Histone methylation versus histone acetylation: new insights into epigenetic regulation.

Post-translational addition of methyl groups to the amino-terminal tails of histone proteins was discovered more than three decades ago. Only now, however, is the biological significance of lysine and arginine methylation of histone tails being elucidated. Recent findings indicate that methylation of certain core histones is catalyzed by a family of conserved proteins known as the histone methyltransferases (HMTs). New evidence suggests that site-specific methylation, catalyzed by HMTs, is associated with various biological processes ranging from transcriptional regulation to epigenetic silencing via heterochromatin assembly. Taken together, these new findings suggest that histone methylation may provide a stable genomic imprint that may serve to regulate gene expression as well as other epigenetic phenomena.

Does using lower limit of normal values enhance the ability of a single bone mineral density measure to predict fractures?

UNLABELLED: Using a single bone mineral density (BMD) measure, we demonstrated that the lower limit of normal (LLN) method is more consistent in predicting osteoporosis fractures than the T-score in white menopausal women from the Study of Osteoporosis Fracture (SOF). INTRODUCTION: In order to circumvent the inconsistencies and limitations with using the T-score when defining osteoporosis, we propose using 95% LLN values derived from centered polynomial models using the NHANES III BMD measures. The main aim of this study was to compare the two methods in prediction of fracture and agreement in osteoporosis classification using cohort data. METHODS: We compared the fracture prediction ability of the two methods using a single BMD measurement in 4,948 white women aged 67-74 years in the SOF employing kappa statistics, sensitivity, and specificity. RESULTS: The T-score provided inconsistent osteoporosis classification (46.6%) across the five hip regions of interest (ROIs) and this was significantly (p<0.0001) reduced when using the LLN method (36.5%). Kappa statistics of incident fracture during 12 years of follow-up related to the prevalence of osteoporosis at baseline was significantly improved using the LLN method compared to using T-score. Sensitivity and specificity for fracture based on a single BMD measurement of different hip ROIs were more consistent using the LLN method. CONCLUSION: The LLN method provides a more consistent and efficient method for osteoporosis fracture prediction than the T-score in 67- to 74-year-old white women.

Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly.

The assembly of higher order chromatin structures has been linked to the covalent modifications of histone tails. We provide in vivo evidence that lysine 9 of histone H3 (H3 Lys9) is preferentially methylated by the Clr4 protein at heterochromatin-associated regions in fission yeast. Both the conserved chromo- and SET domains of Clr4 are required for H3 Lys9 methylation in vivo. Localization of Swi6, a homolog of Drosophila HP1, to heterochomatic regions is dependent on H3 Lys9 methylation. Moreover, an H3-specific deacetylase Clr3 and a beta-propeller domain protein Rik1 are required for H3 Lys9 methylation by Clr4 and Swi6 localization. These data define a conserved pathway wherein sequential histone modifications establish a "histone code" essential for the epigenetic inheritance of heterochromatin assembly.

Investigator Assessment of the Utility of the TruGraf Molecular Diagnostic Test in Clinical Practice.

BACKGROUND: TruGraf v1 is a well-validated DNA microarray-based test that analyzes blood gene expression profiles as an indicator of immune status in kidney transplant recipients with stable renal function. METHODS: In this study, investigators assessed clinical utility of the TruGraf test in patient management. In a retrospective study, simultaneous blood tests and clinical assessments were performed in 192 patients at 7 transplant centers, and in a prospective observational study they were performed in 45 subjects at 5 transplant centers. RESULTS: When queried regarding whether or not the TruGraf test result impacted their decision regarding patient management, in 168 of 192 (87.5%) cases the investigator responded affirmatively. The prospective study indicated that TruGraf results supported physicians' decisions on patient management 87% (39/45) of the time, and in 93% of cases physicians indicated that they would use serial TruGraf testing in future patient management. A total of 21 of 39 (54%) reported results confirmed their decision that no intervention was needed, and 17 of 39 (44%) reported that results specifically informed them that a decision not to perform a surveillance biopsy was correct. CONCLUSIONS: TruGraf is the first and only noninvasive test to be evaluated for clinical utility in determining rejection status of patients with stable renal function and shows promise of providing support for clinical decisions to avoid unnecessary surveillance biopsies with a high degree of confidence. TruGraf is an invaluable addition to the transplant physician's tool kit for managing patient health by avoiding painful and invasive biopsies, reducing health care costs, and enabling frequent assessment of patients with stable renal function to confirm immune quiescence.

