[Effect of metanephric mesenchymal stem cells after renal ischemia reperfusion injury in mice].

OBJECTIVE: To study the effect of metanephric mesenchymal stem cells (MMSCs ) after renal ischemia reperfusion injury (IRI). METHODS: C57BL/6J male mice were divided into control group (n=5) and experimental group (n=30). The control group was given a sham operation, while in the experimental group, a model of renal IRI was established. The experimental group was further divided into two groups according to the material injected through the femoral vein: IRI group (injected with normal saline, 10 ml/kg) and cell therapy group (injected with normal saline containing 5×10(5) MMSCs, 10 ml/kg). Samples of blood and kidney tissues were collected from five mice from each group at 12 h, 24 h and 72 h after IRI. The serum creatinine (SCr) level was detected, and the results of kidney tissue pathological staining and Paller score of renal tubules were analyzed to assess the effect of MMSCs after renal IRI. In addition, the expression of microRNA-26a(miR-26a)in kidney tissues was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and compared among the groups. RESULTS: (1) In both IRI group and cell therapy group, the levels of SCr at 12 h, 24 h and 72 h after operation were all significantly higher than those of the control group, besides, the level of SCr at 24 h was significantly higher than that at 12 h and 72 h (all P<0.05). The levels of SCr at 12 h, 24 h and 72 h were of no significant differences between IRI group and cell therapy group(all P>0.05). (2)Paller scores of renal tubules at 12 h, 24 h and 72 h in both IRI group and cell therapy group were significantly higher than those in the control group, and the scores at 24 h were significantly higher than that at 72 h, while the latter were in turn higher than the scores at 12 h (all P<0.05). In the cell therapy group, Paller score of renal tubules at 24 h(57.2±6.3)was significantly lower than that in IRI group(70.8±14.8) (P<0.05). Histological examination showed renal tubular epithelial cell atrophy, swelling and protein cast in kidney tissues from IRI group at 24 h, compared with the control group and the cell therapy group at the same time.(3) In IRI group and cell therapy group, the levels of miR-26a in the kidney tissues at 12 h and 24 h were significantly lower than those of the control group (P<0.05). In the cell therapy group, the level of miR-26a in the kidney tissues at 24 h (0.416±0.139) and 72 h (1.152±0.239)were significantly higher than that in the IRI group(0.244±0.067, 0.855±0.038, both P<0.05). A negative correlation between the levels of miR-26a and SCr level were found (r=-0.5, P<0.05). CONCLUSION: MMSCs have a certain repairing effect on renal IRI, accompanied by an increase in miR-26a expression.

Association of a miR-34b binding site single nucleotide polymorphism in the 3'-untranslated region of the methylenetetrahydrofolate reductase gene with susceptibility to male infertility.

This study aims to explore the possible associations between a genetic variation in the miR-34b binding site in the 3'-untranslated region (UTR) of the methylenetetrahydrofolate reductase (MTHFR) gene (rs55763075) with male infertility in a Chinese population. Genotype distributions of the rs55763075 single nucleotide polymorphism were investigated by polymerase chain reaction and direct sequencing in a Chinese cohort that included 464 infertile men with idiopathic azoospermia or oligospermia and 458 controls with normal fertility. Overall, no significant differences in the distributions of the genotypes of the MTHFR rs55763075 polymorphism were detected between the infertility and control groups. A statistically significant increased risk of male infertility was found for carriers of the rs55763075 AA genotype when compared with homozygous carriers of the rs55763075 GG genotype in the azoospermia subgroup (OR = 1.721; 95% CI = 1.055-2.807; P = 0.031). Furthermore, we found that rs55763075 was associated with folate and homocysteine levels in patients with idiopathic azoospermia. Our results indicated that the MTHFR 3'-UTR rs55763075 polymorphism might modify the susceptibility to male infertility with idiopathic azoospermia.

Evaluation of the CEL-100 videolaryngoscope(TM) for double-lumen tracheal tube insertion after failure using the Macintosh laryngoscope.

