Neurochemical development of the hippocampal region in the fetal rhesus monkey. II. Immunocytochemistry of peptides,calcium-binding proteins,DARPP-32, and monoamine innervation in the entorhinal cortex by the end of gestation

Material for the study came from one 126 day-old rhesus monkey fetus and two 3 day-old neonates. The immunocytochemical detection of somatostatin, neurotensin (NT), parvalbumin, calbindin D-28K, DARPP-32 as well as tyrosine hydroxylase (TH), dopamine-β-hydroxylase and serotonin (5-HT), was carried out on serial cryostat sections of the entorhinal cortex. The authors reported in a previous paper the precocious differentiation of the entorhinal cortex in rhesus monkey fetuses and featured the conspicuous expression of calbindin D-28K, somatostatin, neurotensin, and the monoaminergic innervation during the first half of gestation. The present study shows distinct temporal profiles of neurochemical development during the second half of gestation: the dense neuropeptidergic innervation remained a constant feature; the three aminergic systems gradually increased in density; parvalbumin, unlike calbindin D-28K, was primarily expressed during the last quarter of gestation. Three othe prominent features of the last quarter of gestation are illustrated: the refinement of the modular neurochemical organization of the lamina principalis externa, the delayed chemoanatomical development of the rhinal sulcus area, and the establishment of a distinct rostrocaudal pattern of neurochemical distribution. In correspondence with the cluster-like organization of the lamina principalis externa, the authors observed in the olfactory, rostral, and intermediate fields of the neonate monkey entorhinal cortex, a particular subset of pyramidal-shaped neurons: located in layer III, they were characterized by fasciculated apical dendrites ascending between the cellular islands of the discontinuous layer II and the coexpression of calbindin D-28K and DARPP-32. Besides, most of the other chemical systems displayed a distinct, area-specific, patcy distribution, except for the homogeneously distributed noradrenergic innervation. In the olfactory and rostral fields, TH positive dopaminergic fibers accumulated on the neuronal islands of layers II-III, and parvalbumin labeled fibers on those of layer III, whereas patches of 5-HT and NT-like reactive terminals were segregated between the cellular islands, overlapping the DARPP-32/calbindin D-28 K labeled dendritic bundles. At the opposite, in the intermediate field, 5-HT positive terminals overlapped the cellular islands of layer II and thin fascicles of dopaminergic fibers ran in the inter island spaces. The somatostatin-LIR innervation was apparently too dense to reveal a patchy distribution that existed at earlier developmental stages. In the cadual field, the patchy pattern was replaced by a predominant bilaminar type of distribution of NT, 5-HT, and TH-like positive afferents. Numerous parvalbumin positive multipolar neurons and basket cells participated to a dense parvalbumin labeled network, extending through layers II-V, whose partial extrinsic origin is open to discussion. The rhinal sulcus, still reduced to a small dimple at E126, appeared fully developed at birth. The fundus of the sulcus was marked by a sharp decrease of the neurotensin and parvalbumin-LIR innervations whereas the density of somatostatin and aminergic terminals increased markedly in the perirhinal cortex. Although a transitory overexpression of some of the neurochemical systems under study might occur during development, their modular organization in the lamina principalis externa of the neonate represents a basic feature of the entorhinal cortex and adds further support to the evidence that neurons of layers II-III that project to different parts of the hippocampal formation, belong to distinct heterogeneous systems. This extensive prenatal development is in line with recent data emphasizing the critical role of limbic structures in early recognition memory in infant monkeys. The question arises, however, in view of the delayed development of the rhinal sulcus area, as to whether the preferential connections of the lateral entohinal and perirhinal cortex with regions of polymodal sensory convergence such as the prefrontal cortex and superior temporal gyrus might be established later than the connections of the rest of the entorhinal cortex.     

Circulating levels of midregional parathyroid hormone-related protein in hypercalcaemia of malignancy

OBJECTIVE: We have developed and evaluated a sensitive radioimmunoassay directed against the midregional part of parathyroid hormone-related protein (PTHrP), which is involved in the syndrome of humoral hypercalcaemia of malignancy. PATIENTS: Midregional PTHrP levels were studied in 41 consecutive inpatients with malignancy and hypercalcaemia, 32 normocalcaemic patients with malignancy, 21 patients with primary hyperparathyroidism, 34 patients with renal failure, and 87 normals. MEASUREMENTS: The assay used an antiserum against the midregional amino acid residues 53-84 of PTHrP and PTHrP(1-86) as label and standard. Midregional PTHrP was stable in serum and plasma and could be measured directly without sample extraction. RESULTS: Normal plasma concentrations ranged from undetectable (< 5 pmol/l) to 21 pmol/l. In renal failure, PTHrP was positively correlated with serum creatinine, but PTHrP elevations of up to 30 pmol/l were found only in severe renal dysfunction with creatinine > 850 mumol/l. In hypercalcaemia caused by solid tumours, midregional PTHrP was elevated in 81% (22 of 27) of patients, ranging from undetectable to 203 pmol/l (median: 40 pmol/l). In these patients serum calcium correlated positively with PTHrP (P < 0.01). Mean PTHrP levels were indistinguishable in subgroups with and without metastatic skeletal disease. The mechanism of hypercalcaemia in 14 patients with haematological malignancy was apparently different, since all but one had normal or only marginally elevated PTHrP levels. In 21 patients with primary hyperparathyroidism midregional PTHrP was normal in 20. The assay was therefore especially useful in distinguishing the latter condition from humoral hypercalcaemia of malignancy as the second major cause of hypercalcaemia. PTHrP was normal in all 32 patients with normocalcaemic malignancy. CONCLUSION: This radioimmunoassay of midregional PTHrP provides high diagnostic sensitivity in the identification of humoral hypercalcaemia of malignancy caused by solid tumours. The assay should therefore be useful in the differential diagnosis of hypercalcaemia.     