Application of TruGraf v1: A Novel Molecular Biomarker for Managing Kidney Transplant Recipients With Stable Renal Function.

TruGraf v1 is a laboratory-developed DNA microarray-based gene expression blood test to enable proactive noninvasive serial assessment of kidney transplant recipients with stable renal function. It has been previously validated in patients identified as Transplant eXcellence (TX: stable serum creatinine, normal biopsy results, indicative of immune quiescence), and not-TX (renal dysfunction and/or rejection on biopsy results). TruGraf v1 is intended for use in subjects with stable renal function to measure the immune status as an alternative to invasive, expensive, and risky surveillance biopsies. MATERIALS AND METHODS: In this study, simultaneous blood tests and clinical assessments were performed in 192 patients from 7 transplant centers to evaluate TruGraf v1. The molecular testing laboratory was blinded to renal function and biopsy results. RESULTS: Overall, TruGraf v1 accuracy (concordance between TruGraf v1 result and clinical and/or histologic assessment) was 74% (142/192), and a result of TX was accurate in 116 of 125 (93%). The negative predictive value for TruGraf v1 was 90%, with a sensitivity 74% and specificity of 73%. Results did not significantly differ in patients with a biopsy-confirmed diagnosis vs those without a biopsy. CONCLUSIONS: TruGraf v1 can potentially support a clinical decision enabling unnecessary surveillance biopsies with high confidence, making it an invaluable addition to the transplant physician's tool kit for managing patients. TruGraf v1 testing can potentially avoid painful and risky invasive biopsies, reduce health care costs, and enable frequent assessment of patients with stable renal function to confirm the presence of immune quiescence in the peripheral blood.

Transcriptional repression of BRCA1 by aberrant cytosine methylation, histone hypoacetylation and chromatin condensation of the BRCA1 promoter.

BRCA1 expression is repressed by aberrant cytosine methylation in sporadic breast cancer. We hypothesized that aberrant cytosine methylation of the BRCA1 promoter was associated with the transcriptionally repressive effects of histone hypoacetylation and chromatin condensation. To address this question, we developed an in vitro model of study using normal cells and sporadic breast cancer cells with known levels of BRCA1 transcript to produce a 1.4 kb 5-methylcytosine map of the BRCA1 5' CpG island. While all cell types were densely methylated upstream of -728 relative to BRCA1 transcription start, all normal and BRCA1 expressing cells were non-methylated downstream of -728 suggesting that this region contains the functional BRCA1 5' regulatory region. In contrast, the non-BRCA1 expressing UACC3199 cells were completely methylated at all 75 CpGs. Chromatin immunoprecipitations showed that the UACC3199 cells were hypoacetylated at both histones H3 and H4 in the BRCA1 promoter compared to non-methylated BRCA1 expressing cells. The chromatin of the methylated UACC3199 BRCA1 promoter was inaccessible to DNA-protein interactions. These data indicate that the epigenetic effects of aberrant cytosine methylation, histone hypoacetylation and chromatin condensation act together in a discrete region of the BRCA1 5' CpG island to repress BRCA1 transcription in sporadic breast cancer.

Garnett Passe and the tonsillectomy gag.

Kevin Kane has written about the painting by Barbara Hepworth of Garnett Passe performing a tonsillectomy, and wondered about the way in which the gag appears to be suspended. This article traces historically the various methods of holding the gag for tonsillectomy, and postulates that what is illustrated in the Hepworth painting is a jack owned by the late Dr Sydney Cocks, who not only was a friend of Passe but who also commenced the discussions with Passe's widow, Barbara, concerning the formation by her of a trust to support young Australian ENT surgeons, which eventually became The Garnett Passe and Rodney Williams Memorial Foundation.

Aberrant methylation of the BRCA1 CpG island promoter is associated with decreased BRCA1 mRNA in sporadic breast cancer cells.