We prospectively evaluated the CEL-100 videolaryngoscope(TM) for insertion of double-lumen tracheal tubes in 48 consecutive patients who had been found to have an unanticipated Cormack and Lehane grade 3 (n=43) and grade 4 (n=5) laryngeal view and in whom two attempts at tracheal intubation using the Macintosh laryngoscope had failed. When the CEL-100 was subsequently employed, the glottic view improved in 45 (94%) patients. The view improved by one grade in 15 (31%) patients and by two grades in 30 (63%) patients, compared with the Macintosh blade (p<0.001). Double-lumen tracheal tube insertion was successful when using the CEL-100 in 43 out of 48 patients (90%; 95% CI 81-98%). This occurred on the first attempt in 27 (56%) patients, 14 (29%) on the second and two (4%) on the third. We conclude that the CEL-100 videolaryngoscope is an effective device in this context, and we therefore propose that this device can be used in circumstances when double-lumen tube insertion proves difficult.

[Willingness and influencing factors related to "centralized slaughtering, fresh poultry listing and marketing" strategy among the household chefs in Guangzhou].

Objective: To study the willingness and influence factors related to "centralized slaughtering, fresh poultry listing and marketing" strategy, among the household chefs, and provide reference for government to adjust and optimize the strategy on avian influenza prevention. Methods: According to the geographical characteristics and regional functions, 6 'monitoring stations' were selected from 12 residential districts of Guangzhou, respectively. Another 21 meat markets which selling live poultry, were selected in each station and 5 household chefs of each market were invited to attend a face to face interview. Basic information, personal cognitive, willingness and influencing factors to the policy were under study. Univariate and multivariate logistic regression methods were used. Results: A total of 664 household chefs underwent the survey and results showed that the rate of support to the "centralized slaughtering, fresh poultry listing and marketing" strategy was 44.6% (296/664). Results from the multi-factor logistic regression showed that those household chefs who were males (OR=1.618, 95% CI: 1.156-2.264, P=0.005), having received higher education (OR=1.814, 95% CI: 1.296-2.539, P=0.001), or believing that the existence of live poultry stalls was related to the transmission of avian influenza (OR=1.918, 95% CI: 1.341-2.743, P<0.001) were factors at higher risk. These household chefs also intended to avoid the use of live poultry stalls (OR=1.666, 95%CI: 1.203-2.309, P=0.002) and accept the "centralized slaughtering, fresh poultry listing and marketing" strategy. Conclusion: Detailed study on this subject and, setting up pilot project in some areas as well as prioritizing the education programs for household chefs seemed helpful to the implementation of the 'freezing-fresh poultry' policy.

[The effect of restraint stress on masseter mechanical hyperalgesia and the activity of neurons and astrocytes in the spinal trigeminal nucleus caudalis in rats].

Objective: To investigate the effect of restraint stress on masseter mechanical hyperalgesia and the activity of neurons and astrocytes in the spinal trigeminal nucleus caudalis (Vc). Methods: The animals were randomly divided into the control group, 1-, 3-, 5-, 7-, 9-, 11-and 14-day stress groups, with 10 rats in each group. The body weight increase and behavior tests were used to testify the animal model. The mechanical sensitivity of masseter of the rat before and after the stress was measured with Von Frey filaments. Histological examinations were used to evaluate the expression of neuronal c-fos and astrocytic glial fibrillary acidic protein (GFAP). Results: Restraint stress resulted in remarkable mechanical allodynia in the masseter muscle. The head withdrawal threshold was significantly lower in the 7-, 9-, 11-and 14-day stress groups ([0.071±0.011], [0.059±0.020], [0.052±0.011], [0.033±0.011] N) than that in the control group ([0.120±0.025] N) (P<0.05). Compared to the control group, the rats in the 1-day stress group showed a significant increase of c-fos in neurons of the Vc and then declined to normal level after 1 week gradually. The GFAP expression in astrocytes of the Vc was significantly increased in the 7-, 9-, 11-and 14-day stress groups (4.3±1.0, 4.5±0.6, 4.6±0.5, 4.8±1.3) compared with the control group (2.0±0.8) (P<0.05). Conclusions: Chronic restraint stress could lower the threshold of mechanical allodynia in the masseter muscle and activate the neurons and astrocytes in Vc. The activation of neurons and astrocytes plays an important role in the masseter hyperalgesia induced by restraint stress in rats.

