Progress in stem cell therapy for major human neurological disorders.

Human neurological disorders such as Alzheimer's disease (AD), Parkinson's disease, stroke or spinal cord injury are caused by the loss of neurons and glial cells in the brain or spinal cord in the Central Nervous System (CNS). Stem cell technology has become an attractive option to investigate and treat these diseases. Several types of neurons and glial cells have successfully been generated from stem cells, which in some cases, have ameliorated some dysfunctions both in animal models of neurological disorders and in patients at clinical level. Stem cell-based therapies can be beneficial by acting through several mechanisms such as cell replacement, modulation of inflammation and trophic actions. Here we review recent and current remarkable clinical studies involving stem cell-based therapy for AD and stroke and provide an overview of the different types of stem cells available nowadays, their main properties and how they are developing as a possible therapy for neurological disorders.

Bcl-XL modulates the differentiation of immortalized human neural stem cells.

Understanding basic processes of human neural stem cell (hNSC) biology and differentiation is crucial for the development of cell replacement therapies. Bcl-X(L) has been reported to enhance dopaminergic neuron generation from hNSCs and mouse embryonic stem cells. In this work, we wanted to study, at the cellular level, the effects that Bcl-X(L) may exert on cell death during differentiation of hNSCs, and also on cell fate decisions and differentiation. To this end, we have used both v-myc immortalized (hNS1 cell line) and non-immortalized neurosphere cultures of hNSCs. In culture, using different experimental settings, we have consistently found that Bcl-X(L) enhances neuron generation while precluding glia generation. These effects do not arise from a glia-to-neuron shift (changes in fate decisions taken by precursors) or by only cell death counteraction, but, rather, data point to Bcl-X(L) increasing proliferation of neuronal progenitors, and inhibiting the differentiation of glial precursors. In vivo, after transplantation into the aged rat striatum, Bcl-X(L) overexpressing hNS1 cells generated more neurons and less glia than the control ones, confirming the results obtained in vitro. These results indicate an action of Bcl-X(L) modulating hNSCs differentiation, and may be thus important for the future development of cell therapy strategies for the diseased mammalian brain.

Fenfluramine-induced increase in preproenkephalin mRNA levels in the striatum: interaction between the serotonergic, glutamatergic, and dopaminergic systems.

Fenfluramine (FE) is a halogenated amphetamine derivative that has been used in the treatment of obesity. It has been suggested that the effects of FE on the striatum are mediated by serotonergic mechanisms. However, several major afferent systems may be involved, and administration of FE may be useful to study interactions between these systems. In this work, the effects of FE on striatopallidal neurons and the possible involvement of the major striatal afferent systems were studied in rats by determination of FE-induced changes in striatal levels of preproenkephalin (PPE) mRNA using in situ hybridization. Injection of FE induced a significant increase (60%) in striatal levels of PPE mRNA. This increase was blocked by pretreatment with the D(1) dopamine receptor antagonist SCH-23390 or with the NMDA glutamate receptor antagonist MK-801, or by lesion of the serotonergic system with 5,7-dihydroxytryptamine or p-chlorophenylalanine. In 6-hydroxydopamine lesioned rats, the lesion-induced increase in PPE mRNA levels was not affected by injection of FE, but was reduced by simultaneous serotonergic deafferentation. The results suggest that the serotonergic, glutamatergic, and dopaminergic system interact to increase striatal PPE mRNA levels after FE administration.

Locomotor-activity-induced changes in striatal levels of preprotachykinin and preproenkephalin mRNA. Regulation by the dopaminergic and glutamatergic systems.

