Apoptosis is a programmed cell death process that helps to regulate both T cell and B cell development. In this study, we have investigated the levels of apoptotic death in cells of the thymuses and spleens (white matter) of autoimmune MRL-lpr/lpr mice with progressive lymphadenopathy and SLE disease activity; we also examined the renal pathology in these animals. Fas is a cell surface receptor, which when activated initiates the sequence of events that lead to apoptosis. In MRL-lpr/lpr mice Fas is defective, so the competency for apoptosis may be reduced. In young animals of advancing age the thymuses enlarged until in 5-month-old females the average weight was three times that at 1 month, and spleen and kidney weights also increased in size disproportionately. At light microscope level apoptotic cells in tissue sections were counted using both routine eosin and haematoxylin staining (to identify them by their morphology) and in situ end-labelling of cells with DNA strand breaks; their presence was further confirmed by electron microscopy. As the mice aged, the numbers of apoptotic cells in thymic cortex, thymic medulla and spleen white pulp areas reduced significantly (P < 0·01–0·001), whereas in BALB/c normal controls they increased significantly (P < 0·05). These changes were coincident with the development of severe lupus, whose activity was assessed by measuring serum anti-ssDNA and anti-dsDNA antibody titres and urinary protein (albumin) level which were elevated significantly by 5 months of age (P < 0·001 for both ssDNA and dsDNA and P < 0·01 for urine albumin) compared with their younger counterparts. Thus, lymphoid organ enlargement, decrease in apoptotic indices, elevated serum anti-ssDNA and anti-dsDNA antibody levels, and impaired renal function coincided with the onset and severity of lupus disease in lpr mice. It seems likely that there is a causal relationship between defective deletion of autoreactive lymphoid cells, imperfect Fas-mediated apoptosis and development of murine SLE.     

Inflammatory cytokines and plasma redox status responses in hypertensive subjects after heat exposure

Hypertension is characterized by a pro-inflammatory status, including redox imbalance and increased levels of pro-inflammatory cytokines, which may be exacerbated after heat exposure. However, the effects of heat exposure, specifically in individuals with inflammatory chronic diseases such as hypertension, are complex and not well understood. This study compared the effects of heat exposure on plasma cytokine levels and redox status parameters in 8 hypertensive (H) and 8 normotensive (N) subjects (age: 46.5±1.3 and 45.6±1.4 years old, body mass index: 25.8±0.8 and 25.6±0.6 kg/m², mean arterial pressure: 98.0±2.8 and 86.0±2.3 mmHg, respectively). They remained at rest in a sitting position for 10 min in a thermoneutral environment (22°C) followed by 30 min in a heated environmental chamber (38°C and 60% relative humidity). Blood samples were collected before and after heat exposure. Plasma cytokine levels were measured using sandwich ELISA kits. Plasma redox status was determined by thiobarbituric acid reactive substances (TBARS) levels and ferric reducing ability of plasma (FRAP). Hypertensive subjects showed higher plasma levels of IL-10 at baseline (P<0.05), although levels of this cytokine were similar between groups after heat exposure. Moreover, after heat exposure, hypertensive individuals showed higher plasma levels of soluble TNF receptor (sTNFR1) and lower TBARS (P<0.01) and FRAP (P<0.05) levels. Controlled hypertensive subjects, who use angiotensin-converting-enzyme inhibitor (ACE inhibitors), present an anti-inflammatory status and balanced redox status. Nevertheless, exposure to a heat stress condition seems to cause an imbalance in the redox status and an unregulated inflammatory response.     

Diminished human immunodeficiency virus type 1 DNA yield from dried blood spots after storage in a humid incubator at 37 degrees C compared to -20 degrees C

