The apoptotic effect of somatostatin analogue SMS 201-995 on human lymphocytes

The antiproliferative effect of a synthetic octapeptide, somatostatin analogue SMS 201-995 (SMS), and its capacity to bind were evaluated on human peripheral blood lymphocytes (PBL) activated by phytohemoagglutinin (PHA).We then addressed our work to investigate if SMS inhibits PHA activation of PBL by a cytostatic rather than a cytotoxic mechanism. Consequently, we studied the cell cycle distribution and the activation of caspase-3, measuring the presence of the cleavage product of poly(ADP-ribose) polymerases (PARP), and we evaluated the presence of apoptotic DNA by using a monoclonal antibody specific for the single-stranded regions of DNA. All our results indicate that SMS induces apoptosis in activated lymphocytes.     

Long-term effects of treatment with SMS 201-995 on sleep apnea syndrome associated with acromegaly

A 60-year-old woman with acromegaly associated with sleep apnea was treated with the somatostatin analogue SMS 201-995 (Sandoz) for several months. Growth hormone levels were normalized and a rapid improvement in sleep apnea was controlled with polygraphic nocturnal monitoring. Hypophysectomy seems to have variable effects on sleep apnea in acromegaly. The origin of obstructive apnea in acromegaly is therefore unclear.     

Induction of experimental allergic encephalomyelitis in a low-susceptible Albino Oxford rat strain by somatostatin analogue SMS 201-995

OBJECTIVE: The effect of the somatostatin analogue SMS 201-995 (octreotide; OCT) on the course of experimental allergic encephalomyelitis (EAE) in the relatively resistant Albino Oxford (AO) strain of rats was studied. METHODS: Animals were actively immunized with bovine brain homogenate in complete Freund's adjuvant. OCT was given subcutaneously in the hind legs on days 7, 8 and 9 after immunization, at a dose of 3 x 5 microg/kg/day. Rats in control groups were treated with saline or were left untreated. EAE was scored clinically and immunophenotypically, estimating by flow cytometry the changes in the popliteal lymph nodes (PLN) and spleen and monitoring immunohistologically the brain sections of rats recovered from disease. RESULTS: In control AO rats, EAE was induced in only 2 of 22 rats (9%). In OCT-treated rats, however, EAE developed in 11 of 20 rats (55%), in comparison with 3 of 17 saline-treated animals (17%) (p <0.05). In PLN of OCT-treated rats during the clinical course of EAE, a decreased proportion of OX8+ cells was seen, followed by increases in OX39+ and W3/25+ cells on days 17 and 26. In spleen, OCT decreased the proportion of OX1+, OX39+ and OX8+ cells (on days 12 and/or 17), and increased the proportion of OX39+ cells on days 26 and 31. In the brain sections of saline-treated rats recovered from EAE, numerous Mac-1+, Mac-3+ and OX8+ cells were found. These cells were, however, absent in OCT-treated rats; instead, several W3/25+ cells were noticed. CONCLUSIONS: These data imply that OCT increases the susceptibility of AO rats to EAE, interfering with specific and/or nonspecific defense mechanisms operating in both the initial and recovery phase of EAE.     

Immunochemical and immunocytochemical characterization of cholinergic markers in human peripheral blood lymphocytes

Cholinergic markers and the expression of M(2)-M(5) muscarinic cholinergic receptor subtypes were investigated in human peripheral blood lymphocytes by Western blot analysis and immunocytochemistry. The totality of peripheral blood lymphocytes express acetylcholine (ACh) immunoreactivity, choline acetyltransferase (ChAT), acetylcholinesterase (AChE), vesicular ACh transporter (VAChT) and M(2)-M(5) muscarinic cholinergic receptor protein immunoreactivity. Western blot analysis performed independently on T and B lymphocytes using anti-ChAT and anti-AChE antibodies revealed labelling of single bands of approximately 68-70 and 70 kDa, respectively, whereas VAChT was bound to two bands of approximately 80 and 45 kDa. The pattern of immunoblotting was similar in membranes of lymphocytes and striatum, used as a reference brain tissue. Western blot analysis using anti M(2)-M(5) receptor antibodies revealed labelling of single bands of approximately 55, 85-90, 50 and 81 kDa, respectively. Confocal laser immunofluorescence showed the localization of ACh and VAChT immunoreactivity in punctiform areas likely corresponding to cytoplasmic vesicles. ChAT and AChE were diffused to the cytoplasm and plasma membrane. Muscarinic receptor immunoreactivity was located in lymphocyte plasma membrane. Although the role of lymphocyte cholinergic system is still unclear, the demonstration of cholinergic markers in T and B human blood lymphocytes supports the view that a cholinergic systems may contribute to the regulation of immune function. The characterization of these cholinergic markers may also contribute to define if their evaluation can be used for assessing the status of brain cholinergic system.     