BRCA1 mRNA is reduced in sporadic breast cancer cells despite the lack of mutations. Because a CpG island is found at the 5' end of the BRCA1 gene, we hypothesized that the decreased BRCA1 mRNA in sporadic breast cancer was associated with aberrant cytosine methylation of the CpG island. We examined BRCA1 mRNA expression in normal human mammary epithelial cells (HMECs), peripheral blood lymphocytes (PBLs) and six sporadic breast cancer cell lines using RT-PCR. The normal breast cells expressed high levels of BRCA1 mRNA. The sporadic breast cancer cell lines and PBLs expressed lower levels of BRCA1 mRNA ranging from a 3-16-fold decrease compared to the normal breast cells. We identified a 600 bp region of the BRCA1 CpG island that possessed strong promoter activity (approximately 40-fold above control), and determined the cytosine methylation patterns of the 30 CpG sites within this region by sodium bisulfite genomic sequencing. The HMECs, PBLs and five of the sporadic breast cancer cell lines were largely unmethylated. However, one sporadic breast cancer cell line, UACC3199, was > or = 60% methylated at all 30 CpG sites (18 sites were 100% methylated) and was associated with an eightfold decrease in BRCA1 mRNA compared to normal breast cells. These findings suggest that aberrant cytosine methylation of the BRCA1 CpG island promoter may be one mechanism of BRCA1 repression in sporadic breast cancer.

Outcomes of vitrectomy for advanced diabetic retinopathy at Groote Schuur Hospital, Cape Town, South Africa.

BACKGROUND: Present limitations in primary and secondary prevention of diabetic retinopathy mean that many patients with diabetes present with advanced retinal complications, often requiring surgery (vitrectomy). OBJECTIVES: To determine the outcomes of vitrectomy for advanced diabetic retinopathy and to examine context-specific risk factors that may influence outcomes and decisions affecting resource allocation. METHODS: This was a retrospective cohort study of 124 vitrectomies with up to 6 months' follow-up. RESULTS: Visual acuity was 6/60 or worse in the better eye in 23.4% of patients at presentation. The mean visual acuity of the listed eye was 2/60. The fellow eye was considered inoperable in 20.2% of cases. Visual function declined significantly in 26.2% of patients while awaiting surgery. The average waiting time until surgery was 2.9 months (range 1 day - 9 months). Epiretinal membranes were present in 93.6% of cases, and posterior iatrogenic breaks occurred in 49.2%. Silicone oil was used in 24.2%. Visual acuity improved in 54.9%, was unchanged in 30.1%, and worsened in 14.0% of cases at 6 months. Patients with poorer vision at surgery were more likely to improve (odds ratio 2.15; p=0.048). Factors associated with a worse visual outcome were increased age at surgery (p=0.042) and posterior iatrogenic retinal breaks (p=0.007). Renal dysfunction was not associated with worse visual outcomes. CONCLUSION: Vitrectomy improved or stabilised vision in 85.0% of cases, although outcomes were unpredictable. A long waiting time to surgery contributed to patient morbidity. The presence of renal dysfunction did not predict poorer visual outcomes.

Gonorrhea incidence and HIV testing and counseling among adolescents and young adults seen at a clinic for sexually transmitted diseases.

OBJECTIVE: To determine whether HIV testing and posttest counseling may be associated with an increase in gonorrhea incidence among adolescents and young adults seen at a clinic for sexually transmitted diseases (STD). DESIGN: A historical cohort study with the collection of longitudinal data on the patients' HIV testing and counseling experience. SETTING: Delgado STD clinic of New Orleans, Louisiana, a public ambulatory primary care center that serves mainly the economically disadvantaged Black population. PATIENTS: A record-based inception cohort of 4031 patients aged 15-25 years diagnosed at the clinic between June 1989 and May 1991 with a first lifetime gonorrhea infection. INTERVENTION: Routine confidential HIV tests and posttest counseling sessions experienced at the clinic during follow-up. OUTCOME MEASURE: Incidence rate of reported gonorrhea reinfection. RESULTS: Of the patients, 51.5% were tested once for HIV antibodies and 25.9% twice or more. Formal posttest counseling occurred after 8.5% of the 4665 HIV-negative and 44.0% of the 49 HIV-positive tests. In the most pessimistic of several models controlling for history of gonorrhea, HIV testing and counseling history, and other potential confounding factors, a significantly lower rate of gonorrhea reinfection was observed after a first HIV-negative test than before [adjusted relative risk (RR), 0.66; 95% confidence interval (CI), 0.59-0.74; P < 0.00011. As compared with the pretest period, significantly higher rates of gonorrhea were observed after respectively a second (RR, 1.18; 95% CI, 1.01-1.37; P = 0.03) and a third (RR, 1.52; 95% CI, 1.22-1.88; P = 0.0001) HIV-negative test. No significant association was found between HIV-positive testing and any variation in gonorrhea rate (RR, 0.95; 95% CI, 0.56-1.62; P = 0.85). Posttest counseling for HIV-negative and HIV-positive results were followed respectively by a significantly higher rate of gonorrhea (RR; 1.27; 95% CI, 1.09-1.48; P = 0.002) and a non-significantly lower rate of gonorrhea (RR, 0.53; 95% CI, 0.17-1.60; P = 0.85). CONCLUSION: Our results do not exclude the possibility of a modest increase in gonorrhea incidence after routine HIV testing and counseling in an STD clinic. Nevertheless, this conclusion holds only under the least favorable assumptions and applies solely to a minority of patients.

