Effect of temperature on the metabolism of proteoglycans in explants of bovine articular cartilage

The turnover of proteoglycans in bovine articular cartilage was determined in explant cultures, maintained at 32 degrees C or 37 degrees C. Both the rate of proteoglycan synthesis and the release of newly synthesized proteoglycans were decreased in cultures incubated at 32 degrees C compared to 37 degrees C. At both temperatures the newly synthesized proteoglycans were similar in hydrodynamic size and chain length of the glycosaminoglycans. However, the ratio of 6-sulfated disaccharides over 4-sulfated disaccharides of the newly synthesized glycosaminoglycans, differed less from the endogenous ratio at 32 degrees C than at 37 degrees C. At both temperatures, the incorporated 35S-sulfate is released from explants in two pools. Twenty-three percent of the 35S-radiolabel was released into the culture medium during an initial short phase (t1/2 = 1.1 day at 32 degrees C, 1.3 day at 37 degrees C), 77% had a much longer half-life. The lowered temperature markedly decreased the release of 35S-sulfate with a slow turnover (t1/2 = 60 days at 32 degrees C, 38 days at 37 degrees C).     

Sensitivity of preincubated lymphocytes to suppressor regulation

DNA synthesis of human peripheral blood lymphocytes increases if Con A is added to the culture after 24 h preincubation at 37 degrees C. During preincubation the mitogen reactive lymphocytes lose their sensitivity to the suppressive effect of autologous mitomycin-treated mononuclear leukocytes, and of supernatants of autologous preincubated cells. The reactive lymphocytes preincubated at 4 degrees C retain their sensitivity to the suppressive effect of regulatory cells and their supernatants. It is assumed that the enhancement of mitogen response after preincubation at 37 degrees C is caused by a decrease of suppressor regulation of human lymphocytes. Prostaglandins may be regarded as one of the mediators of the suppression.     

Subpopulations of lymphocytes in human thymomas.

Lymphocyte populations in six normal thymuses and ten thymomas were examined. The majority of lymphocytes from both thymus and thymoma differ from peripheral T lymphocytes in their capacity to form E-rosettes resistant to incubation at 37 degrees C. Low percentages of T lymphocytes bearing receptors for the Fc portion of IgG (TG) and IgM (TM) were found in normal thymus. In contrast, lymphocytes from five out of nine thymomas showed remarkable percentages of TM cells. Compared with normal thymocytes, lymphocytes from seven out of ten thymomas responded vigorously to mitogens. The possible origin and nature of thymoma lymphocytes are discussed.     

Enhancement of mitogen-induced lymphocyte proliferation after preincubation is due to altered sensitivity to prostaglandins

Twenty-four hour preincubation at 37 degrees enhances the mitogen-induced DNA synthesis of human lymphocytes. PGE1, given simultaneously with Con A at the start of lymphocyte culture, inhibits the DNA synthesis. After 24 h preincubation of cells, PGE1 fails to decrease the DNA synthesis. Similarly, preincubation abolished the effect of indomethacin increasing DNA synthesis of freshly separated lymphocytes. The intracellular cAMP level of human mononuclear leukocytes rapidly decreases during in vitro incubation at 37 degrees C. PGE1 elevates the intracellular cAMP of freshly separated lymphocytes to 45 times of its starting level. After 24 h preincubation of cells at 37 degrees C, PGE1 elevates cAMP to a lesser extent. This change of PGE1 action may explain the fact that the effect of exogenous PGE1 and endogenous prostaglandins (the production of which can be inhibited by indomethacin) diminishes after incubation of lymphocytes. It is very likely that the change of the effect of prostaglandins produced in lymphocyte cultures and the spontaneous decrease of intracellular cAMP level explains the enhancement of mitogen-induced lymphocyte proliferation after 24 h preincubation at 37 degrees C.     

Temperature dependence of antigen-specific rosette formation by lymphocytes from immunised mice.

Spleen cell suspensions from mice undergoing a secondary response to sheep erythrocytes (SRBC) contained about one tenth as many specific antigen-binding, rosette-forming cells (RFC) when they had been washed at 37 degrees C instead of 4 degrees C before rosetting. This difference was correlated with the presence of IgG anti-SRBC antibody in the serum, and the 37 degrees C washings of immunised spleen cells could passively allergise non-immune spleen cells at 4 degrees C for specific rosette formation which was inhibitable by anti-mouse F(ab)2 serum. The RFC from actively immunised mice were lymphocytes and not macrophages by morphological and cytochemical criteria. It is suggested that the 37 degrees C-labile RFC are lymphocytes to which IgG antibodies bind in the cold. These data indicate that in the use of antigen-binding cell assays to monitor immunological responses, it is necessary to wash lymphocytes at 37 degrees C before testing.     