Growth hormone rebound after cessation of sms 201-995 treatment in acromegaly

We studied a 42-year-old woman who had persistent active acromegaly despite conventional therapies. She was treated for 6 months with SMS 201-995. Her mean plasma growth hormone GH values decreased during treatment from 9.1 +/- 1.2 to 6.6 +/- 1.2 micrograms/L. One month after the withdrawal of SMS 201-995, the plasma GH level increased to 24.4 micrograms/L (P less than 0.001). This elevation was clinically silent and transitory, as GH levels decreased 8 months later to 6.9 +/- 1.3 micrograms/L. Furthermore, at the beginning of therapy, her intractable headache was completely relieved; however, it progressively resumed under therapy. In conclusion, cessation of SMS 201-995 may be followed in some acromegalic patients by a rebound of plasma GH levels. This rebound suggests that SMS 201-995 decreases GH levels by an inhibition of its release from the pituitary. Furthermore, SMS 201-995 may relieve intractable headache in some acromegalic patients, but tolerance to the analgesic effect may develop.     

Dopamine D1-like receptor subtypes in human peripheral blood lymphocytes.

Molecular biology studies have shown that human peripheral blood lymphocytes express a dopamine D5 receptor, whereas no information is available on dopamine D receptor, the other dopamine D1-like receptor subtype. Radioligand binding assay investigations with the nonsubtype selective dopamine D1-like receptor antagonist [3H]SCH 23390 as radioligand have suggested the presence of a dopamine D5 receptor in human peripheral blood lymphocytes. However, so far no evidence was provided as whether or not human peripheral blood lymphocytes express a dopamine D1 receptor. In this study, we have investigated dopamine D1 and D5 receptor mRNA and the influence of antibodies against dopamine D1 and D5 receptors on [3H]SCH 23390 binding to intact human peripheral blood lymphocytes. The two receptors were also analyzed by immunocytochemistry. Dopamine D5 receptor, but not D1 mRNA, was detected in human peripheral blood lymphocytes. Anti-dopamine D5 receptor antibodies, but not anti-dopamine D1 receptor antibodies, significantly decreased [3H]SCH 23390 binding to human peripheral blood lymphocytes. A dark-brown immunoreactivity was visualized in cytospin centrifuged human peripheral blood lymphocytes exposed to anti-dopamine D5, but not to anti-dopamine D1 receptor antibodies. These data collectively indicate that dopamine D5 receptor is the only dopamine D1-like receptor subtype expressed by human peripheral blood lymphocytes.     

Structural determinants of the binding of gliadin fragments to human peripheral blood lymphocytes

A series of alpha-gliadin fragments, structurally related to alpha-gliadin-(43-49), were synthesized. The effect of these fragments and beta-casomorphin and naloxone on the steady-state binding of [125I]-alpha-gliadin-(43-49) to human peripheral blood lymphocytes was investigated. In an attempt to correlate the binding data with the conformation of the peptides, their circular dichroism spectra were measured in both trifluorethanol and aqueous solution. It was found that there is a striking correlation between the results of the binding studies and the chiroptical properties of the gliadin fragments. The presence of N-terminal tyrosine and the tendency of the peptides to adopt periodic, 310 helix-like secondary structure appear to be crucial for the binding to human peripheral blood lymphocytes.     

Alprazolam enhances the antibacterial activity exerted by normal human peripheral blood lymphocytes.

The effects of two benzodiazepines, diazepam and alprazolam, have been evaluated on the in vitro antibacterial activity exerted by human peripheral blood lymphocytes (PBL). Results demonstrate that diazepam has no influence on this PBL function, while alprazolam is able to enhance this activity in six out of nine normal donors considered. The possible therapeutical implications of these data are discussed.     

Identification of dopamine plasma membrane and vesicular transporters in human peripheral blood lymphocytes.