The Unhealthy Lifestyle Factors Associated with an Increased Risk of Poor Nutrition among the Elderly Population in China.

OBJECTIVES: The associations between nutritional status and lifestyle factors have not been well established. This study aimed to investigate the prevalence of poor nutrition and to examine the relationships between nutritional status and unhealthy lifestyle and other related factors among the elderly. METHODS: This cross-sectional study was conducted in Liaobu Town, Dongguan city, China. A total of 708 community-dwelling older adults aged ≥60 years were recruited by stratified random sampling. Data on sociodemographic characteristics, health and lifestyle factors, and the Mini Nutritional Assessment (MNA) scores were collected using structured questionnaires via face-to-face interviews. A multivariate logistic regression model was constructed to identify the risk factors of poor nutrition. RESULTS: The prevalence of malnutrition among the elderly adults in this study was 1.3%, and 24.4% were at risk of malnutrition (RM). Poor nutrition was significantly associated with female gender, older age, lower education, a high number of self-reported chronic diseases, and hospitalization in the last year. Unhealthy lifestyle factors associated with poor nutrition included current smoking status, higher alcohol consumption, lack of physical activity, longer duration of sitting, negative attitude towards life, and a poor family relationship. CONCLUSIONS: While the prevalence of malnutrition was low, RM was high in the elderly population in China. The determinants of malnutrition were explored and the relationships between nutritional status and unhealthy lifestyle factors were examined. The results of this study provide information for future longitudinal studies with multi-factorial interventional design in order to determine the effects of the causal relationships.

[Clinical characteristics and prognosis in 12 patients with adult T cell leukemia/lymphoma confirmed by HTLV-1 provirus gene detection].

Objective: To analyze the clinical characteristics and prognosis of adult T cell leukemia/lymphoma (ATLL). Methods: Peripheral blood samples from patients who were suspected as ATLL from March, 2013 to July, 2015, were collected for HTLV-1 provirus genes detection in genomic DNA extraction by PCR. Cases showing positive results were confirmed as ATLL. Clinical and laboratory characteristics, therapeutic outcomes and survival evaluation were collected. Results: 12 out of 23 suspected patients were confirmedly diagnosed as ATLL through HTLV-1 provirus genes detection by PCR. Eight patients were male and four patients were female. Median age was 51 (range 28-66) years old. All of those patients came from coastal cities of Fujian province where a HTLV-1 epidemic area locates. In the subtype classification of these 12 ATLL, 11 patients were classified as acute type and one case as lymphoma type ATLL. As one of the clinical characteristics of ATLL, ' flower cells ', with typical or atypical morphology had been observed in a high rate (81.8%). Clinical symptom such as hepatomegaly, splenomegaly and lymphadenectasis were detected in most of patients, and hypercalcemia and elevated LDH were also noted commonly. The ATLL cells immunophenotype were typical, and the major subtype was CD4+ CD8- type. Confection of hepatitis B virus was detected in a high rate (54.5%). Ten patients received chemotherapy, and 2 cases in complete remission after chemotherapy received allogeneic hematopoietic stem cell transplantation. At the end of the follow-up, 7 cases died, 4 cases survived, 1 case was lost, and the median survival was 2.8 (0.9-10.8) months. We found a case had HTLV-1 provirus negative after transplantation. Conclusion: In the coastal area of Fujian Province, ATLL is not rare. Characteristics of those ATLL are typical. But prognosis is still unsatisfactory.

Further studies on the immunization of chickens with temperature-sensitive Mycoplasma gallisepticum mutant.

Newly hatched chickens were immunized with a temperature-sensitive (TS) Mycoplasma gallisepticum (MG) mutant (TS 100). Immunized chickens resisted challenge with the virulent S6 strain. The dose of TS MG needed for protection was less than 3.3 X 10(4) colony-forming units. After immunization with TS 100, chickens were subjected to a variety of virus infection and immunosuppressive treatments. Neonatal bursectomy or thymectomy, infectious bursal disease virus infection, and infectious bronchitis virus vaccination or a combination of infectious bronchitis virus and Newcastle disease virus vaccination did not contribute to the development of air-sac lesions in TS MG-immunized chickens. Infectious bronchitis virus vaccination enabled MG to migrate from the nasal cavity to the trachea but not to the air sacs. The TS MG vaccine appears to be a safe immunogen.