The mechanisms by which dopaminergic and glutamatergic inputs interact to regulate striatal neuropeptide expression during physiological motor activity are poorly understood. In this work, striatal expression of preprotachykinin (PPT) and preproenkephalin (PPE) mRNA was studied by in situ hybridization in rats killed 2 h after treadmill running (36 m/min for 20 min). Treadmill running induced a significant increase in the levels of both PPT (60% increase) and PPE (90% increase) mRNA in the striatum of normal rats. The increase in the level of PPT mRNA was blocked in rats previously subjected to nigrostriatal deafferentation (i.e., 6-hydroxydopamine lesion) or pretreated with D1-receptor antagonist SCH-23390 (0.1 mg/kg), the D2-receptor antagonist eticlopride (0.5 mg/kg), or the N-methyl-D-aspartate (NMDA) glutamate receptor antagonist MK-801 (0.1 mg/kg). The running-induced increase in the level of PPE mRNA was blocked in rats pretreated with SCH-23390 or MK-801. Rats subjected to nigrostriatal deafferentation or pretreated with eticlopride showed an increase in PPE mRNA levels (around 150% and 40% increase, respectively), that was enhanced by running (around 230% and 160% increase, respectively). These results suggest that locomotor activity increases, in a NMDA receptor dependent fashion, the excitatory influence of the corticostriatal glutamatergic system on the two populations of striatal projection neurons, as reflected by increases in the levels of PPT and PPE mRNA. The results obtained after dopamine depletion or injection of dopamine receptor antagonists suggest that a concomitant increase in dopamine release may enhance PPT mRNA level in striatonigral neurons via D1 receptors, and reduce PPE mRNA level in striatopallidal neurons via D2 receptors. Additionally, levels of dopamine and glutamate may be regulated by other complex indirect mechanisms.

Sprouting of the serotonergic afferents into striatum after selective lesion of the dopaminergic system by MPTP in adult mice.

Neonatal destruction of the nigrostrial dopaminergic (DA) system with 6-hydroxydopamine leads to serotonergic (5-HT) hyperinnervation of the striatum. However, it is not clear whether this occurs in adult animals. We investigated whether serotonergic sprouting occurs in adult mice subjected to bilateral lesion of the DA system by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The effects of the lesion were evaluated using a new rotarod test and immunohistochemistry. One hundred days after the last MPTP lesion, there was a clear bilateral serotonergic hyperinnervation throughout the striatum. Additionally, those mice showing the highest reductions in striatal tyrosine hydroxylase (TH) immunoreactivity and in rotarod performance showed the highest density of serotonergic innervation (116% increase). The functional consequences of this process in Parkinson's disease and secondary parkinsonism remain to be clarified.

Interaction between the serotonergic, dopaminergic, and glutamatergic systems in fenfluramine-induced Fos expression in striatal neurons.

Fenfluramine (FE) is a halogenated amphetamine derivative used in the treatment of obesity and thought to induce serotonin (5-HT) release from nerve terminals and to reduce re-uptake. However, other pathways may also be involved. In this work, the effects of FE on the major striatal afferent systems, and the possible interactions of these systems in FE-induced striatal expression of Fos, were studied by lesion of the serotonergic and/or dopaminergic system and administration of NMDA glutamate (MK-801) or D1 dopamine (SCH-23390) receptor antagonists. Both the D1 and NMDA receptor antagonists suppressed Fos expression in response to FE almost entirely. FE-induced Fos expression was also dramatically reduced 24 h after 6-hydroxydopamine (6-OHDA) lesion of the dopaminergic system. However, the reduction was not so marked after chronic 6-OHDA lesion, probably due to compensatory changes. Chronic (5,7-dihydroxytryptamine injection, 4 weeks before) or acute (p-chlorophenylalanine injection) lesion of the serotonergic system led to a marked reduction in Fos expression in response to FE (decrease of about 50%). After simultaneous chronic lesion of both serotonergic and dopaminergic systems, a considerable number of Fos-positive nuclei were still observed (decrease of about 70% in the dorsal and dorsomedial regions). The FE-induced expression of Fos was almost totally suppressed (decrease of about 95% in the dorsal and dorsomedial regions) after simultaneous acute lesion. Our results indicate that FE-induced striatal expression of Fos is due in large measure to DA release and dopaminergic stimulation of D1 receptors. However, concurrent stimulation of NMDA glutamate receptors also appears to be essential, and 5-HT release (although not indispensable) doubles striatal Fos expression.

Time course of striatal, pallidal and thalamic alpha 1, alpha 2 and beta 2/3 GABAA receptor subunit changes induced by unilateral 6-OHDA lesion of the nigrostriatal pathway.

