Lack of prognostic significance of the monoclonal antibody Ki-S1, a novel marker of proliferative activity,in node-negative breast carcinoma

In a series of 205 node-negative breast cancers (NNBC), we determined staining by the novel antibody Ki-S1, a marker of tumor cell proliferation, in order to test its association with other prognostic variables and its prognostic significance. Ki-S1 was determined in routinely formalin-fixed paraffin-embedded tumor samples. Ki-S1 gave a nuclear staining in the majority of the carcinomas (188 of 205), with percentages of reacting nuclei ranging from 2% to 90% (median value of 7%). In 107 tumors frozen sections were available to also assess the Ki-67 antibody. Among these, 94 had a nuclear staining of cancer cells ranging from 5% to 80% (median value of 7%). In 46 tumors we also determined the MIB-1 antibody. The percentage of MIB-1 nuclear staining ranged from 1% to 50% (median value of 20%). There was no significant relationship between Ki-S1 and the other two cell kinetic markers. Ki-S1 labeling was significantly associated only with tumor size (p = 0.03).With a median follow-up of 6 years, Ki-S1 had no significant prognostic value for either relapse-free survival (RFS) or overall survival (OS)(Ki-S1 as continuous logarithmic variable; p = 0.86 and p = 0.23, respectively). For RFS the following variables had a significant prognostic value: Ki-67 ( 10% vs > 10%; p = 0.037); progesterone receptor (PgR) expression (– vs +/++; p = 0.041); tumor size (pT1 vs pT2–3; p = 0.042) and grading (GI vs GII–III; p = 0.047). For OS, tumor size (p = 0.0044), age (continuous variable; p = 0.0060), and Ki-67 (p = 0.043) were significantly prognostic.In multivariate analysis (final model), only tumor size retained a significant and independent prognostic value for RFS (p = 0.0042). For OS, both tumor size (p = 0.0029) and age ( 55 years vs > 55 years; p = 0.041) retained significance in the multivariate model.In conclusion, Ki-S1 does not seem to have prognostic relevance in this series of NNBC. Possible hypotheses to explain this observation are discussed.     

Interrelationship between retinal ischaemic damage and turnover and metabolism of putative amino acid neurotransmitters,glutamate and GABA

Conditions causing a reduction of oxygen availability (anoxia), such as stroke or diabetes, result in drastic changes in ion movements, levels of neurotransmitters and metabolites and subsequent neural death. Currently, there is no clinically available treatment for anoxia induced neural cell death resulting in drastic and permanent central nervous system dysfunction. However, there have been some exciting developments in experimentally induced anoxic conditions where several classes of drugs appear to significantly reduce neural cell death. This report aims to provide the foundations for understanding both the basic mechanisms involved in retinal ischaemic damage and experimental treatments used to prevent such damage. We discuss the normal release, actions and uptake of the fast retinal neurotransmitters, glutamate and GABA, in the vertebrate retina. Immunocytochemistry is used to demonstrate that both glutamate and GABA are found in the macaque retina. Following this is a discussion on how ischaemia may enhance neurotransmitter release or disrupt its uptake, thus causing an increase in extracellular concentration of these neurotransmitters and subsequent neuronal damage. The mechanisms involved in glutamate neurotoxicity are reviewed, because excess glutamate is the likely cause of retinal ischaemic damage. Finally, the mechanisms behind four possible modes of treatment of neurotransmitter toxicity and their advantages and disadvantages are discussed. Hopefully, further research in this area will lead to the development of a rational therapy for retinal, as well as cerebral ischaemia.Abbreviations -KG -ketoglutarate - AAT aspartate amino transferase - AC amacrine cell - ACL amacrine cell layer - BC bipolar cell - CNS central nervous system - EAA excitatory amino acids - G'ase glutaminase - GABA -amino butyric acid - GABA-T GABA transaminase - GAD glumatic acid decarboxylase - GC ganglion cell - GCL ganglion cell layer - GDH glutamate dehydrogenase - gj gap junction - GS glutamine synthetase - HC horizontal cell - ILM inner limiting membrane - INL inner nuclear layer - IPC inter-plexiform cell - IPL inner plexiform layer - IS inner segment of photoreceptor - NFL nerve fibre layer - NMDA N-methyl-D-aspartate - OLM outer limiting membrane - ONL outer nuclear layer - OPL outer plexiform layer - OS outer segment of photoreceptor - Ox acetateoxaloacetate - RL receptor layer - SSAD succinate semi-aldehyde decarboxylase - Succinate SA succinate semi aldehyde - TCA cycle tricarboxylic acid cycle     