Immunization of chickens with temperature-sensitive mutants of Mycoplasma gallisepticum.

Temperature-sensitive (TS) mutants of the S6 strain of Mycoplasma gallisepticum (MG) were used to immunize newly hatched chickens. Immunized chickens developed antibodies to the wild-type (WT) S6 strain as demonstrated by serologic tests. MG was recovered from nasal cavities but not from the lower respiratory tract of the immunized chicks. Three weeks after intranasal immunization, chickens were challenged via the air sacs with the virulent S6 strain. Immunized chickens were significantly better protected from development of air-sac lesions than were controls.

Active and passive immunization of ducks against duck viral enteritis.

The newly isolated Sheridan-83 strain of duck enteritis virus (DEV) was apathogenic to ducks. Inoculation of ducks with this virus resulted in the production of antibodies that enabled the ducks to resist challenge with the virulent LA strain of DEV. The minimum protective dose of the Sheridan-83 virus was determined to be less than 10 TCID50. Solid immunity lasted 1-2 months. Passive transfer of antibodies to susceptible ducks also significantly protected them from lethal challenge.

Serological survey of infectious bursal disease virus serotypes 1 and 2 in California turkeys.

The prevalence of two infectious bursal disease (IBD) viruses--serotype 1, of chicken origin, and serotype 2, of turkey origin--was studied in California turkeys. Serum samples were collected from 15 turkey flocks representing nine counties. The virus-neutralization test was used. Overall, only 15% of 342 samples were positive for serotype 1, whereas 89% were positive for serotype 2. However, 12 out of 15 flocks had a least one sample positive for serotype 1, and 14 out of 15 flocks had at least one sample positive for serotype 2. Flocks with antibodies to serotype 1 had low geometric mean titers (GMTs) (1.5 to 8.8) and low-to-medium prevalence rates (5 to 53.3%). Flocks with antibodies to serotype 2 had high GMTs (36.8 to 2048) and high prevalence rates (60 to 100%). Results of this study lead us to question the efficacy of IBD vaccination in turkeys.

Antibody-mediated resistance against duck enteritis virus infection.

Vaccination of ducks with an apathogenic strain of duck enteritis virus resulted in protection against challenge with the virulent Lake Andes strain of duck enteritis virus by intramuscular inoculation or contact exposure. Antisera produced in the vaccinated ducks were able to transfer resistance against the challenge strain to recipient ducks. Antisera against duck enteritis virus were cytotoxic for duck enteritis virus-infected duck embryo fibroblasts in the presence of guinea pig complement. These in vivo and in vitro data suggest that the humoral immune mechanism plays a role in protecting ducks from duck enteritis virus infection.

[Synthesis of N-(4-carbomethoxy-4-phthalimidobutanoyl)-N-substituted glycines, proline and pyroglutamic acid].

This paper reports the synthesis of eleven N-(4-carbomethoxy-4-phthalimidobutanoyl)-N-substituted glycines (VII1-9), proline (VII10) and pyroglutamic acid (VII11) expected to have inhibitory activity on angiotensin converting enzyme. All of the compounds mentioned above and the corresponding t-butyl esters of VII1-9 were not reported in the literature previously. The structures were confirmed through their IR, 1HNMR, MS spectra and elemental analysis. In preliminary test in rats, compounds VII8, VII9 and VII10 showed marked hypotensive activity.

Modulation of liver intracellular C3 in mice by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Earlier studies from this laboratory have shown that the complement system, especially the component C3, in female B6C3F1 mice is suppressed following TCDD exposure in vivo. However, the direct exposure of TCDD in vitro does not affect the C3-producing capacity of two types of hepatoma cells, as well as mouse primary hepatocytes. To investigate the effect of TCDD on C3 production by the liver following in vivo exposure, liver intracellular C3 levels and pro-C3, the precursor of the secreted C3, were examined in the present study. The results demonstrated that there was a dose-dependent increase of liver intracellular C3 levels (from 138% to 175% of control) immediately following TCDD (from 10 to 40 micrograms/kg) exposure. This increase was rapid (4 h after exposure), but transient (less than 2 h), and was not accompanied by an alteration of serum C3 levels. Studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the increase in liver intracellular C3 levels resulted from, at least partially, an increase in intracellular pro-C3. Serum C3 levels did not decrease until d 3 after exposure, when both liver intracellular C3 levels and pro-C3 in TCDD-treated mice were not different from those of the control mice. These results indicated that the modulation of liver intracellular C3 by TCDD did not correlate with the suppression of serum C3 levels following exposure.