Immunocytochemical techniques were used to investigate the distribution and abundance of GABAA receptor subunits (alpha 1, alpha 2 and beta 2/3) in the brains of unilaterally 6-OHDA-lesioned rats. Three and 7 days after lesion, the alpha 2-subunit was significantly more abundant in the lesion-ipsilateral striatum than in the lesion-contralateral striatum; by 4 weeks after lesion, however, no significant between-side differences were observed. Three and 7 days after lesion, the alpha 1-subunit was significantly less abundant in the lesion-ipsilateral globus pallidus than in the lesion-contralateral side; again, this difference disappeared within 4 weeks of lesion. Similarly, alpha 1 was initially less abundant in several relay thalamic nuclei on the lesioned side while alpha 2 was initially more abundant in intralaminar thalamic nuclei on the lesioned side. There were no significant between-side changes for the beta 2/3-subunits. Comparison of non-lesioned and 6-OHDA-lesioned rats revealed significant differences in brain areas which also showed differences on comparison of the lesioned and non-lesioned sides of 6-OHDA-lesioned rats. These results suggest that there is an early adaptation to the lesion, achieved through changes in GABAA receptor abundance. That some of these changes are no longer apparent after 4 weeks is due not only to partial reversion of the changes in the lesioned side but also to compensatory changes in the non-lesioned side.

Treadmill running induces striatal Fos expression via NMDA glutamate and dopamine receptors.

Several non-physiological stimuli (i.e. pharmacological or electrical stimuli) have been shown to induce Fos expression in striatal neurons. In this work, striatal Fos (i.e. Fos-like) expression was studied after physiological stimulation, i.e. motor activity (treadmill running at 36 m/min for 20 min). In rats killed 2 h after the treadmill session, Fos expression was observed in the medial region of the rostral and central striatum, and in the dorsal region of the caudal striatum. Fos expression was prevented by pretreatment with the non-competitive N-methyl-D-aspartate (NMDA) glutamate receptor antagonist MK-801 (0.1 mg/kg) or the D1 dopamine receptor antagonist SCH-23390 (0.1 mg/kg), but not by pretreatment with the D2 receptor antagonist eticlopride (0.5 mg/kg). Thirty-six hours after 6-hydroxydopamine lesion, a considerable reduction in treadmill-induced Fos expression was observed in both sides; however, Fos expression in the lesioned striatum was higher than in the contralateral intact striatum. Several weeks after unilateral 6-hydroxydopamine lesion of the nigrostriatal system, treadmill-induced Fos expression was significantly, but not totally, reduced in the lesioned striatum. Corticostriatal deafferentation also led to considerable reduction in treadmill-induced Fos expression. The present results indicate that exercise induces striatal Fos expression and that, under physiological stimulation, concurrent activation of D1 and NMDA receptors is necessary for such expression to occur. Reduction of Fos expression is practically absolute after acute blockage of these receptors, but not after lesions, possibly due partially to compensatory changes.

Effects of lesions of the nigrostriatal pathway and of nigral grafts on striatal serotonergic innervation in adult rats.

Neonatal destruction of the nigrostriatal dopaminergic system leads to serotonergic hyperinnervation of the striatum. However, it is not clear whether this occurs in adult animals. We investigated whether serotonergic sprouting occurs in adult animals, and also studied the effects of prior or subsequent implantation of dopamine-rich intrastriatal grafts. One group of adult rats received maximal 6-hydroxydopamine lesions. Other rats received maximal lesions and intrastriatal grafts 2 months later, or vice versa. The lesioned non-grafted rats showed clear serotonergic hyperinnervation throughout the striatum ipsilateral to the lesion. Intrastriatal grafts did not prevent or revert this serotonergic hyperinnervation, and were themselves densely innervated by serotonergic fibers. Serotonergic neurons usually present in the grafted cell suspension also contributed to the serotonergic innervation of the graft and the surrounding striatum.

GABA(A) receptor subunit expression in intrastriatal striatal grafts comparison between normal developing striatum and developing striatal grafts.