Light and Electron Microscopic Localization of the Neutral Metalloendopeptidase EC 3.4.24.16 in the Mesencephalon of the Rat

The topographic and cellular distribution of the neurotensin-hydrolysing neutral metalloendopeptidase 24.16 (EC 3.4.24.16) was examined by light and electron microscopic immunohistochemistry in adult rat mesencephalon. Light microscopic immunoradioautography revealed a ubiquitous distribution of the enzyme throughout the midbrain with a relative enrichment of grey matter areas including the substantia nigra, ventral tegmental area, interfascicular nucleus, interpeduncular nucleus, rostral and caudal linear raphe nuclei, central grey and superficial grey of the superior colliculus. Peroxidase - antiperoxidase immunocytochemistry revealed two distinct cellular patterns of immunostaining: (1) weakly labelled neuronal perikarya more or less uniformly distributed throughout the grey matter, and (2) intensely immunoreactive glial cells heterogeneously distributed across the mesencephalon. Areas exhibiting dense concentrations of endopeptidase 24.16-containing glial cells corresponded to those displaying enhanced immunoreactivity in immunoradioautographs, suggesting that a major proportion of brain endopeptidase 24.16 is associated with glia. Electron microscopic examination of the substantia nigra and ventral tegmental area confirmed the association of the enzyme with a subpopulation of neurons and allowed identification of labelled glial cells as protoplasmic astrocytes. In neurons, endopeptidase 24.16 immunoreactivity was distributed heterogeneously within the cytoplasm of perikarya, dendrites and axons. Reaction product was also characteristically associated with restricted zones of the plasma membrane and underlying neuroplasm. In astrocytes, endopeptidase 24.16 immunostaining was densely and uniformly distributed throughout the cytoplasm of cell bodies and processes. Many of these processes were in direct contact with endopeptidase 24.16-immunopositive neuronal elements. The present results demonstrate that within the midbrain, endopeptidase 24.16 is both intracytoplasmic and membrane-associated in neurons and predominantly intracytoplasmic in glia. The presence of a large number of immunostained elements within areas of the midbrain known to display high levels of neurotensin and/or neurotensin receptors, together with the demonstrated catabolic activity of the enzyme on neurotensin in vitro, is consistent with a role of endopeptidase 24.16 in the functional inactivation of endogenous neurotensin in this region of the brain.     

Calbindin-D28K (CaBP28k)-like Immunoreactivity in Ascending Projections

This study concerns the involvement of calbindin-D28K (CaBP28k)-containing neurons in ascending spinal projections to the brainstem (nucleus of the solitary tract, lateral reticular nucleus area), pontine (parabrachial area) and mesencephalic (periaqueductal grey) structures. All these central structures are important in the processing of visceroception and visceronociception and all are targets for spinal efferents from similar areas. CaBP28k controls the excitability of cells by acting on intrinsic calcium metabolism. Results refer to the caudal spinal areas where the visceroceptive regions are concentrated. Experiments were performed through a double labelling approach that combined the retrograde transport of a protein - gold complex to identify the projection cells and immunohistochemistry to identify the CaBP28k-positive cells. The caudal spinal cord is rich in both CaBP28k-containing and projection cells. Cells colocalizing the protein and the retrograde tracer were quite numerous, with a particularly high concentration in the superficial layers of the dorsal horn (laminae I and outer II) and the lateral spinal nucleus. The other spinal areas containing immunoreactive projection cells were the reticular part of the neck of the dorsal horn, the medial laminae VII and VIII, lamina X and the sacral parasympathetic nucleus. The superficial layers and the neck of the dorsal horn are targets for nociceptive, visceroceptive and thermal inputs; the sacral parasympathetic column and lamina X are involved in visceroceptive integration. A functional role for the lateral spinal nucleus has not yet been established. Quite similar results were obtained for each of the ascending pathways under study. The high incidence of CaBP28k in spinal pathways suggests that calbindin has a major role in controlling the excitability of spinal cells subserving the processing of visceroception and/or visceronociception information to supraspinal levels. The participation of CaBP28k-immunoreactive cells in spinal ascending tract cells largely outnumbers those previously reported for various neuropeptides (Leah et al., Neuroscience, 24, 195 - 207, 1988)     

Progesterone Receptor-Containing Neurons in the Guinea-Pig Mediobasal Hypothalamus have Axonal Projections to the Medial Preoptic Area