Collecting whole blood on filter paper simplifies the processing, transport, and storage of specimens used for the diagnosis of human immunodeficiency virus type 1 (HIV-1) and other tests. Specimens may be collected in tropical or rural areas with minimal facilities for handling specimens. To compare simulated tropical conditions with freezer storage, we examined the stability of HIV-1 DNA in dried blood spots (DBS) stored in humid heat and at −20°C. DBS were created by spotting 50-μl aliquots of whole blood on 903 filter paper. DNA was extracted from DBS at baseline and after 2, 6, or 12 months of storage at −20°C or at 37°C with ~85% humidity. The DNA was tested undiluted or diluted using the Amplicor HIV-1 DNA PCR (Roche), version 1.5. Each reaction was scored positive, negative, or indeterminate based on optical density. Results were compared between storage conditions and over time. A total of 1,832 reactions from 916 DBS were analyzed, including 100 DBS at baseline, 418 stored at −20°C, and 398 stored at 37°C. A chi-square test showed fewer positive reactions for DBS stored at 37°C (55%) than for those stored at −20°C (78%) (P < 0.0001). Samples stored at −20°C showed little change in the probability of detection of HIV-1 DNA over time; the odds ratio (OR) was 0.93 after storage for 1 year. Samples stored at 37°C demonstrated a significant change in detection at 1 year (OR, 0.29). We conclude that exposure of DBS to 37°C and high humidity impaired the recovery of HIV-1 DNA from DBS, whereas DNA recovery was preserved when DBS were stored frozen.     

Increased immunoglobulin production in silver catfish (Rhamdia quelen) exposed to agrichemicals

Fish vaccination has been increasingly exploited as a tool to control pathogen infection. The production of immunoglobulin following vaccination might be affected by several factors such as management procedures, water temperature, and the presence of xenobiotics. In the present study, we aimed to investigate the kinetics of immunoglobulin production in silver catfish (Rhamdia quelen) inoculated with inactivated Aeromonas hydrophila and kept at two different water temperatures (17.4±0.4° or 21.3±0.3°C). The effect of a second antigen inoculation and exposure of fish to sublethal concentrations of the herbicides atrazine and glyphosate at 10% of the lethal concentration (LC_50-96h) on specific serum antibodies were also investigated. Antibodies to A. hydrophila were detected as early as 7 days post-inoculation and increased steadily up to 35 days. The kinetics of antibody production were similar in fish kept at 17.4±0.4° and 21.3±0.3°C, and reinoculation of antigen at 21 days after priming failed to increase specific antibody levels. Intriguingly, we found that, in fish exposed to atrazine and glyphosate, the secretion of specific antibodies was higher than in non-exposed inoculated fish. These findings are important for the design of vaccines and vaccination strategies in Neotropical fish species. However, because atrazine and glyphosate are widespread contaminants of soil and water, their immune-stimulating effect could be harmful, in that fish living in herbicide-contaminated water might have increased concentrations of nonspecific antibodies that could mediate tissue injury.     

Effect of incubation temperature on mitogen responses of lymphocytes from adult peripheral blood and from cord blood

Lymphocytes from normal adults, when cultured with phytohaemagglutinin (PHA) or Concanavalin A (Con A) at 39°C, showed an enhancement and earlier onset of ³H-thymidine incorporation compared with cultures incubated at 37°C. In contrast, the peak responses of cord blood lymphocytes incubated at either 35°C or 39°C did not differ significantly from those incubated at 37°C. Cultures of adult lymphocytes showed an exponential rise and fall in ³H-thymidine incorporation, which was much more rapid at 39°C than at 37°C. However, the kinetics of thymidine incorporation into mitogen-stimulated cord blood lymphocytes incubated at 39°C were similar to those at 37°C. The following results suggested that temperature acted predominantly on the proliferative phase of the transformation response. Firstly, by inhibiting binding of Con A using methyl-α-D-mannopyranoside, it was found that activation of adult lymphocytes took place within the same time period at both 39°C and 37°C. Secondly, cultures incubated at 37°C for 3 days, and labelled for 4 hr at either 37°C or 39°C showed no significant difference in ³H-thymidine uptake, whereas cultures incubated at 39°C for 3 days, and labelled for 4 hr at 37°C showed significantly higher responses than those both incubated and labelled at 37°C. Thirdly, the major increase in thymidine uptake occurred after incubation at 39°C for the second and third days of culture. These findings were consistent with a shortening of the cell cycle at the higher temperature. Thus, the failure of cord blood lymphocytes to show increased thymidine uptake after incubation at 39°C apparently reflects an insensitivity to temperature of certain of the metabolic pathways involved in cell replication in the neonate.