Expression of the alpha1, alpha2 and beta2/3 GABA(A) receptor subunits in maturing cell-suspension striatal grafts and in normal developing striatum was studied by immunocytochemistry. During normal postnatal development, the alpha1 subunit was present in the striatum only at very low density, while the alpha2 and beta2/3 subunits were present with a patchy distribution, in some patches at high density. Double-staining techniques indicated that DARPP-32 (a marker of striatal projection neurons) was not colocalized with alpha1, but was present in some beta2/3-positive areas and all alpha2-positive areas. In striatal grafts, alpha1 immunoreactivity was first detected 2 weeks post-grafting (p.g.), and by 3-10 weeks p.g. the pattern was similar to that observed in mature grafts (1 year p.g.), in which alpha1-immunopositive patches surrounding DARPP-32-positive (i.e. striatum-like) areas are observed. Alpha2 and beta2/3 immunoreactivity was observed within the first week p.g., and by 3-10 weeks p.g. was similar to that observed in mature grafts (i.e. immunoreactivity throughout the graft but with patches of different intensity). During graft maturation there was a marked decline in alpha2 immunoreactivity in DARPP-32-negative areas, as is observed during normal development of the globus pallidus and ventral pallidum. Interestingly, alpha1- and beta2/3-positive fibers (perhaps mostly dendrites) entered DARPP-32-positive patches from DARPP-32-negative areas. This study indicates that the time course of expression of GABA(A) receptor subunits in grafted striatal neurons, closely matches that of morphological maturation of the transplant, that of the development of functional synaptic activity and that of GABA(A) receptor subunit immunoreactivity in normal developing striatum. Our results also suggest that there are significant interactions between DARPP-32-positive and DARPP-32-negative areas with respect to the expression of GABA(A) receptors, and support the suggestion that miniature 'striatopallidal systems' may develop within grafts; such interactions may be important for the functional integration of striatal grafts with the host brain.

Intrathalamic implants of GABA-releasing polymer matrices reduce motor impairments in rats with excitotoxically lesioned striata.

Current models of basal ganglia disorders suggest that the choreoathetosis is the end result of reduced GABAergic inhibition of the motor thalamus. GABA-releasing polymer matrices or control matrices without GABA were implanted either unilaterally or bilaterally in the vicinity of the ventromedial thalamic nucleus of normal rats and of rats with unilateral or bilateral excitotoxic striatal lesions (rat model of Huntington's disease), to study the effects of these GABA-releasing matrices on amphetamine-induced rotational behavior (unilateral implants in unilaterally lesioned rats) and on overnight spontaneous locomotor activity (bilateral implants in bilaterally lesioned rats). Unilateral implants led to a reduction (about 25%) in motor asymmetry; the response was transitory, probably because of the exhaustion of GABA release by the matrix. Some rats showed a more marked and permanent reduction of motor asymmetry, but this was probably due to lesion of the ventromedial nucleus or its thalamocortical projection. Bilateral implants of GABA-releasing matrices (but not control matrices) led to a marked (about 65%) but again transitory reduction in the locomotor hyperactivity induced by bilateral striatal lesion. These results suggest that implantation of a GABA-releasing source may be an effective alternative to intrathalamic fetal-tissue grafts or lesions as an experimental approach to the treatment of hyperkinetic movement disorders.

GABAA receptor alpha 1-subunit-immunopositive neurons in the rat striatum.

The morphological characteristics and distribution of striatal neurons expressing the GABAA receptor alpha 1-subunit were investigated following immunolabeling with an alpha 1-subunit-specific antibody. alpha 1-Immunopositive striatal neurons are relatively scarce. Those located in the dorsal striatum are small and have rounded somata and numerous dendrite varicosities. alpha 1-Immunoreactive neurons with these characteristics were likewise observed in the ventral striatum, which also contained large pyramidal neurons with smooth dendrites and polygonal neurons with spiny dendrites. A fourth neuron type (oval neurons with dendrites oriented in bipolar fashion) was found in the lateral striatum. All four neuron types were observed in the rostral, central and caudal striatum. Double-labeling experiments using an antibody specific for DARPP-32 (a cell-level indicator of dopamine D1-like receptors) and the alpha 1 antibody showed a complete lack of colocalization of these two markers in striatal neurons.

Previous dopaminergic innervation is not necessary for the development of dopamine supersensitivity in rat striatal neurons.

The development of supersensitivity to dopamine by striatal neurons is widely considered to be a response to interruption of dopaminergic synaptic function. However, it is not clear whether pre-existing dopaminergic innervation is in fact necessary for supersensitivity to develop. In this study, rat foetal striatal cells were obtained prior to their innervation by dopaminergic fibres, and grafted into the cortex or thalamus of adult rats. In the absence of dopaminergic innervation, the grafts developed and became organized into striatum-like and non-striatum-like patches. Both in animals with intrathalamic and in animals with intracortical grafts, systemic administration of a low dose of apomorphine (0.25 mg/kg) induced intense fos expression in striatum-like patches not innervated by dopaminergic fibres. These results indicate that pre-existing dopaminergic innervation is not required for the development of supersensitivity, and that dopaminergic innervation is necessary to develop an adequate synergistic interaction between D1 and D2 receptors.