The location and number of progesterone receptor-containing neurons in the mediobasal hypothalamus that project to the medial preoptic area were determined by combining retrograde fluorescent tract tracing with progesterone receptor immunocytochemistry. Injections of the retrograde tract tracer Fluoro-gold were made in the preoptic area of female guinea-pigs ovariectomized and primed with estradiol. After 5 days survival to allow for retrograde transport, tissue sections were incubated with monoclonal antibodies to the progesterone receptor to detect the presence of progesterone receptor-immunoreactive neurons. Cell bodies were labelled with Fluoro-gold throughout the arcuate nucleus. These neurons were not concentrated in any particular area of the nucleus but were diffusely distributed bilaterally. Retrogradely-labelled neurons were also observed in the ventrolateral and ventromedial nuclei mainly contralateral to the injection site. Progesterone receptor immunofluorescence labelled a subpopulation (7% to 10%) of these retrogradely-labelled cells particularly in the arcuate nucleus, including the median eminence. The double-labelled cells were more numerous in the anterior two-thirds of the arcuate nucleus. Although our estimates of the proportion of hypothalamic progesterone receptor-immunoreactive neurons that sent axons directly to the medial preoptic area were low, (about 0.35%), these neurons may be part of a neural circuit involved in the regulation of reproductive processes.     

Luteinizing Hormone-Releasing Hormone Neurons which are R-etrogradely Labeled After Peripheral Fluoro-Gold Administration in the Male Ferret

This study identified luteinizing hormone-releasing hormone (LHRH)-producing neurons which have access to fenestrated capillaries in prepubertal male European ferrets. Fluoro-Gold was injected intraperitoneally to retrogradely label neurons with terminals outside the blood-brain barrier. LHRH neurons were identified by immunofluorescence using a secondary antibody tagged with tetramethylrhodamine isothiocyanate. Cell bodies which demonstrated both tetramethylrhodamine isothiocyanate and Fluoro-Gold fluorescence were defined as LHRH-producing neurons with axon terminals in regions containing fenestrated capillaries. The total number and neuroanatomical distribution of immunopositive (LHRH +) cells concurred with previous studies in the ferret in which cell bodies were diffusely distributed from rostral forebrain through caudal diencephalon, with approximately 70% of the LHRH + cell bodies located in retrochiasmatic hypothalamus. In the present study, an average of 59.8% of all LHRH+ neuronal perikarya also contained Fluoro-Gold. The majority of Fluoro-Gold filled LHRH+ neurons demonstrated only faint to moderate amounts of Fluoro-Gold when compared to other Fluoro-Gold filled neurosecretory neurons. This limited uptake of Fluoro-Gold may be due to a relative inactivity of LHRH neurons projecting outside the blood-brain barrier. Double-labeled LHRH + neurons were dispersed throughout the entire population of LHRH+ cell bodies and no apparent nuclear groups of double-labeled neurons were found. This observation suggests that the LHRH+ neurons responsible for neurosecretion into the median eminence coexist with the LHRH+ neurons responsible for intracerebral neurotransmission or neuromodulation. One distinguishable population of LHRH + neurons was consistently observed in all the brains. Only 26% of total LHRH+ perikarya within the caudal arcuate nucleus contained Fluoro-Gold, while at least 50% of LHRH+ neurons in other structures, including the rostral arcuate nucleus, contained Fluoro-Gold. Thus, in the prepubertal male ferret, the majority of LHRH cell bodies located in the caudal arcuate nucleus may be differentially regulated and/or involved in non-neuroendocrine functions.     

Brain Spectrins 240/235 and 240/235E: Differential Expression During Development of Chicken Dorsal Root Ganglia in vivo and in vitro

Brain spectrin, a membrane-related cytoskeletal protein, exists as two isoforms. Brain spectrin 240/235 is localized preferentially in the perikaryon and axon of neuronal cells and brain spectrin 240/235E is found essentially in the neuronal soma and dendrites and in glia (Riederer et al., 1986, J. Cell Biol., 102, 2088 - 2097). The sensory neurons in dorsal root ganglia, devoid of any dendrites, make a good tool to investigate such differential expression of spectrin isoforms. In this study expression and localization of both brain spectrin isoforms were analysed during early chicken dorsal root ganglia development in vivo and in culture. Both isoforms appeared at embryonic day 6. Brain spectrin 240/235 exhibited a transient increase during embryonic development and was first expressed in ventrolateral neurons. In ganglion cells in situ and in culture this spectrin type showed a somato - axonal distribution pattern. In contrast, brain spectrin 240/235E slightly increased between E6 and E15 and remained practically unchanged. It was localized mainly in smaller neurons of the mediodorsal area as punctate staining in the cytoplasm, was restricted exclusively to the ganglion cell perikarya and was absent from axons both in situ and in culture. This study suggests that brain spectrin 240/235 may contribute towards outgrowth, elongation and maintenance of axonal processes and that brain spectrin 240/235E seems to be exclusively involved in the stabilization of the cytoarchitecture of cell bodies in a selected population of ganglion cells.     