Disordered salivary immunoglobulin secretion and sodium transport in human chronic graft-versus-host disease.

Whole saliva samples and lip biopsies were collected from 12 allogeneic bone marrow transplant recipients who developed extensive chronic graft-versus-host disease (GVHD) and from 10 healthy allogeneic and syngeneic recipients without GVHD. Six of ten biopsies from patients with chronic GVHD had lichenoid stomatitis or sialadenitis, or both, with sialodochitis. Seven of nine biopsies from patients free of chronic GVHD were entirely normal, and two had either mild glandular or mucosal changes. Salivary gland involvement in chronic GVHD was associated with decreased or absent levels of salivary IgA and inorganic phosphate, decreased salivary flow rates, and increased concentrations of salivary sodium, albumin, and IgG. The most striking abnormalities were found in patients with histologic evidence of sialadenitis. In contrast, marrow transplant recipients without chronic GVHD had normal salivary immunoglobulin and electrolyte levels. Secretory IgA deficiency may contribute to the frequent sinobronchial infections observed in patients with chronic GVHD.

The impact of primary health care services on under-five mortality in rural Niger.

BACKGROUND: Despite large investments in basic primary health care in sub-Saharan Africa over the past two decades, quantifying the contribution of national programme efforts to the reduction of infant/child mortality in the region has proven difficult. This study takes advantage of the phased implementation of the national Rural Health Improvement Program in Niger and conveniently timed survey data to reassess programme impact on under-five mortality during the 1980-1985 period. METHODS: Health service use and under-five mortality rates for children born in the 5 years prior to the 1985 survey are compared for three groups of villages: villages served by a dispensary, villages served by village health teams (VHT), and villages without access to modern primary care services. Multi-level regression analyses using both household- and community-level variables are undertaken in estimating the magnitude of effects. RESULTS: Children residing in villages proximate to health dispensaries were approximately 32% less likely to have died during the study period than children without access to modern health services. Village health teams were not, however, associated with significantly lower mortality probabilities. Formal test for endogeneity indicated that these effects were not the result of non-uniform/non-random allocation of resources. CONCLUSIONS: The findings are largely supportive of the key premise underlying selective primary health care interventions - that packages of basic services can be effectively mounted nationally in poor countries and have a significant impact over a short time period. In Niger, less than optimal implementation of VHT appears to have reduced the magnitude of the impact achieved.

PIP: The phased implementation of Niger's Rural Health Improvement Program, in conjunction with a 1985 Ministry of Health survey, facilitated quantification of the contribution of primary health care interventions to the reduction of infant and child mortality. During 1978-84, over 8000 health workers were trained and deployed to unserved villages; in addition, 45% of rural villages were provided with primary care services through dispensaries or village health teams. The 35 rural clusters covered in the survey were grouped into three categories: 1) villages located 5 km or less from a dispensary; 2) those located over 5 km from a dispensary, but with a village health team; and 3) villages located more than 5 km from a dispensary and with no health team. Mothers residing near a dispensary were two to five times more likely than their counterparts in the other two groups to have received prenatal care for the most recent birth, had the delivery attended by trained health personnel, received nutrition and health education, and know how to prepare oral rehydration solution. Children in the dispensary-proximate villages were three times more likely to have been at least partially immunized and to have a health card and twice as likely to have had their most recent diarrhea episode treated by a health worker. The unadjusted proportions of infants and children who died in the five years preceding the survey were 0.191 in villages served by a dispensary, 0.203 in villages served by a health team, and 0.267 in villages with neither resource. Multivariate analyses indicated that significantly lower mortality was associated with the presence of a dispensary, maternal literacy, and the existence in the community of farm machinery or access to seeds to plant the next crop. Overall, these findings confirm the significance of primary health care services, especially treatment of infantile diarrhea and tetanus, to reducing under-five mortality in sub-Saharan Africa.