The combined influence of age of embryo and temperature and duration of incubation on the replication and yield of avian infectious bronchitis (IB) virus in the developing chick embryo

Two experiments were undertaken to investigate the combined influence of the age of embryo at infection and the temperature and duration of incubation on the replication and yield of the Beaudette strain of IB virus lin allantoic fluid and the chorio allantoic membrane. In general a higher titre of virus was found in the allantoic fluid and chorioallantoic membrane of living embryos than of dead ones and with continued incubation of dead embryos the titre of virus fell still further. For embryos of the same age at infection peak titres were similar for those incubated at 32 degrees C and 37 degrees C but were greater than for the corresponding embryos at 42 degrees C. The range of titres for embryos of all ages associated with duration of incubation was narrower for those incubated at 32 degrees C than at 37 degrees C and 42 degrees C and a longer survival period was observed for embryos incubated at 32 degrees C. Peak titres of virus for all age groups were observed after 24 h at 32 degrees C and after 12 h at 37 degrees C and 42 degrees C and then declined. The highest titre for virus in allantoic fluid, in both experiments, was in the 10 d old embryos at 32 degrees C harvested after 24 h. For the chorioallantoic membrane the highest titre in the first experiment was in 12 d old embryos at 32 degrees C, harvested after 24 h, while in the second it was also in 12 d old embryos but at 37 degrees C and harvested after 12 h. The lowest titre of all was in 10 d old embryos incubated at 42 degrees C. It is considered that the maximum total yield of virus from the allantoic fluid and chorio allantoic membrane of the same embryo will be in those infected at 10 - 12 d of age incubated at 32 degrees C to 37 degrees C.     

Clinical data obtained through coagulation testing suggests that hypothermia exerts influence on a patient's blood coagulation reaction

It often takes longer to achieve hemostasis when performing an operation on a patient in cerebral hypothermia therapy than in a normal patient, despite the lack of abnormal clinical blood coagulation findings, and this study was conducted to investigate the cause of delays in coagulation in patients with hypothermia. In this study, 93 samples of plasma were collected at our center from 10 patients(7 men and 3 women; mean age, 33.7) who were in cerebral hypothermia therapy with a urinary bladder temperature maintained at 32-34 degrees C. Each sample was divided into two, and PT(prothrombin time) and APTT(activated partial thromboplastin time) were measured at the normal analysis temperature of 37 degrees C in one sample, and at the hypothermia temperature of 32-34 degrees C in the other sample. The results showed that PT and APTT tended to shorter at 37 degrees C, than those measured at 32-34 degrees C. Thus, we suggest that it is necessary to regulate the temperature of patients with accidental hypothermia or in whom hypothermia therapy is performed.     

Limited clonal heterogeneity of antigen-specific T cells localizing in the pleural space during mycobacterial infection.

To detect possible differences in phenotype and fine specificity for mycobacterial antigens between CD4-positive T cells from peripheral blood (PB) and from inflammatory sites, we identified four patients presenting with a mycobacterial pleural exudate (PE) rich in PPD-specific lymphocytes and with a negative skin test to tuberculin purified protein derivative (PPD) and a negative proliferative response of PB lymphocytes to PPD at the same time. Several weeks after chemotherapy, these patients converted to PPD responsiveness in the periphery, and PPD-specific clones could be generated from PB at this stage. The phenotypic comparison of PE lymphocytes and concomitant PB lymphocytes obtained before treatment showed an increase of CD8 cells and a high frequency of HLA-DR-positive activated T cells in PE. The frequency of tetanus toxoid-specific and Candida albicans-specific proliferating T cells was lower than that of PPD-specific cells in PE but not in PB. PPD-specific clones were derived initially from PE and from PB once the patients had converted to PPD responsiveness. The two sets of clones from each patient were compared for proliferative response to mycobacterial antigen clusters of defined molecular weight ranges. A large number of PE-derived clones (36%) responded to a fraction of 27 to 35 kDa, whereas only one clone from PB responded to the same fraction. The purified antigen P32 (32 kDa), a soluble mycobacterial protein, stimulated PE-derived clones that were responsive to the 37- to 27-kDa fraction but did not stimulate PB-derived clones. The data demonstrate that PE- and PB-derived lymphocytes differ both in phenotype and in fine specificity, suggesting a limited clonal heterogeneity of T cells localizing at the inflammatory site in tuberculous patients without a PPD response in the periphery. Therefore T cells compartmentalized at inflammatory sites provide information that is different from that provided by T cells in the periphery.     

