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1.
Neuropharmacology ; 95: 83-99, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25724083

RESUMO

RATIONALE: The role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. EXPERIMENTAL APPROACH: LSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs). RESULTS: Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-ß,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPßS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,ß-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 >> P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R. CONCLUSIONS: Purines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology.


Assuntos
Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Receptores Purinérgicos/metabolismo , Plexo Submucoso/efeitos dos fármacos , Plexo Submucoso/fisiologia , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Colectomia , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/fisiologia , Humanos , Imuno-Histoquímica , Purinérgicos/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Imagens com Corantes Sensíveis à Voltagem
2.
Am J Physiol Gastrointest Liver Physiol ; 294(2): G554-66, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18079280

RESUMO

Based on findings in rodents, we sought to test the hypothesis that purinergic modulation of synaptic transmission occurs in the human intestine. Time series analysis of intraneuronal free Ca(2+) levels in submucosal plexus (SMP) from Roux-en-Y specimens was done using Zeiss LSM laser-scanning confocal fluo-4 AM Ca(2+) imaging. A 3-s fiber tract stimulation (FTS) was used to elicit a synaptic Ca(2+) response. Short-circuit current (I(sc) = chloride secretion) was recorded in mucosa-SMP in flux chambers. A distension reflex or electrical field stimulation was used to study I(sc) responses. Ca(2+) imaging was done in 1,222 neurons responding to high-K(+) depolarization from 61 surgical cases. FTS evoked synaptic Ca(2+) responses in 62% of recorded neurons. FTS caused frequency-dependent Ca(2+) responses (0.1-100 Hz). FTS Ca(2+) responses were inhibited by Omega-conotoxin (70%), hexamethonium (50%), TTX, high Mg(2+)/low Ca(2+) (< or = 100%), or capsaicin (25%). A P2Y(1) receptor (P2Y(1)R) antagonist, MRS-2179 or PLC inhibitor U-73122, blocked FTS responses (75-90%). P2Y(1)R-immunoreactivity occurred in 39% of vasoactive intestinal peptide-positive neurons. The selective adenosine A(3) receptor (AdoA(3)R) agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IBMECA) caused concentration- and frequency-dependent inhibition of FTS Ca(2+) responses (IC(50) = 8.5 x 10(-8) M). The AdoA(3)R antagonist MRS-1220 augmented such Ca(2+) responses; 2-Cl-IBMECA competed with MRS-1220. Knockdown of AdoA(1)R with 8-cyclopentyl-3-N-(3-{[3-(4-fluorosulphonyl)benzoyl]-oxy}-propyl)-1-N-propyl-xanthine did not prevent 2-Cl-IBMECA effects. MRS-1220 caused 31% augmentation of TTX-sensitive distension I(sc) responses. The SMP from Roux-en-Y patients is a suitable model to study synaptic transmission in human enteric nervous system (huENS). The P2Y(1)/Galphaq/PLC/inositol 1,3,5-trisphosphate/Ca(2+) signaling pathway, N-type Ca(2+) channels, nicotinic receptors, and extrinsic nerves contribute to neurotransmission in huENS. Inhibitory AdoA(3)R inhibit nucleotide or cholinergic transmission in the huENS.


Assuntos
Sistema Nervoso Entérico/fisiologia , Receptores Purinérgicos/fisiologia , Transmissão Sináptica/fisiologia , Compostos de Anilina , Cálcio/metabolismo , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/metabolismo , Estimulação Elétrica , Sistema Nervoso Entérico/efeitos dos fármacos , Corantes Fluorescentes , Humanos , Microscopia Confocal , Fibras Nervosas/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Obesidade/metabolismo , Quinazolinas/farmacologia , Receptores Purinérgicos/efeitos dos fármacos , Receptores Purinérgicos P2/fisiologia , Receptores Purinérgicos P2Y1 , Plexo Submucoso/citologia , Plexo Submucoso/efeitos dos fármacos , Plexo Submucoso/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Triazóis/farmacologia , Fosfolipases Tipo C/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Xantenos
3.
Infect Immun ; 70(8): 4292-301, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12117938

RESUMO

Experimental and clinical studies suggest that influenza A virus promotes Streptococcus pneumoniae-induced otitis media; however, the mechanism underlying this synergistic interaction has not been completely defined. In this study, glycoconjugate expression patterns were evaluated on the cell surface in the chinchilla eustachian tube (ET) lumen of a cohort challenged intranasally (i.n.) with S. pneumoniae type 6A, which is predominantly transparent and a cohort with an antecedent influenza A virus infection, followed by i.n. inoculation with S. pneumoniae. The labeling patterns obtained with six lectin probes revealed that the binding of Bandeiraea simplicifolia lectin II, succinylated wheat germ agglutinin, and peanut agglutinin were significantly increased in the lumenal surface of the ET in the cohort infected with both pathogens compared to the cohort inoculated with only S. pneumoniae, which indicated that N-acetylglucosamine (GlcNAc) and D-galactose residues were exposed. A significant decreased labeling with Sambucus nigra agglutinin in the combined influenza A virus and pneumococcus infection cohort suggested that there were few sialic acid residues remaining in the ET epithelium. In addition, the colonial opacity of S. pneumoniae during the disease course was examined. The opaque phenotype was predominant among the pneumococcus isolates from the middle-ear fluid in the cohort infected with the both pathogens. Together, these data suggest that the synergic effect of influenza A virus and S. pneumoniae on the changes of the carbohydrate moieties in the ET epithelium and that the selection of the opaque variant may facilitate the pneumococcal invasion of the middle ear.