Plasma membrane dopamine transporter (DAT), vesicular monoamine transporters (VMAT) type-1 and -2 and the expression of the dopaminergic markers dopamine and tyrosine hydroxylase were assessed in membranes and/or in cytospin centrifuged human peripheral blood lymphocytes. The radiolabeled DAT ligand [3H]GBR12935 was bound to peripheral lymphocytes in a manner consistent with the specific binding to a dopamine uptake system, with a dissociation constant similar to that found in striatum, but with a lower density of binding sites. On the other hand, no specific binding occurred in cerebellum used as a test tissue not expressing DAT. Western blot analysis using antibodies raised against amino or carboxy terminus of DAT or against VMAT-1 or VMAT-2 revealed labeling of single bands of approximately 76, 55 or 68 KDa, respectively, displaying similar migration characteristics in lymphocytes and test tissues used for comparison. Immunofluorescence revealed that anti-dopamine, anti-tyrosine hydroxylase, anti-DAT, anti-VMAT-1 and anti-VMAT-2 antibodies labeled the total population of cytospin-centrifuged lymphocytes mounted on microscope slides. Confocal laser microscopy demonstrated that dopamine and VMAT-2 immunoreactivity was developed mainly in cytoplasmic punctiform areas likely corresponding to vesicles and to a lower extent was associated to plasma membrane. Tyrosine hydroxylase immunoreactivity was diffused to cytoplasm and to plasma membrane of lymphocytes, whereas DAT and VMAT-1 immunoreactivity were located almost exclusively in lymphocyte plasma membrane and cytoplasm, respectively. Lymphocyte DAT characterized in this study has probably functional relevance as [3H]dopamine was taken up by intact lymphocytes and uptake was inhibited specifically by compounds known to affect dopamine transport. These findings indicate that human peripheral blood lymphocytes possess DAT plasma membrane and VMAT-1 and VMAT-2 transporters. Increasing evidence indicates that dopamine transporter changes may be related to neuronal injury. In view of this assessment of lymphocyte DAT and VMAT transporters can be considered for identifying pathologies characterized by impaired dopaminergic neurotransmission.     

Age-dependent changes in the expression of dopamine receptor subtypes in human peripheral blood lymphocytes

The pharmacological profile and the density of dopamine D₃ and D₅ receptor subtypes expressed by human peripheral blood lymphocytes of subjects of different ages (ranging from 20 to 75 years) were assessed using radioligand binding techniques. Dopamine D₃ receptor was assayed with [³H]7-hydroxy-N,N-di-n-propyl-2-aminotetraline ([³H]7-OH-DPAT) as a ligand. Dopamine D₅ receptor was assayed using [³H][R]-(+)-(-chloro-2,3,4,5,tetrahydro-5-phenyl-1H-3-benzazepin-al-hemimaleate) ([³H]SCH 23390) as a ligand. The affinity and the pharmacological profile of [³H]7-OH-DPAT and [³H]SCH 23390 at dopamine D₃ and D₅ receptor, respectively, were similar in subjects of different ages. The density of dopamine D₃ receptor binding sites was slightly decreased in subjects of 30–39 years in comparison with younger individuals. A remarkable loss of dopamine D₃ receptor was then found between 40 and 49 years of age in comparison with younger subjects. A further slight decrease was noticeable between 50 and 59 years of age. The number of [³H]7-OH-DPAT binding sites was then stabilized after 60 years of age. The density of dopamine D₅ receptor binding sites did not show age-dependent changes. The above findings indicate the occurrence of a decline in the density of lymphocyte dopamine D₃ but not D₅ receptor between adult and mature subjects. The possibility that dopamine D₃ receptor assay in peripheral blood lymphocytes may represent a tool for investigating dopamine receptor function in aging and age-related neurological disorders is discussed.     

Nerve growth factor receptor gene expression in human peripheral blood lymphocytes in aging.

Nerve growth factor (NGF) has a modulating effect on immune function, which may occur as a consequence of binding to the NGF receptor (NGF-R). To determine if mRNA for the gene coding for p75NGFR (low affinity NGF-R) is present in lymphocytes, Northern blot analysis of mRNA from human peripheral blood lymphocytes (PBL) and purified T lymphocytes was initiated using cDNA probe for human p75NGFR. p75NGFR mRNA was present in PBL and T lymphocytes, and the mRNA in response to phytohemagglutinin stimulation showed maximum levels at 14 hr of stimulation. p75NGFR mRNA content when analyzed in PBL and T cells from volunteers of various ages showed that p75NGFR mRNA expression does not change with the age of the cell donor.     

Autosomal dominant cerebrovascular amyloidosis: Properties of peripheral blood lymphocytes

Selected Properties of Peripheral blood lymphocytes (PBLs) from five ambulatory affected individuals of a kindred with autosomal dominant cerebrovascular amyloidosis were studied. The percentage of PBLs bearing surface membrane immunoglobulin (SmIg + cells) was increased in the patient group (30 ± 3% versus 20 ± 2%; p < 0.05). The Percentage of PBLs forming early and total E-rosettes was comparable in patient and control groups. Mitogenic response to concanavalin A (Con A) was suggestively reduced in the patient group, measured both by total ³Hthymidine incorporation and by comparison of stimulation indices. Mitogenic response to phytohemagglutinin and pokeweed was comparable in the two groups. Capping of Con A by PBLs was significantly reduced in the patient group compared with the controls (13 ± 1% versus 26 ± 2%; p < 0.01). The findings of reduced Con A response and increased SmIg⁺ cells support the hypothesis that immune dysfunction contributes to the development of amyloidosis. The reduced capping suggests altered membrane properties in this autosomal dominant disorder.     

