Midline correction by extraction of the remaining mandibular canine: myth or reality.

Standard of practice does not always equal standard of care. When faced with a problematic situation, the dentist will either treat from experience, or else depend on what has been suggested by others. In some cases, anecdotal information may not be applicable. Recommended clinical procedures have not always been supported by adequate research. Although midline correction has been expressed as a certainty, it does not always occur. Patience on the part of the practitioner will allow the child to be observed periodically, and at the same time allow the dentition to develop naturally. Orthodontic treatment can always be used, once the permanent canines erupt.

Interleukin 1 beta mediates stress-induced immunosuppression via corticotropin-releasing factor.

Intracerebroventricular (icv) infusion of human interleukin 1 beta (IL-1) into intact and adrenalectomized rats impairs immune function. Using antibody to IL-1 as well as an inhibitor of IL-1 action, we sought to determine if endogenous IL-1 in the central nervous system has a physiological role in mediating the immunosuppressive effects of stress. Compared with freely moving controls, rats given intermittent electric shock to the tail for 40 min exhibited a fall in T lymphocyte proliferation and natural killer (NK) cell cytotoxicity of 33% and 38%, respectively; however, when pretreated with icv human IL-1 monoclonal antibody, which significantly crossreacts with rat IL-1, the decrement was attenuated to 14.6% and 15%, respectively. When rats were pretreated with icv alpha-MSH, which blocks many IL-1 effects, shock-induced suppression of 42% in both T lymphocyte proliferation and NK cytotoxicity were blunted to 33% and 31%, respectively. Similar results were found in adrenalectomized rats. These findings suggest that endogenous IL-1 is a physiologically relevant mediator of the immune response to stress. As IL-1 has been reported to release CRF, which we have shown always plays a significant role in stress-induced immunomodulation, we then assessed the relationship of IL-1 and CRF in immunosuppression. Infusion of icv IL-1 caused a decrease of 35% in T lymphocyte proliferation and 34% in NK activity, but pretreatment with CRF antibody icv attenuated IL-1 suppression of T lymphocyte proliferation and NK activity to 10% and 8%, respectively. Comparable results were observed in adrenalectomized rats. These findings suggest that CRF antibody is able to block the immunosuppressive effects of IL-1. To further examine the interaction of CRF in mediating stress-induced immunosuppression, we found that animals pretreated with icv CRF antibody, shocked and then given icv IL-1, had a decrement in T lymphocyte proliferation and NK cytotoxicity of 24% and 21%, respectively, demonstrating that the immunosuppressive effect of icv IL-1 is blocked when central CRF has been neutralized by prior administration of icv CRF antibody. In contrast, animals pretreated with icv IL-1 antibody, shocked and then given icv CRF, had decrements of 38% and 40%, respectively, showing that icv CRF does act even when central IL-1 has been neutralized by prior administration of icv IL-1 antibody. Thus, we conclude there is a sequential relationship between two of the known mediators of stress-induced immunosuppression, with release of central IL-1 followed by that of CRF.

Receptor-mediated immunomodulation by corticotropin-releasing factor.

In both normal and adrenalectomized rats, exogenous corticotropin-releasing factor (CRF) suppresses immune function, and stress-induced immunosuppression can be partially reversed with either CRF antibody or CRF antagonist, suggesting a role for CRF in immunomodulation. We now report binding of CRF to human monocyte-macrophages and T-helper lymphocytes but not to T-suppressor or B lymphocytes. Bound CRF was displaced by synthetic CRF as well as CRF antagonist. CRF binding at these sites was accompanied by increases in the concentration of cAMP (but not cGMP) in the cells, with minimal and maximal effective CRF doses of 10(-13) and 10(-6) M for the monocyte-macrophage and 10(-12) M and 10(-8) M for the T-helper cell. Production of cAMP in response to CRF was effectively inhibited by CRF antagonist in both cell types. Moreover, rat splenocyte proliferation induced by interleukin 2(IL-2; 110 IU) was blocked by CRF, half-maximally at a CRF dose of 2.2 x 10(-10) M and completely at 3.5 x 10(-9) M. Finally, when CRF was added together with a 50-fold molar excess of CRF antagonist the IL-2 effect was fully restored. This demonstration of specific, physiologically relevant CRF receptors on two key immunocytes, the monocyte-macrophage and the T-helper lymphocyte, along with in vitro immunosuppression concomitant with CRF binding reinforces the growing body of evidence for a prominent role for CRF in immunomodulation.