Delineation of IgM-receptor bearing human T and B lymphocytes using a direct plaque forming cell (PFC) assay.

Based on the observation that binding of IgM cytophilic antibodies to lymphocytes is temperature dependent, a direct plaque forming cell (PFC) assay was developed to detect IgM-receptor bearing human peripheral blood T and B lymphocytes. Lymphocytes were passively sensitized with IgM anti-SRBC molecules at 4 degrees C, added to SRBC monolayers then incubated at 37 degrees C with guinea pig complement to develop the plaques. The PFC assay has methodological advantages over rosetting methods which demonstrate IgM receptors, and under certain conditions is more sensitive than these rosette techniques. A mean of 17% of freshly isolated uncultured lymphocytes, enriched for B cells, formed direct plaques while a mean of 3% of T-enriched preparations formed direct plaques. However, if the lymphocytes were preincubated with vibrio cholerae neuraminidase (VCN) these figures increased to 46% and 35% respectively. The specificity of plaque formation by VCN-treated lymphocytes was established. SRBC sensitized with a F(ab')2 preparation of an IgG anti-SRBC reagent failed to bind to VCN-treated lymphocytes, inclusion of IgM, but not other Ig molecules in the test medium, inhibited plaque formation, and, most important, plaque formation by T and B cells was inhibited by F(c)5 mu but not by Fab mu fragments. These results indicate that T and B lymphocytes express IgM-class specific membrane receptors, that these receptors may be hidden on normal lymphocytes but are revealed by treatment with VCN and that the IgM receptor on VCN-treated lymphocytes is F(c)mu specific. These findings are discussed briefly with regard to other and partly contradictory data obtained after overnight in vitro lymphocyte culture. As demonstrated by direct PFC assay, the B cell IgM receptor is trypsin sensitive.     

B/T cell ratio of rabbit peripheral blood lymphocytes. Influence of separation technique on results

Spontaneous in vitro T cell rosette formation at room temperature leading to enrichment of B cells has been reported. We tested 10 individual New Zealand White rabbits sequentially, separating the lymphocytes either at room temperature or at 37 degrees C. T cells are lost constantly at room temperature but to a lesser extent at 37 degrees C. The determination of the yield of lymphocytes after Ficoll separation gives the best control for the accuracy of the results. If lymphocytes are separated at 37 degrees C and if the yield of lymphocytes is greater than 45%, the variation in T cells is small and their number is constant between 62 and 74%. These data show that the reported wide range of T cells in rabbit peripheral blood is due to methodological errors and not inherent in the rabbit.     

Temperature dependent replication of porcine parvovirus isolates

The replication of four porcine parvovirus isolates, NADL-8, NADL-2, KBSH, and Kresse, in swine testes cells were compared at temperatures of 32, 37, and 39 degrees C. Replication of the Kresse isolate was restricted at 32 and 37 degrees C as evidenced by progeny virus, virus polypeptide and viral DNA synthesis, but not at 39 degrees C. In contrast, replication of KBSH was restricted at 39 degrees C, but not at 37 or 32 degrees C. Findings from this study support the contention that replication of KBSH and Kresse isolates are temperature dependent.     