Time course of striatal changes induced by 6-hydroxydopamine lesion of the nigrostriatal pathway, as studied by combined evaluation of rotational behaviour and striatal Fos expression.

Changes taking place after unilateral 6-hydroxydopamine lesion of the dopaminergic nigrostriatal system have been studied by performing spontaneous, amphetamine-induced and apomorphine-induced rotational behaviour testing and tyrosine hydroxylase (TH) and Fos protein immunohistochemistry in the same rats. Apomorphine at a low dosage (0.25 mg/kg) induced contraversive rotation and supersensitive striatal Fos expression that were detected 24-48 h post-lesion and gradually increased in magnitude. Twenty-four hours after lesion, both high (5 mg/kg) and low doses (0.5 mg/kg) of D-amphetamine induced contraversive rotation and intense striatal Fos activation on the denervated side; however, only the higher dose induced Fos on the normal side. Two, 3 and 4 days after lesion, 0.5 mg/kg amphetamine induced contraversive rotation, but 5 mg/kg induced transitory contraversive rotation which switched to ipsiversive. In the normal striatum, only high doses of amphetamine induced Fos, but Fos induction in the denervated striatum was similar with both doses: areas showing severely decreased TH immunoreactivity still showed considerable Fos immunoreactivity, and some areas still showing TH immunoreactivity had higher Fos density than in the normal side. Seven and 14 days after lesion the loss of TH immunoreactivity and apomorphine-induced supersensitive Fos expression were more evenly distributed, and amphetamine induced only ipsiversive rotation and a low density of Fos-positive nuclei in the denervated striatum. These results indicate that the severe and progressive loss of dopaminergic terminals is counteracted by an early and rapidly progressing dopamine supersensitivity, together with a higher susceptibility to drug-induced dopamine release. This explains the apparently paradoxical contraversive rotation induced by amphetamine during the first week post lesion. However, experiments involving successive drug injections indicated that only the first amphetamine injection releases dopamine from the lesioned terminals.

Semiquantitative immunocytochemical analysis of GABAA receptor subunit expression in the rat neostriatum.

A semiquantitative immunogold technique was used to investigate the levels of expression of specific GABAA receptor subunits in different regions (dorsolateral, dorsomedial and ventromedial regions) of the rat striatum. The results indicate that the subunits studied can be classified into three groups on the basis of their labelling density in the striatum: alpha 1 and alpha 3 (labelling density of less than 100 gold particles per 1000 microns2), gamma 2 and delta (between 100 and 200 particles per 1000 microns2), and alpha 2 and beta 2/3 (more than 300 particles per 1000 microns2). The alpha 1 and alpha 3 subunits are about 35% more abundant in the dorsal than in the ventral striatum, while the beta 2/3 and gamma 2 subunits are about 40% more abundant in the medial than in the lateral striatum. The alpha 2 and delta subunits did not show significant regional differences in abundance. The present data are consistent with the possibility that there are regional variations in the relative abundances of different GABAA receptor subtypes in the rat striatum.

Cortical stimulation induces Fos expression in striatal neurons via NMDA glutamate and dopamine receptors.

Cortical electrical stimulation has been shown to induce dense and widespread Fos expression throughout the ipsilateral and contralateral striatum. This raises interest for studying the mechanisms underlying the regulation of striatal neuron activity by cortical afferents, and for elucidating the interactions with other systems. However, the receptors mediating cortical-stimulation-induced expression of Fos in striatal neurons have not been identified. This was studied in the work reported here by stimulating the cortex after administration of glutamate or dopamine receptor antagonists, or after 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal dopaminergic system. Pretreatment with the non-competitive N-methyl-D-aspartate (NMDA) glutamate receptor antagonist MK-801 led to a marked reduction in the stimulation-induced density of Fos-immunoreactive nuclei in both the medial (about 80% reduction) and lateral (about 50-60% reduction) striatum. Preadministration of the D1-selective dopamine antagonist SCH-23390 alone or in combination with the D2-selective dopamine antagonist eticlopride led to a reduction in the stimulation-induced density of Fos-positive nuclei of about 60-65% in the lateral striatum, but no significant change in the medial region. The effects of 6-OHDA lesion were less pronounced, and the stimulation-induced density of Fos-immunoreactive nuclei decreased by only about 25% in the lateral region. These results indicate that both dopamine and NMDA glutamate receptors are involved in the induction of Fos by cortical stimulation, and support the hypothesis that cortex-dopamine interactions in the lateral striatum may be functionally different from those in the medial striatum.