T lymphocytes in peripheral blood from patients with neurological diseases

Absolute and relative numbers of T lymphocytes in peripheral blood were determined in 19 different groups of patients with neurological diseases, using the E-rosette test. A significantly decreased total number of T lymphocytes was found in the following groups: acute Guillain-Barré syndrome (GBS), active multiple sclerosis (MS), and malignant cerebral tumor. A less pronounced and not significant reduction was observed in patients with amyotrophic lateral sclerosis (ALS). In all other groups of patients normal total numbers of T lymphocytes were found, thus indicating that the decrease in T lymphocyte counts in GBS, MS and malignant cerebral tumors is not due to nonspecific injury of nerve tissue.     

Catecholamine levels in peripheral blood lymphocytes from multiple sclerosis patients

Circumstantial evidence suggests the involvement of sympathoadrenergic mechanisms in the progress of multiple sclerosis (MS).We studied peripheral blood lymphocytes from MS patients. The levels of dopamine (DA), norepinephrine (NE), epinephrine (E) and their metabolites in extracts of lymphocytes from 58 MS patients and 19 healthy controls were measured by using capillary electrophoresis. The MS patients were divided into clinical subgroups: a laboratory-supported definitive (first-attack) MS group, and a relapsing-remitting (RR) group in remission.The peripheral blood lymphocyte level of epinephrine was significantly higher in the first-attack MS patients (p=0.028) than in the controls. However, the norepinephrine levels were significantly (p=0.027) lower in the RR patients in remission. The catecholamines are known to be able to affect the lymphocyte activity, both by stimulation and by immunosuppression. Our results suggest that the catecholamines are important regulators of lymphocyte activation in MS, and of potential importance as concerns new diagnostic and therapeutic methods.     

Dopamine transporter immunoreactivity in peripheral blood lymphocytes in Parkinson's disease

Summary. There is increasing interest in the identification of biological markers for the early diagnosis of Parkinson's disease (PD). Previous studies indicate changes of dopamine content, tyrosine hydroxylase immunoreactivity and dopamine receptors in peripheral blood lymphocytes (PBL) in PD. Here we demonstrate a reduction of dopamine transporter immunoreactivity in PBL in the early clinical stages of the disease. These findings contribute to our understanding of the peripheral dopamine system in PD. Received November 16, 2000; accepted March 19, 2001     

Reduced dopamine in peripheral blood lymphocytes in Parkinson's disease.

The early clinical symptoms of Parkinson's disease (PD) may be difficult to perceive and are frequently overlooked. Thus, interest has focused on the identification of biological or instrumental markers that may contribute to the early diagnosis of PD, with the aim of introducing neuroprotective therapies at the very start of illness. Impairment of nigrostriatal dopamine transmission can be visualized in vivo by functional imaging techniques, but these are rather complex and expensive examinations, available only in selected institutions. Here we show that dopamine content and tyrosine hydroxylase immunoreactivity are reduced in peripheral blood lymphocytes (PBL) in the early stages of PD. These data suggest that PBL may represent a simple and useful tool with which to identify precociously dopamine impairment in PD.     

Radioligand binding assay of M1-M5 muscarinic cholinergic receptor subtypes in human peripheral blood lymphocytes.

Analysis of lymphocyte muscarinic cholinergic receptors using quantitative techniques such as radioligand binding assay is made difficult due to the low density of these sites and the lack of subtype-specific selectivity of most available muscarinic ligands. In this study, a combined kinetic and equilibrium labeling technique recently developed for brain tissue was used for labeling the five muscarinic cholinergic receptor subtypes in intact human peripheral blood lymphocytes. No specific muscarinic M1 receptor binding was detectable in human peripheral blood lymphocytes using [3H]-pirenzepine as a ligand. Labeling of M2-M5 muscarinic receptors using [3H]N-methyl-scopolamine (NMS) by occluding various receptor subtypes with muscarinic antagonist and mamba venom resulted in the labeling of M2-M5 receptors in brain as well as in human peripheral blood lymphocytes. The relative density of different receptor subtypes was M3 > M5 > M4 > M2. The development of a reproducible technique for assaying muscarinic cholinergic receptor subtypes expressed by human peripheral blood lymphocytes may contribute to clarify their role in lymphocyte function.