Corticotropin-releasing factor modulates the immune response to stress in the rat.

We examined the role of CRF, a key mediator of the endocrine response to stress, in modulating immunosuppression during the subacute stress of intermittent electrical shock over 1 h. Administration of shock to intact rats resulted in a 74% decrement in T-lymphocyte proliferation and a 59% decrease in natural killer cytotoxicity. Similar suppression of these two parameters of immune function in response to shock was noted in adrenalectomized rats as well. The immunosuppressive effects of this shock were significantly and comparably blunted when both intact and adrenalectomized animals were pretreated 1) iv with either a highly potent polyclonal CRF antibody or a specific CRF antagonist or 2) intracerebroventricularly with either a high affinity monoclonal antibody to CRF or a specific CRF antagonist. An immunomodulatory role for CRF is further supported by the findings that administration of exogenous CRF, either iv (10 micrograms/animal) or intracerebroventricularly (1 microgram/animal), resulted in significant decrements in lymphocyte proliferation and natural killer cytotoxicity, similar to those seen with the stress paradigm. Our observations indicate that CRF plays a significant role in modulating the immune response to subacute stress, largely by adrenal-independent mechanisms.

Structural characterization and localization of corticotropin-releasing factor in testis.

To sequence and thereby definitively characterize corticotropin-releasing factor (CRF)-like material from a representative peripheral tissue, CRF was obtained from 76 ovine testes. The novel extraction procedure involved use of an immunoaffinity column to which a high-affinity CRF monoclonal antibody was attached as well as fast protein liquid chromatography. The complete sequence was elucidated by gas-phase sequencing, carboxyamidopeptidase digestion and cyanogen bromide cleavage. Aside from microheterogeneity at position 39, all the other amino acids were identical to ovine hypothalamic CRF. Additionally, in immunohistochemical studies in the rat, CRF was localized to the Leydig cell. These findings along with related observations by ourselves and others are compatible with the hypothesis that CRF plays a significant local role, possibly by paracrine or autocrine mechanisms.

Corticotropin-releasing factor levels in the peripheral plasma and hypothalamus of the rat vary in parallel with changes in the pituitary-adrenal axis.

Impressive evidence has emerged indicating that immunoassayable and bioassayable CRF, which is immunoneutralizable, is present not only in the hypothalamus but in many peripheral tissues as well. Using highly specific and sensitive RIAs and immunoaffinity chromatography to investigate whether this extrabrain CRF circulates in the rat, we found low but clearly measurable levels in peripheral plasma (mean, 11.4 +/- 0.8 pg/ml). Immunological findings were corroborated by fast protein liquid chromatography, which resolves peptides by both hydrophobicity and ionic charge. With this approach the major immunoreactive peak was eluted at the position of synthetic rat CRF standard. To assess whether levels of peripheral plasma CRF-like immunoreactivity (CRF-LI) vary in parallel with those of hypothalamic CRF-LI, we performed studies with low and high dose dexamethasone administration and withdrawal, adrenalectomy, and hypophysectomy. Seven days after oral administration of dexamethasone, there was a decrement in the levels of peripheral plasma and hypothalamic CRF-LI. Depending on the dose, recovery was also found 7 days after cessation of the treatment. After either adrenalectomy or hypophysectomy, there were increments in the levels of CRF-LI in both peripheral plasma and hypothalamus. Thus, concentrations of CRF-LI in the peripheral plasma and in the hypothalamus vary in parallel in response to alterations in the pituitary-adrenal axis.

[Changes of growth hormone-releasing factor (GRF) in rat peripheral blood--effect of hypophysectomy].

Accumulating evidence has emerged indicating that growth hormone-releasing factor (GRF) is present not only in the hypothalamus but in other tissues as well. Using highly specific and sensitive radioimmunoassays and immunoaffinity chromatography, we found low but clearly measurable GRF-like immunoreactivity (GRF-LI) levels in rat peripheral plasma. In order to verify the immunological findings, the peripheral plasma GRF-LI was characterized using fast protein liquid chromatography. The immunological peak was eluted at the position of synthetic rat GRF standard. These findings demonstrate that rat peripheral plasma GRF is immunologically and chromatographically indistinguishable from authentic rat GRF. Moreover, we performed studies with hypophysectomy to assess whether peripheral plasma GRF-LI changes in physiological status. At 4 weeks after hypophysectomy, there was a significant (p less than 0.05) increment in the rat plasma GRF-LI [12.4 +/- 0.5 (+/- SEM) pg/ml in hypophysectomized rats as opposed to 5.8 +/- 0.4 pg/ml in sham-operated control rars]. On the other hand, hypothalamic GRF-LI fell significantly as compared that of controls (36.1 +/- 1.0 vs. 78.3 +/- 3.0 pg/mg wet weight tissue). A similar pattern of changes in GRF-LI at 10 weeks after hypophysectomy was also revealed. The source of rat peripheral plasma GRF has not yet been elucidated. Our results, however, may suggest that GRF levels are modulated by negative feedback at the level of the hypothalamus by a pituitary factor, presumably growth hormone (GH) and that hypothalamic GRF release exceeds its synthesis in hypophysectomized rats.