Genetic stability determinants of temperature sensitive, live attenuated respiratory syncytial virus vaccine candidates

An intranasally delivered, live attenuated, temperature sensitive (ts) respiratory syncytial virus vaccine candidate, rA2cp248/404/1,030DeltaSH, exhibits a low level of genetic instability in clinical studies, in contrast to the relatively high stability of two similar candidates, cpts248/404 and rA2cp248/404DeltaSH. The latter strains, containing two ts mutations (248ts and 404ts), are partially growth restricted at 37 degrees C, whereas, rA2cp248/404/1,030DeltaSH contains an additional ts mutation (1,030ts) that increases attenuation and partially restricts virus growth at 35 degrees C. Since the maximum human airway temperature is 35.5 degrees C, we investigated whether growth restriction at 35 degrees C contributes to genetic instability of rA2cp248/404/1,030DeltaSH in vitro. We conducted in vitro passage studies with the three strains at 32 degrees C (a fully permissive growth temperature) and 35 degrees C (restrictive for only rA2cp248/404/1,030DeltaSH). Instability of the ts phenotype was observed only in rA2cp248/404/1,030DeltaSH passaged at 35 degrees C, and corresponded with reversion at the 248ts or 1,030ts mutation sites, as observed in clinical studies. This study indicates that ts mutations that partially restrict replication at physiologic temperatures may contribute to genetic instability of viruses in vivo. In vitro passage studies performed at appropriate temperatures can be used to assess genetic stability and to prioritize ts vaccine candidates for clinical evaluation.     

Interferons from mouse lymphocytes produced at various temperatures (26 degrees C and 37 degrees C)

The study concerns the synthesis and properties of interferons induced at various temperatures (26 degrees C and 37 degrees C) with Newcastle Disease Virus (NDV) in mouse lymphocytes from peritoneal cavity. The results obtained revealed differences in the kinetics of their production. IFN synthesis at 37 degrees C proceeded fast while at 26 degrees C this process was slower and longer lasting. Besides, it was shown that both interferons differ in antigenic properties, molecular weight--IFN (37 degrees C)--26,000, IFN (26 degrees C)--40,000, and resistance to temperature (56 degrees C).     

Global analysis of outer membrane proteins from Leptospira interrogans serovar Lai

Recombinant leptospiral outer membrane proteins (OMPs) can elicit immunity to leptospirosis in a hamster infection model. Previously characterized OMPs appear highly conserved, and thus their potential to stimulate heterologous immunity is of critical importance. In this study we undertook a global analysis of leptospiral OMPs, which were obtained by Triton X-114 extraction and phase partitioning. Outer membrane fractions were isolated from Leptospira interrogans serovar Lai grown at 20, 30, and 37 degrees C with or without 10% fetal calf serum and, finally, in iron-depleted medium. The OMPs were separated by two-dimensional gel electrophoresis. Gel patterns from each of the five conditions were compared via image analysis, and 37 gel-purified proteins were tryptically digested and characterized by mass spectrometry (MS). Matrix-assisted laser desorption ionization-time-of-flight MS was used to rapidly identify leptospiral OMPs present in sequence databases. Proteins identified by this approach included the outer membrane lipoproteins LipL32, LipL36, LipL41, and LipL48. No known proteins from any cellular location other than the outer membrane were identified. Tandem electrospray MS was used to obtain peptide sequence information from eight novel proteins designated pL18, pL21, pL22, pL24, pL45, pL47/49, pL50, and pL55. The expression of LipL36 and pL50 was not apparent at temperatures above 30 degrees C or under iron-depleted conditions. The expression of pL24 was also downregulated after iron depletion. The leptospiral major OMP LipL32 was observed to undergo substantial cleavage under all conditions except iron depletion. Additionally, significant downregulation of these mass forms was observed under iron limitation at 30 degrees C, but not at 30 degrees C alone, suggesting that LipL32 processing is dependent on iron-regulated extracellular proteases. However, separate cleavage products responded differently to changes in growth temperature and medium constituents, indicating that more than one process may be involved in LipL32 processing. Furthermore, under iron-depleted conditions there was no concomitant increase in the levels of the intact form of LipL32. The temperature- and iron-regulated expression of LipL36 and the iron-dependent cleavage of LipL32 were confirmed by immunoblotting with specific antisera. Global analysis of the cellular location and expression of leptospiral proteins will be useful in the annotation of genomic sequence data and in providing insight into the biology of Leptospira.     

Cytotoxic antibodies in multiple sclerosis

Serum cold cytotoxic activities against allocells: total lymphocytes, B lymphocytes and monocytes were detected in 12 of 21 multiple sclerosis (MS) patients at 15 degrees C using a microcytotoxicity technique. Cytotoxic activity was found at 37 degrees C in certain MS patients. This activity was weak or absent in non MS patients and in healthy controls. Tests with autocells were positive in 6 MS patients. Cerebrospinal fluid cytotoxic activity was found in MS as well as in non-MS diseases; at 37 degrees C CSF produced lysis of monocytes in the absence of complement. No correlation was found between cytotoxic activity and parameters of clinical disease. Our results suggest that there may be a wide variety of cytotoxic antibodies in MS. Their significance is unknown; it may be that they have an effect on certain lymphocyte subsets as it has been suggested in other diseases.     