Growth hormone receptor expression in the Dunning R 3327 prostatic carcinoma of the rat.

The Dunning R3327 rat carcinoma is an important model for human prostate adenocarcinoma. In the present study this tumor was further characterized by immunohistochemical demonstration of receptors for growth hormone (GH-R). Weak GH-R immunoreactivity was present in the secretory epithelial cells of the tumor acini. Large epithelial cells which were localized at the periphery of the acini and large cells in the stroma, which are probably derived from the epithelium ("Large neoplastic epithelial cells"), displayed a strong staining with one of the monoclonal antibodies (Mab 263) to GH-R. The presence of GH-R receptors in proliferating prostatic tumor cells supports the concept that GH reacts directly on prostate target tissue to facilitate tumor cell growth.     

Coexistence of GABA and glutamate in mossy fiber terminals of the primate hippocampus: an ultrastructural study.

One of the links in the trisynaptic circuit of the hippocampus is the synapse between the mossy fibre terminals of dentate granule cells and CA3 pyramidal cells of Ammon's horn. This synapse has been physiologically characterized as excitatory, and there is pharmacological and immunohistochemical evidence that mossy fibre terminals utilize glutamate as a neurotransmitter. This study demonstrates the presence of GABA-immunoreactivity in mossy fibre axons and terminals of the monkey at the electron microscopic level. We combined Golgi impregnation to identify CA3 pyramidal neurones, with postembedding immunocytochemistry to characterize the inputs to the identified cells. GABA immunoreactivity was present in mossy fibre terminals that made synaptic contact with complex embedded spines of identified Golgi-impregnated CA3 pyramidal neurones. GABA immunoreactivity could be demonstrated in serial sections of the same mossy fibre terminals by using 3 different antisera raised against GABA. In serial sections, the mossy fibre terminals were shown to be immunoreactive for both glutamate and GABA. In contrast, glutamate immunoreactivity but not GABA immunoreactivity was found in other terminals that did not have the morphological characteristics of mossy fibre terminals. GABA immunoreactivity in mossy fibre terminals was also demonstrated in a human surgical specimen of hippocampus. The coexistence of an "excitatory" amino acid and of an "inhibitory" amino acid in the same "excitatory" nerve terminal raises the possibility of corelease of the two transmitters, suggesting that the control of hippocampal neural activity is more complex than hitherto suspected.     

Glycine-receptor immunoreactivity in retinal bipolar cells is postsynaptic to glycinergic and GABAergic amacrine cell synapses.

Glycinergic innervation of the synaptic terminals of mixed rod-cone bipolar cells in the goldfish retina was investigated by electron microscopical immunocytochemistry with presynaptic and postsynaptic markers for glycinergic neurons: a monoclonal antibody (mAb 7A) against the 93 kDa subunit of the strychnine-sensitive glycine receptor and polyclonal antisera against a glycine/BSA conjugate. Conventional "glycinergic" synaptic contacts, made by amacrine cell processes, accounted for 7-10% of the input to the bipolar cell terminals, whether determined by glycine receptor immunoreactivity (GlyR-IR) or glycine-IR. In addition to the conventional synapses, the large bipolar cell terminals in the proximal inner plexiform layer (type Mb) gave rise to spinules (spine-like protrusions) that invaginated into presynaptic amacrine cell processes. Although 85% of the spinules were GlyR-IR, no spinules were postsynaptic to glycine-IR processes; yet 86% of the spinules were postsynaptic to GAD-IR processes, suggesting that the GlyR-IR spinules were postsynaptic to GABAergic terminals. Furthermore, a single amacrine cell process could make two synapses with an Mb terminal: a GlyR-IR contact onto a spinule and a conventional synapse that was not GlyR-IR. We suggest that glycinergic innervation of bipolar cell terminals involves conventional glycinergic synapses as well as an unconventional situation in which GABA and glycine may interact in as yet undetermined manner, perhaps by potentiation.