Growth hormone-releasing factor releases ACTH from an AtT-20 mouse pituitary tumor cell line but not from normal pituitary cells.

Corticotropin-releasing factor (CRF) and both human pancreatic growth hormone-releasing factor (hp-GRF) and rat hypothalamic GRF (rh-GRF) stimulated ACTH release from neoplastic AtT-20 mouse pituitary tumor cells in a dose-dependent fashion, with CRF inducing a 10-fold increase and GRF a maximal increment of approximately one-half that of CRF. Neither rh-GRF nor hp-GRF induced ACTH release in normal anterior pituitary cells. Pretreatment with either dexamethasone or somatostatin prior to the addition of rh-GRF inhibited the increase in ACTH release. Both ovine CRF and rh-GRF stimulated adenosine 3,5-monophosphate production in AtT-20 cells. The weak but clearly discernible effect of GRF on ACTH release from AtT-20 cells may be due to an abnormality in the AtT-20 cell receptor.

A physiological role of E and F series prostaglandins in the regulation of TSH secretion.

The regulation of TSH secretion by E1, E2, E1 alpha and F2 alpha prostaglandins was studied by means of a monolayer culture system of dispersed rat anterior pituitary cells which was appropriately responsive to TRH, T3 and SRIF. PGEs and Fs induced significant increases in basal TSH release of the order of 30% at 10(-9) or 10(-8) to 10(-5) or 10(-4) M. Only PGEs accentuated the TSH release induced by a half maximal dose of TRH (10(-9) M) of the order of 60% in a dose dependent manner (10(-9) to 10(-6) M of PGEs), whereas PGFs did not. SRIF (10(-8) or 10(-9) M) alone failed to alter basal TSH release but did completely inhibit the TSH response to TRH (10(-9) M). SRIF also significantly inhibited both the increase in basal TSH release and the accentuation of the TSH response to TRH induced by PGEs (10(-6) M) but did not diminish the enhancement of basal TSH release induced by PGFs (10(-6) M). 7-oxa-13-prostynoic acid (PY1), a prostaglandin antagonist, which can act as an agonist in some systems, itself exhibited agonistic properties of PGEs with respect to basal and TRH induced TSH release. PY1 failed to inhibit the TSH release induced by all PGs, but partially inhibited the accentuated TSH response to TRH induced by PGEs. Indomethacin, PG synthetase inhibitor, did not affect basal or TRH induced TSH release in our system. These data suggest that PGs of the E and F series probably modulate TSH release via different mechanisms and that the PGE effect on basal TSH release differs from its augmentation of TRH induced TSH response. It is speculated that these effects of PGs may have physiological significance.

Corticotrophin-releasing factor in extra-hypothalamic brain of the mouse: demonstration by immunoassay and immunoneutralization of bioassayable activity.

Corticotrophin-releasing factor (CRF) bioactivity has been described in the extra-hypothalamic brain, but its relationship to hypothalamic CRF has remained questionable. Of the seven regions of the mouse brain examined, highest concentrations of CRF-like immunoreactivity (CRF-LI) and bioassayable CRF activity were present in the median eminence and hypothalamus. However, substantial CRF-LI and bioassayable CRF activity were also seen in brain extracts from the amygdala, thalamus, frontal cortex, pons medulla and cerebellum. Bioactivity was largely neutralized by prior incubation with heat-inactivated antiserum to ovine CRF. These findings, in conjunction with previous immunocytochemical evidence, strongly suggest that a substance closely resembling hypothalamic CRF is present in the extrahypothalamic brain of the mouse.

Levels of human and rat hypothalamic growth hormone-releasing factor as determined by specific radioimmunoassay systems.