Intrathalamic striatal grafts survive and affect circling behaviour in adult rats with excitotoxically lesioned striatum.

Current models of basal ganglia disorders suggest that choreoathetosis is the end result of reduced GABAergic inhibition of the motor thalamus. Graft-derived release of GABA from intrastriatal striatal grafts has also been reported. In the present work, cell suspension grafts from embryonic day 14-15 rat striatal primordia were implanted close to the ventromedial thalamic nucleus to investigate whether they can develop and survive in this ectopic location, and whether they induce changes in the circling behaviour of the host. The grafts were implanted either in normal rats or in rats whose striatum had been lesioned with ibotenic acid. These grafts were implanted either ipsilateral or contralateral to the lesioned striatum. Additionally, some rats received intrastriatal grafts, and lesioned but non-grafted rats and lesioned rats that had received injections of saline or of cell suspensions from fetal spinal cord in the thalamus were used as control. Four to eight months after transplantation, circling behaviour after amphetamine or apomorphine injection was evaluated. Serial sections were stained with Cresyl Violet and studied immunohistochemically with antibodies against DARPP-32 (dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein, as striatal marker), Fos protein, glutamate decarboxylase (67,000 mol. wt), glutamate decarboxylase (65,000 mol. wt) and GABA. Cresyl Violet sections showed that the intrathalamic striatal grafts developed into tissue masses resembling those observed in intrastriatal striatal grafts. DARPP-32 immunohistochemistry revealed that the grafts were composed of DARPP-32 immunoreactive (striatum-like) and DARPP-32-negative patches. The intrathalamic grafts of rats which had received a low dose of apomorphine (0.25 mg/kg) 2 h before perfusion showed clusters of intensely Fos-immunoreactive nuclei throughout the transplant, indicating that these cells had developed dopamine receptors and supersensitivity to dopamine agonists. Double Fos and DARPP-32 immunohistochemistry revealed that the Fos-positive nuclei were located in the striatum-like areas. Finally, the intrathalamic grafts also contained neurons immunoreactive to GABA and glutamate decarboxylase (65,000 and 67,000 mol. wt). Rats that had received intrathalamic grafts contralateral to the lesioned striatum (i.e. contralateral to the lesion-induced turning direction) showed a significant reduction of circling both after amphetamine (78% reduction) or apomorphine (77% reduction) injection. Rats that had received grafts ipsilateral to the lesioned striatum showed a 75% decrease in amphetamine-induced circling, but no significant change in apomorphine-induced circling. No significant drug-induced circling was observed in non-lesioned and grafted rats. Sham grafting (saline) or grafting of weakly GABAergic tissue (fetal spinal cord) had no significant effects on lesion-induced circling behaviour.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of the inter-alpha-trypsin inhibitor polymorphism in west Africa: description of a new allele, ITI*7.

Inter-alpha-trypsin inhibitor (ITI) phenotypes were classified in the West African population of Cabo Verde by polyacrylamide gel isoelectric focusing, followed by immunofixation and silver staining. Gene frequencies of the alleles ITI*1, ITI*2, ITI*3, and ITI*4 were calculated to be 0.532, 0.153, 0.307 and 0.002, respectively. A new rare allele, ITI*7, was found, providing evidence for further genetic variability of the ITI protein. The ITI*7 allele frequency has been determined to 0.006. The assumption that allele ITI*3 may be used to characterize populations of African origin is supported by our data.

Distribution of ITI phenotypes in the Iberian Peninsula: Negative correlation between ITI*3 frequencies and latitude in human populations.

The molecular phenotypes of ITI were analysed in samples from seven populations of the Iberian Peninsula (Galicia, Central Portugal, Castilla-Leon, Castilla-La Mancha, Extremadura, Western Andalusia, and Eastern Andalusia) by means of IEF in ultrathin layer polyacrylamide gels followed by immunofixation-silver staining. The observed ITI*3 frequencies exhibit the highest values thus far reported in European populations. Regression analysis between ITI*3 frequencies and latitude reveals a negative correlation (r = -0.8798, P < 0.001). © 1994 Wiley-Liss, Inc.