Mouse Hepa 1c1c7 hepatoma cells produce complement component C3; 2,3,7,8-tetrachlorodibenzo-p-dioxin fails to modulate this capacity.

Previous studies from this laboratory have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreased complement component C3 levels in female B6C3F1 mouse serum following in vivo acute or subchronic exposure (White et al., 1986). Since TCDD is a hepatotoxic compound and more than 90% of serum C3 is produced by the liver, studies were undertaken using mouse Hepa 1c1c7 (Hepa 1) hepatoma cell line to determine if TCDD acts directly on hepatocytes to inhibit C3 production. The C3-producing capacity of Hepa 1 cells was first examined. When confluent Hepa 1 cell monolayers were cultured in 24-well plates with serum-free medium, a detectable amount of C3 (14.1 +/- 0.8 ng/ml) was secreted as early as 1 h after culture and reached a plateau at 12 h (68.3 +/- 4.9 ng/ml). Furthermore, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that the molecular weight of C3 in culture supernatant corresponded to that present in mouse serum. Human recombinant IL-1 beta (hrIL-1 beta), a known inducer of complement C3, at doses as low as 1 unit/ml increased the C3 production to 158% of control after 24 h of incubation. The effect of hrIL-1 beta was dose dependent, and the maximum tested dose of 10 units/ml increased C3 production to 256% of control. When cells were directly exposed to TCDD at concentrations from 10(-10) to 10(-6) M, there was no inhibitory effect on production of C3. TCDD also failed to block the stimulatory effect of 10 units/ml hrIL-1 beta added to the culture 1 h later. To verify that cultured Hepa 1 cells were able to respond to TCDD, 7-ethoxyresorufin O-deethylase (EROD) activity was measured under the same conditions. TCDD dose-dependently increased EROD activity of Hepa 1 cells at 24 h following exposure. The activity reached 56.7 +/- 3.0 pmol/min/mg protein with 10(-9) M TCDD, compared with 10.7 +/- 1.7 pmol/min/mg protein of vehicle-exposed cells. Our results indicate that the direct interaction of TCDD with Hepa 1 cells does not affect their C3-producing capacity, although EROD activity, a characteristic response mediated by the cellular TCDD/Ah receptor, was induced. The lack of effect of TCDD in vitro suggests that the decrease of serum C3 levels observed in vivo may result from an indirect effect of TCDD on hepatocytes.

Production of complement component C3 in vivo following 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to decrease serum complement C3 levels in female B6C3F1 mice but failed to alter C3 production when added in vitro to either hepatoma cells (both human and mouse hepatoma cells) or mouse primary hepatocytes (Lin and White, 1993a). It has also been demonstrated that mouse liver intracellular C3 levels were not affected following TCDD exposure in vivo, while serum C3 levels were suppressed (Lin and White, 1993b). Therefore, further studies were undertaken to investigate the mechanism by which TCDD modulates newly synthesized serum C3 in vivo. Mouse serum C3 was depleted by an intravenous injection of 50 anti-complement units (ACU)/kg cobra venom factor (CVF). This dose of CVF depleted serum C3 levels to 9% of control at 24 h after treatment. Subsequently, serum C3 levels returned to 19% and 75% of the control level on d 3 and d 5. The recovery of serum C3 was then monitored following an acute oral exposure to 20 micrograms/kg TCDD. In mice exposed to both TCDD and CVF, serum C3 levels reached 15% and 69% of control on d 3 and d 5 after treatment; these results were not significantly different from those of mice treated with CVF alone. Furthermore, when the radiolabeled amino acid [3H]leucine was injected intravenously into either vehicle- or TCDD-treated mice, the incorporation of this labeled precursor into both C3 and other secreted plasma proteins was not inhibited by TCDD. These results demonstrated that TCDD did not decrease newly synthesized C3 in vivo. These studies provide additional support for the concept that TCDD does not act directly on hepatocytes to suppress C3 production. The lower serum C3 levels observed in vivo following TCDD exposure is not the result of a decrease in C3 production by hepatocytes.