Polyclonal antibodies to synthetic human pancreatic growth hormone-releasing factor [hpGRF(1-44)NH2] and rat hypothalamic growth hormone-releasing factor [rhGRF(1-43)OH] were produced in rabbits by injecting these weak immunogens, coupled to thyroglobulin and emulsified with complete Freund's adjuvant in the presence of activated charcoal, directly into the spleen. A subsequent booster injection by the conventional intramuscular route resulted in high-titer antibodies, which at a 1:20,000 dilution were used to develop highly sensitive and specific radioimmunoassays for these peptides. By using antibodies with an apparent Ka of 3.3 X 10(-12) (human) and 7.7 X 10(-11) (rat), the sensitivity of these assays in both human and rat was found to be less than 1 fmol. The antibody to hpGRF(1-44)NH2 is directed against the COOH-terminal region of the molecule, as shown by its crossreactivity with various hpGRF analogues: 140% with hpGRF(30-44)NH2; 1%-2% with hpGRF(1-37)OH, hpGRF(1-40)OH, and hpGRF(1-40)NH2; and none with hpGRF(1-29)NH2. Serial dilutions of human and rat hypothalamic extracts demonstrated parallelism with the corresponding species-specific standard and 125I-labeled tracer. There was no crossreactivity with other neuropeptides, gastrointestinal peptides, or hypothalamic extracts of other species. The hypothalamic content in fmol/mg (wet weight) of tissue was 3.6 +/- 0.2 for the human and 11.1 +/- 5.5 for the rat. Age-related changes in hypothalamic GRF content were present in rats, with a gradual increase from 2 to 16 weeks and a correlation between increasing body weight and GRF content. These radioimmunoassays will serve as important tools for understanding the regulation of growth hormone secretion in both human and rat.

The effects of centrally administered neuropeptides on the development of gastric lesions in the rat.

Centrally administered neuropeptides were investigated for their effects on the development of gastric lesions in rats. Thyrotropin releasing hormone (TRH), vasoactive intestinal peptide (VIP) and gonadotropin releasing hormone (LHRH) produced gastric lesions acutely, with TRH demonstrating the most pronounced effect in terms of incidence and severity. Ten-fold higher doses of the same peptides administered intravenously produced none or very few gastric lesions. Moreover, pretreatment with atropine partially inhibited their production. Corticotropin releasing factor (CRF) exhibited only mild ulcerogenic effects, and the gastric lesions induced with this peptide developed more slowly than with TRH, VIP and LHRH. Although ulcerogenic in their own right, none of these four neuropeptides significantly potentiated the potent ulcerogenic effects of cold-restraint stress. Since other neuropeptides, including somatostatin, human pancreatic growth hormone releasing factor (hpGRF), substance P, bombesin, and neurotensin, had no demonstrable effects on gastric mucosa, we can conclude that the lesions were not a general effect of intracisternal administration of neuropeptides. The results suggest that within the central nervous system, there are several neuropeptides that play a significant role in the development of gastric lesions via, at least in part, vagal-dependent mechanisms.

Evidence for a role of endogenous corticotropin-releasing factor in cold, ether, immobilization, and traumatic stress.

The role of corticotropin-releasing factor (CRF) in four model stresses (cold, ether, immobilization, and trauma) was examined in the guinea pig by using passive immunoneutralization with anti-CRF antiserum. Plasma corticotropin levels were measured at various times after exposure to stress, and groups treated with CRF antiserum were compared with those treated with normal rabbit serum. Of the four stresses tested, ether had the most pronounced effect on corticotropin secretion. Treatment with anti-CRF inhibited most of the ether-induced corticotropin secretory response, the difference between the normal serum- and the anti-CRF antiserum-treated groups being significant at 5 and 10 min (P less than 0.01). Corticotropin responses to cold stress in the two groups differed at the 0.05 level of significance at 10 and 20 min. After administration of trauma (leg fracture), a statistically significant difference (P less than 0.01) between the two groups also was evident, albeit only at 20 min. During immobilization, corticotropin levels differed significantly from control only in the normal serum-treated group but not in the anti-CRF-treated group. These findings show that CRF antiserum was effective in reducing corticotropin levels, indicating that CRF has an important role in mediating corticotropin response to stress. The fact that neutralization was incomplete might be due to an inability of the antiserum to sufficiently neutralize the endogenous CRF or, more likely, reflects the contribution of additional mediators, notably catecholamines and vasopressin, of corticotropin release upon stress.