Sequelae of limited amputation.

Ninety patients underwent toe amputations because of vascular disease; 21% required higher amputation and 21% healed without further surgery (i.e., revascularization). Of 60 patients who required bypass surgery, 52 underwent successful first amputations and eight required higher amputations. No difference was seen between diabetic and nondiabetic patients in eventual limb salvage; however, men fared better than women. Without bypass surgery, 11 of 30 patients required a higher level of amputation. No patient's toe amputation site healed with an ankle-to-brachial index of less than 0.35. The judicious use of toe amputation remains an important tool in the surgeon's quest for limb salvage.

Hemodynamic assessment of high-compression hosiery in chronic venous disease.

Graded high-compression support hosiery have long been recognized as a physiologically significant mode of therapy for chronic venous disease because of their effects on the hemodynamics of venous return. Photoplethysmography (PPG) in the noninvasive vascular laboratory is now recognized as a quick, simple, and noninvasive measurement technique, which correlates well with ambulatory venous pressure in the postphlebitic limb with chronic venous insufficiency. The purpose of this study was to evaluate the hemodynamic effects, as measured by PPG, of 40 mm Hg graded compression support hosiery in the treatment of patients with a documented history of hospital-treated thrombophlebitis. Fifty lower extremities among 38 patients with a documented history of deep vein thrombosis and chronic venous insufficiency were matched against 50 control extremities among patients without disease. All 50 lower extremities in the study group had abnormal noninvasive venous studies, including Doppler ultrasound examination, phleborheography, and PPG (mean, 5.9. seconds). Thus these patients were ascertained to have incompetent deep venous systems, but with normal arterial flow as documented by ankle:brachial ratios. After application of 40 mm Hg gradient compression stockings to the study group, PPG measurements in all 50 limbs initially converted to normal (20.6 seconds). Abnormal PPG measurements were converted to normal in postphlebitic limbs with the application of graded compression stockings in the 29 patients who wore the prescribed hosiery; 21 patients did not wear the gradient stockings after the initial evaluation(s) and were not found to have improved PPG measurements. It can be concluded that such gradient stockings should be associated with a reduction in ambulatory venous pressure, which may, in turn, lead to clinical prevention or improvement of the various sequelae associated with chronic venous hypertension.

Cardiovascular interactions between methionine-enkephalin and substance P in the conscious dog.

Interactions between the undecapeptide Substance P (SP) and the pentapeptide methionine-enkephalin (Met5-ENK) have been described in isolated organ systems and in baroreceptor reflex mechanisms. Previous studies in our laboratory have demonstrated that systemically injected Met5-ENK simultaneously increases mean systemic arterial pressure (MAP) and heart rate (HR) in the conscious, chronically instrumented dog. We have now evaluated cardiovascular interactions between SP and Met5-ENK. In this model, SP injected intravenously produces a rapid and transient decrease in MAP and increase in HR over the dose range from 1.0 to 10.0 ng/kg. SP does not appear to appreciably alter subsequent responses to Met5-ENK. At SP doses of 1.0 ng/kg, the peak hemodynamic response to SP and Met5-ENK (35 micrograms/kg) given together appears to represent a simple summation effect of both drugs on HR and MAP. However, at higher SP doses (5.0 ng/kg), the SP response predominates and is little altered by the presence of Met5-ENK. Thus, Met5-ENK does appear capable of modulating the hemodynamic responses to SP over certain dose ranges.

Methionine-enkephalin facilitates the cardiovascular response to epinephrine in the conscious dog.