Immune complexes in Takayasu''s arteritis.

We examined sera and Fc receptor-bearing lymphocytes from peripheral blood of patients with Takayasu's arteritis for the purpose of investigating the presence of immune complexes (IC). IC in sera were assayed by solid-phase conglutinin-binding test. Seven of 29 patients exceeded the normal range of circulating IC. IC combined with Fc receptors were estimated by enumerating EA-RFC. EA-RFC of lymphocytes from patients with Takayasu's arteritis were 13.0% and those of normal controls were 29.1%. Low EA-RFC in the patient group increased significantly when lymphocytes were incubated with EA after rising lymphocytes with medium at 37 degrees C. On the contrary, EA-RFC from healthy subjects did not increase after rinsing cells. These findings indicate that IC combined with Fc receptors and hindered EA rosette formation and that rinsing cells with medium at 37 degrees C removed IC from Fc receptors. Comparable results were obtained by a membrane immunofluorescence method using FITC-conjugated anti-human immunoglobulin. In order to confirm that EA rosette formation was really blocked by IC, lymphocytes from a healthy donor were incubated with heat-aggregated human IgG. Incubating cells with IgG aggregates caused reduction of EA-RFC and these lymphocytes restored their ability to form rosettes with EA by rinsing cells with medium at 37 degrees C. In conclusion, we could confirm the presence of IC both in sera and on lymphocyte Fc receptors in some cases of Takayasu's arteritis.     

Replication of parainfluenza type 3 virus in alveolar macrophages: evidence of in vivo infection and of in vitro temperature sensitivity in virus maturation.

Approximately 2% of cultured alveolar macrophages (AM), originally lavaged from the lungs of parainfluenza type 3 virus (PI-3V)-infected calves, were observed to contain viral antigen (by fluorescent antibody method) or viral nucleocapsids (by electron microscopy). Plaque assays, however, indicated that virus titers were generally low when cultures were incubated at 37 degrees C for 10 days. AM, obtained from "in vivo infected" and "noninfected" calves, were found to be equally susceptible to further in vitro PI-3V infection when cultures were incubated at 37 degrees C. AM that were obtained from the lungs of normal calves, cultured at 37 degrees C, and inoculated with PI-3V were observed to produce relatively high virus titers when the incubation temperature was shifted down to 32 degrees C. Results from hemagglutinin assays showed that considerable amounts of hemagglutinin were detected when AM cultures were incubated at 32 degrees C, but only limited amounts were detected at 37 degrees C. Results from electron microscopic examinations at both temperatures substantiated the results of plaque and hemagglutinin assays. The PI-3V, isolated from AM cultures incubated at 32 degrees C, grew well in Madin-Darby bovine kidney cells at 32 degrees C, but little virus was produced at 37 degrees C. In contrast, parent PI-3V grew equally well at both temperatures. The results are discussed in terms of host susceptibility, temperature-sensitivity and virus maturation, and surface viral antigens and persistent viral infection.     

Effect of mild hypothermia on the expression of aquaporin family in cultured rat astrocytes under hypoxic condition

Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens, that increase plasma membrane water permeability in secretory and absorptive cells. In this study, we investigated the effect of mild hypothermia on the expression of AQP4, AQP5 and AQP9 in rat astrocytes cultured under hypoxic conditions. At 37 degrees C, a marked decrease in the expression of AQP4, AQP5 and AQP9 mRNAs was observed. However, at 32 degrees C (mild hypothermia), the expression of AQP5 mRNA was restored to its basal level. Interestingly, under mild hypothermia AQP4 mRNA expression transiently decreased and then increased about two-fold; while AQP9 mRNA expression decreased the same as at 37 degrees C. The changes in the expression of AQP4 and AQP9 proteins were confirmed by Western blot analysis. The restoration of the AQP4 and AQP5 expression at 32 degrees C from the hypoxia-induced decrease at 37 degrees C may play an important role in the reduction of brain edema under hypothermic conditions.