Levels of corticotropin releasing factor-like immunoreactivity in mammalian hypothalamic and extrahypothalamic brain tissue as determined with a monoclonal antibody to the ovine material.

A monoclonal antibody to ovine corticotropin releasing factor (CRF) has been produced by fusion of a non-producing plasmacytoma cell line P3U1 with spleen cells of Balb/c mice immunized with the synthetic 41 amino acid peptide coupled covalently with rabbit myosin by a heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate. A total immunizing dose of 500 micrograms resulted in a highly specific, high-affinity antibody with a Ka of 0.15 x 10(12) M-1, which was used to establish a specific RIA with a sensitivity of 10 pg/tube. Levels of corticotropin releasing factor-like immunoreactivity (CRF-LI) in a pg/mg of hypothalamic tissue ranged from 4-10 in ovine, 2.5-8 in bovine, 47.5-67.5 in mouse and 2.3-20 in human tissue. Moreover, CRF-LI was widely distributed in extrahypothalamic mouse brain at concentrations approximately one half those seen in hypothalamus.

Dopamine stimulates somatostatin release from perifused hypothalamic cells.

We studied the release of immunoreactive somatostatin (IR-SRIF) from hypothalamic cells that were obtained from rats and dispersed with the aid of collagenase. Twenty-four hours after dispersion, cells were placed in a column supported by a matrix of preswollen Biogel P2 and perifused. Fractions were collected on ice and subsequently assayed for SRIF. SRIF release was stimulated markedly by potassium depolarization (KCl, 56 mM), by the Na+-K+-ATPase inhibitor ouabain (10(-4) M), and by dopamine at concentrations as low as 10(-11) M. The stimulatory effects of membrane depolarization were calcium dependent and were not observed in the absence of exogenous calcium in the perifusion medium or in the presence of EDTA (0.05 M). Metoclopramide, the dopamine antagonist, abolished the stimulatory effect of dopamine. In conclusion, release of IR-SRIF by dispersed rat hypothalamic cells can be studied in a simple perifusion apparatus. Release is stimulated by membrane depolarization in a calcium-dependent manner and by dopamine at physiological concentrations.

Somatostatin release from dispersed hypothalamic cells - effects of membrane depolarization, calcium and glucose deprivation.

We have developed a new short term in vitro system to examine hypothalamic somatostatin (SRIF) release. Hypothalamic cells were obtained from normal rats after trypsin or collagenase aided dispersion and released immuno-reactive (IR) SRIF which eluted in 3 molecular weight (MW) forms on gel chromatography. The smallest MW form, which constituted the major peak, co-eluted with synthetic cyclic 1-14 SRIF on gel and reverse phase high pressure liquid chromatography (HPLC). After 24 h in culture in medium containing heat inactivated fetal calf serum, cell viability was demonstrated by two techniques, (1) vital staining with trypan blue, and (2) incorporation of 32Pi into phospholipids. SRIF release was also studied at this time which was optimal in terms of responsivity of the cells to depolarizing stimuli. SRIF release increased in a time dependent manner, over 3 h. Membrane depolarization, induced either by potassium chloride 56 mM or ouabain (the Na+, K+-ATPase inhibitor) 10(-6) M or greater, markedly stimulated SRIF release. Incubation at 4 degrees C, or in the presence of EDTA 0.05 M or verapamil, the calcium channel blocker, 50 microM abolished these stimulatory effects. Glucose deprivation was induced by the addition of 2-deoxy-D-glucose (2-DG) to the experimental medium. 2-DG, at concentrations of up to 200 mg%, had no significant effect on SRIF release during incubation periods of up to 1 h.

Distribution, biosynthesis, and physiological role of corticotropin-releasing factor in the human: an overview.

Our findings to date indicate that: A peptide resembling oCRF is present in human and mammalian hypothalamus. oCRF is present in human lumbar cerebrospinal fluid. oCRF concentrations do not differ in CSF from normal individuals and from patients with Cushing's syndrome. oCRF appears to be synthesized via a large oligopeptide precursor. An oCRF-like molecule (oCRF-LI) is present in hypothalamic brain tissue. We have also observed more tentative evidence of low levels of oCRF-LI outside of the brain. oCRF is likely to be a central mediator of stress in its multiple forms. We believe that oCRF is clearly of major physiological importance, but that many critical unanswered questions remain. Probably, the most fascinating of these, which we are only beginning to comprehend, concerns the functions of CRF in extrahypothalamic brain as well as the CRF which appears to be present outside the brain.