Methionine-enkephalin (ME) is present in high concentrations in the adrenal medulla; it is co-stored with catecholamines in chromaffin vesicles, and released together with catecholamines during adrenal stimulation. We have examined the interactions of intravenously administered bolus doses of ME and epinephrine (EPI) in the conscious dog. EPI, 1.0 microgram/kg, increased mean arterial pressure (MAP) from 104 +/- 6 to 130 +/- 11 mm Hg, while reflexly reducing heart rate(HR) from 103 +/- 13 to 83 +/- 13 beats/min (bpm). ME, 5.0 micrograms/kg, increased MAP from 106 +/- 7 to 122 +/- 7 mm Hg and increased HR from 111 +/- 12 to 139 +/- 14 bpm. EPI and ME administered together increased MAP in apparently additive fashion from 106 +/- 6 to 153 +/- 12 mm Hg, and also increased HR from 102 +/- 10 to 114 +/- 17 bpm. ME, 1.0 microgram/kg, exerted a similar effect. Thus, in these concentrations, ME exerts a co-operative influence upon the EPI cardiovascular response in the conscious, neurologically intact dog, probably by inhibiting baroreceptor reflexes. These findings suggest a possible role for ENK as an excitatory stress hormone.

Systemic methionine-enkephalin evokes cardiostimulatory responses in the human.

The cardiovascular effects of bolus doses of methionine-enkephalin (Met5-ENK) (1 to 100 micrograms/kg) were studied in 9 subjects in whom, at cardiac catheterization for evaluation of chest pain, patent coronary arteries were found. Met5-ENK produced a simultaneous increase in blood pressure and heart rate beginning within 20 sec, reaching maximal values between 30 and 40 sec, and then terminating by 60 sec. Heart rate, systolic, diastolic, and mean arterial blood pressures increased significantly (p less than 0.0005); pulse pressure remained unchanged. Positive dose-effect relationships were observed for heart rate (p less than 0.002), systolic, diastolic, and mean arterial blood pressures (p less than 0.05). Naloxone (0.5 mg/kg), given to 4 subjects, prevented the heart rate and blood pressure changes associated with Met5-ENK administration, demonstrating that the cardiovascular changes were mediated by opiate receptors. Subjects also described cutaneous paresthesias which were not prevented by naloxone pretreatment. These data suggest a role for peripheral enkephalins in cardiovascular regulation.

Comparison of prolonged in vivo inhibitory activity of several potent bombesin (BN) antagonists on BN-stimulated amylase secretion in the rat.

New BN analogues designed to be competitive receptor antagonists at the BN/gastrin releasing peptide receptor(s) can exhibit diverse properties ranging from full antagonist, partial agonist or weak agonist activity, depending on the assay system and animal species employed. Here we evaluate the following 3 antagonists which have the most potent receptor affinities in several in vitro assay systems and are representative of 3 main classes of BN antagonists for their in vivo effects on pancreatic amylase secretion in the rat: [D-Cpa6,Phe14,psi 13-14]BN(6-14), [D-Phe6]BN(6-13) propylamide, and [D-Phe6]BN(6-13) methyl ester. After injection in the rat, the methyl ester was clearly the most potent antagonist and completely inhibited BN-stimulated amylase release at the 20 nmol/kg (IV bolus) for about 2 h. In contrast, the propylamide analogue at the 200 nmol/kg (IV bolus) dose produced incomplete inhibition of amylase release. Inhibition was transient and lasted for only about 1 h, possibly reflecting the significant agonist activity of this latter peptide in the rat pancreatic amylase secretion test in vitro. The psi-analogue, while being the longest acting analogue, was also incapable of lowering amylase to basal level at 50 times the BN dose, suggesting that it is a mixed agonist-antagonist in vivo as was also previously shown in vitro in the rat.

Identification by microinjection of TRH-sensitive sites in the cat's brain stem that mediate respiratory, temperature and other autonomic changes.

Cats were prepared with an array of stereotaxically implanted guide tubes, the tips of which rested just above selected structures in the brain stem. Thyrotropin-releasing hormone (TRH) was microinjected in a volume of 0.5 micronl at 347 individual sites scattered throughout the hypothalamus and mesencephalon Polypnea, hypothermia, vocalization, salivation, defecation and vasodilation were evoked by 10-20 ng of TRH injected only at loci in the mesencephalon, principally in the reticular substance. TRH failed to lower body temperature when it was infused at the same sites in the anterior hypothalamus at which norepinephrine produced its characteristic hypothermia. These results suggest that the TRH-induced hypothermia is a secondary effect of tachypnea which results from the action of the tripeptide on the mesencephalic respiratory-autonomic mechanism.