Effect of alcohol on expression of heat shock protein 73 in monolayers of hepatocytes from alcohol-exposed rats.

Alcohol dramatically reduced loss of heat shock proteins (HSP73) and prevented morphological damage in monolayers of hepatocytes prepared from alcohol-fed rats. The monolayers were treated with 0, 5, 25, or 100 mM alcohol and triplicate samples were assayed at 24, 48, 72, and 96 hr after exposure. The content of HSP73 was measured by PAGE electrophoresis and Western blotting with a mouse monoclonal anti-HSP70 IgG antibody. HSP72 is not expressed under these conditions. Damage to the hepatocytes, quantified by leakage of lactate dehydrogenase (LDH), was also decreased by 100 mM alcohol. Although the initial 100 mM alcohol concentration decreased logarithmically to 1.7 mM over the first 24 hr, the effect of alcohol on HSP73 loss, LDH leakage, and morphological damage was most pronounced at 96 hr.

Interactive effects of pertussis toxin and the phorbol ester tumour promotor, phorbol dibutyrate, on T-lymphocyte mitogenesis and the expression of phenotypic determinants.

The B oligomer of pertussis toxin serves as a weak mitogen in the T lymphocyte, an effect which is associated with an early rise in cytosolic free calcium concentrations, as monitored by Fura-2 fluorescence. Upon co-administration of phorbol dibutyrate, a phorbol ester tumour promotor which activates protein kinase C, pertussis toxin-induced proliferation was synergistically enhanced, as measured by the increased uptake of [3H]thymidine, into cellular DNA. Although phorbol ester co-administration has often been associated with an inhibition of Ca2+-mobilizing pathways, phorbol dibutyrate pretreatment had no inhibitory effect on the pertussis toxin-induced calcium flux and may actually have enhanced this response slightly. Flow cytometric analysis of cell populations expanded by the combined regimen did not provide evidence for the preferential expansion of cells bearing either CD4 or CD8, the T-cell determinants representative of the helper-inducer and cytotoxic-suppressor subsets, respectively. Pertussis toxin and phorbol dibutyrate appear, therefore, to elicit polyclonal stimulation, rather than the selective activation of a given lymphocyte subset. Expression of the transferrin receptor, a marker for nutrient uptake, and CD25, the Tac component of the interleukin-2 (IL-2) receptor, was, however, synergistically enhanced in cells activated by the co-treatment procedure.

Suppression of the murine gut mucosal IgA response to cholera toxin with oral cyclosporine.

The gut mucosal immune system may be a primary target for many ingested chemicals. Methods have been developed to examine the effects of chemicals on the systemic humoral immune response; however, studies to evaluate various methods of assessing the local gut mucosal immune response in a toxicology assay have been limited. The objectives of this study were to examine the effects of the known immunosuppressive compound, cyclosporine (CYS), on the generation of a cholera toxin (CT)-specific gut mucosal IgA response and evaluate the methods used to measure the gut IgA response. Groups of female B6C3F1 mice were left untreated or were treated daily, p.o., with corn oil (vehicle) or CYS at doses of 10 and 50 mg/kg for 20 days. On Days 3 and 13, mice were sensitized p.o. with CT. On Day 21, mice were terminated, gut washings were collected, and lamina propria lymphocytes were extracted from gut tissue with collagenase treatment. Cholera toxin-specific IgA in the gut washings was measured by an ELISA. The numbers of CT-specific IgA (CT-IgA) and total IgA antibody-forming cells (spot-forming cells, SFC) obtained from the lamina propria were determined by the ELISPOT method. A dose of 50 mg/kg CYS produced a significant decrease in the amount of CT-IgA in gut washings. This dose also decreased the number of cells recovered from the lamina propria by at least 50%. The amount of CT-specific SFC/million lamina propria cells decreased with a dose of 10 mg/kg CYS, whereas 50 mg/kg CYS did not alter the response.(ABSTRACT TRUNCATED AT 250 WORDS)