Separating the wheat from the chaff

Received from the Harrisburg Hospital Family Practice Residency Program, Harrisburg, Pennsylvania. Dr. Slawson is currently at the University of Virginia, Charlottesville, Virginia.     

Interleukin 12 and B7-1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression

Stimulation of antitumor immune mechanisms is the primary goal of cancer immunotherapy, and accumulating evidence suggests that effective alteration of the host–tumor relationship involves immunomodulating cytokines and also the presence of costimulatory molecules. To examine the antitumor effect of direct in vivo gene transfer of murine interleukin 12 (IL-12) and B7-1 into tumors, we developed an adenovirus (Ad) vector, AdIL12–B7-1, that encodes the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 under control of the murine cytomegalovirus promoter. This vector expressed high levels of IL-12 and B7-1 in infected murine and human cell lines and in primary murine tumor cells. In mice bearing tumors derived from a transgenic mouse mammary adenocarcinoma, a single intratumoral injection with a low dose (2.5 × 10⁷ pfu/mouse) of AdIL12–B7-1 mediated complete regression in 70% of treated animals. By contrast, administration of a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Interestingly, coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated at this dose. Significantly, cured animals remained tumor free after rechallenge with fresh tumor cells, suggesting that protective immunity had been induced by treatment with AdIL12–B7-1. These results support the use of Ad vectors as a highly efficient delivery system for synergistically acting molecules and show that the combination of IL-12 and B7-1 within a single Ad vector might be a promising approach for in vivo cancer therapy.     

Benzodiazepines for acute pain in children

When pediatric pain is refractory and unresponsive to appropriate use of analgesic agents, there might be additional physical or psychologic dimensions of the pain that are not addressed by the analgesics. In addition to appropriate analgesic therapy, the psychologic needs of the child should be directly addressed and appropriate adjunctive physical modalities employed. Although benzodiazepines lack direct analgesic effects, they can reduce the distress associated with acute pain states by decreasing anxiety, insomnia, and muscle spasms that can be associated with acute pain. Intermediate or long-acting benzodiazepines in modest doses can be useful adjunctive agents when used short term for the treatment of selected acute pain complaints. In the highly distressed school-age child or adolescent with pain complaints relatively unresponsive to appropriate care, judicious use of benzodiazepines is worthy of consideration.     

The Local Anesthetic n-Butyl-p-Aminobenzoate Selectively Affects Inactivation of Fast Sodium Currents in Cultured Rat Sensory Neurons

Background: Aqueous suspensions of the local anesthetic n-butyl-p-aminobenzoate (BAB), epidurally applied in terminal cancer patients, resulted in a sensory blockade, lasting up to several months. To investigate the mechanism of action on the cellular level, the effect of 100 micro Meter BAB on Sodium sup + action potentials and on Sodium sup + currents in dorsal root ganglion neurons from neonatal rats was studied.

Methods: Small neurons grown in cell culture were selected for patch-clamp measurements. Both Sodium sup + action potentials, evoked by current pulses of increasing amplitude (current clamp) and Sodium sup + currents, activated at different membrane potentials (voltage clamp), were investigated in the absence and presence of 100 micro Meter BAB. The local anesthetic was applied by external perfusion for 2 or 10 min.

Results: In the presence of 100 micro Meter BAB, either the firing threshold was raised or the action potential was abolished. The maximal peak conductances, underlying the fast sodium current INa,F and the slow sodium current INa,S, were not changed. However, the inactivation of INa,F Was increased by BAB. The sigmoid inactivation curve shifted 12 mV toward hyperpolarizing membrane voltages, whereas no changes were found for the inactivation of the slow Sodium sup + current. Only at short exposure times of 2 min, the effects of BAB could be reversed during a 10-min wash-out.     

Effect of glutamine on tumor and host growth

Background: Oral glutamine supplementation has been found to support gastrointestinal mucosal growth and increase intestinal and systemic toxicity after chemotherapy and radiation therapy. Glutamine is also an important nutrient for rapidly proliferating tumor cells. However, it is not clear whether long-term glutamine supplementation in the tumor-bearing host has a selective benefit for host growth or tumor cell proliferation. Methods: To study the effect of glutamine in tumor-bearing animals, 30 Lewis/Wistar rats with subcutaneous mammary tumor implants (MAC-33) were randomized to receive a 3% glutamine- or 3% glycerine-enriched (control) diet for 25 days. Results: No significant difference was found in carcass weight, primary tumor weight, or spontaneous pulmonary metastasis with glutamine supplementation. Tumor cell cycle kinetics (aneuploidy, %S and %S [synthetic] + G₂/M [growth fraction]) were similar between glutamine-supplemented and control animals. A trophic effect of glutamine on distal ileal mucosa was seen with increased DNA content (344±68 vs. 184±38 µg/100 mg tissue) (p<0.05) and RNA content (435±44 VS. 335±30 µg/100 mg tissue) (p=0.06) compared with control animals. No detectable differences were observed in liver or muscle, or in tumor DNA, RNA, or protein content. Conclusions: These findings confirm the trophic effect of glutamine on small intestinal mucosa and suggest that glutamine can be administered to the tumor-bearing host over a long period of time without significantly stimulating tumor growth kinetics or metastasis.