Assuntos
Acetilglucosamina/metabolismo , Tuba Auditiva/metabolismo , Galactose/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Otite Média/metabolismo , Lectinas de Plantas , Infecções Pneumocócicas/metabolismo , Animais , Metabolismo dos Carboidratos , Membrana Celular/metabolismo , Chinchila , Modelos Animais de Doenças , Orelha Média/metabolismo , Orelha Média/microbiologia , Orelha Média/virologia , Humanos , Influenza Humana/complicações , Lectinas/metabolismo , Nasofaringe/microbiologia , Otite Média/complicações , Otite Média/microbiologia , Aglutinina de Amendoim/metabolismo , Fenótipo , Infecções Pneumocócicas/complicações , Infecções Pneumocócicas/microbiologia , Ácidos Siálicos/metabolismo , Streptococcus pneumoniae/crescimento & desenvolvimento , Aglutininas do Germe de Trigo/metabolismo
4.
Microb Pathog ; 31(6): 309-17, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11747378

RESUMO

Six different lectin probes were used to examine alterations of the cell surface carbohydrates in the chinchilla eustachian tube (ET) lumen subsequent to the intranasal (i.n.) challenge with the Streptococcus pneumoniae parent strain, D39, or its isogenic derivative, DeltaNA1, which is deficient in neuraminidase NanA. The labelling pattern revealed that the binding of wheat germ agglutinin (WGA), Erythrina cristagalli lectin (ECL), peanut agglutinin (PNA), Bandeiraea simplicifolia lectin II (BSL II) and succinylated wheat germ agglutinin (SWGA) were increased in the lumenal surface of the ET in the D39 inoculated cohort compared to the uninfected control, which indicated that N-acetylglucosamine (GlcNAc) and D-galactose residues were exposed. Concurrently, decreased labelling with Sambucus nigra agglutinin (SNA) indicated that there were few sialic acid residues remaining in the ET epithelium subsequent to i.n. inoculation with D39. The DeltaNA1 neuraminidase deficient mutant, however, did not induce any significant changes in the lectin labelling patterns, and was comparable to that of the control cohort. We propose that products of the nanA gene have a significant impact on the changes of the carbohydrate moieties in the ET epithelium and may be responsible for the previously reported increased ability of the D39 parent to colonize the nasopharynx and invade the middle ear.


Assuntos
Carboidratos/química , Tuba Auditiva/patologia , Neuraminidase/genética , Infecções Pneumocócicas/patologia , Streptococcus pneumoniae/genética , Animais , Carboidratos/análise , Chinchila , Modelos Animais de Doenças , Células Epiteliais/química , Células Epiteliais/patologia , Tuba Auditiva/química , Histocitoquímica , Lectinas , Mutagênese Insercional , Neuraminidase/deficiência , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/enzimologia
5.
J Neurotrauma ; 15(7): 555-61, 1998 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9674558

RESUMO

Dendrites were transected from murine spinal neurons. Unlesioned neurons showed dark nucleolar and patchy cytoplasmic jun immunostaining. By 0.5 and 2 h, most lesioned neurons stained intensely throughout the soma. However, at 24 h only dead neurons displayed intense somal staining, and 100% of the surviving cells stained like unlesioned controls. Correlation of immunostaining patterns with viability, injury, and death suggests jun gene expression may influence the survival of neurons after physical injury.


Assuntos
Genes Precoces/fisiologia , Genes jun/fisiologia , Degeneração Neural/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-jun/biossíntese , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , Animais , Biomarcadores , Morte Celular/fisiologia , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Expressão Gênica , Camundongos , Microscopia de Contraste de Fase , Degeneração Neural/patologia , Neurônios/patologia , Medula Espinal/embriologia , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia , Fatores de Tempo
6.
J Neurotrauma ; 13(12): 809-18, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9002066

RESUMO

Hypothermia has been reported to be beneficial in CNS physical injury and ischemia. We previously reported that posttraumatic cooling to 17 degrees C for 2 h increased survival of mouse spinal cord (SC) neurons subjected to physical injury (dendrite transection) but that cooling below 17 degrees C caused a lethal NMDA receptor-linked stress to both lesioned and uninjured neurons. The present study tested whether cooling below 17 degrees C increases extracellular levels of excitatory amino acids (EAA). SC cultures were placed at 10 degrees C or 37 degrees C. Glutamate (Glu) and aspartate (Asp) levels were higher in the medium of the cooled cultures after 0.5 h (23 +/- 4 nM/microgram vs. 4 +/- 1 nM/microgram and 4 +/- 1 nM/microgram vs. 1 +/- 0 nM/microgram, respectively). The concentration of each EAA then declined and reached a plateau at 2-4 h that was still significantly higher than control levels (p < 0.0001, two-factor ANOVA, three cultures per group). Other amino acids (glycine, asparagine, glutamine, serine) showed an opposite pattern, with higher levels in the 37 degrees C group. Both NMDA and non-NMDA antagonists prevented the lethal cold injury. Survival of SC neurons cooled at 10 degrees C for 2 h and rewarmed for 22 h was 58% +/- 25% in the control group, 94% +/- 5% in the CNQX-treated group, 97% +/- 5% in the DAPV-treated group, and 99% +/- 2% in the group treated with both antagonists [p < 0.0006, one factor ANOVA, five cultures (> 120 neurons) per group]. These results show that death of neurons cooled to 10 degrees C is caused by elevated extracellular Glu and Asp and requires activation of both the NMDA and non-NMDA receptor subtypes.


Assuntos
Temperatura Baixa , Aminoácidos Excitatórios/fisiologia , Traumatismos da Medula Espinal/etiologia , 2-Amino-5-fosfonovalerato/farmacologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Ácido Aspártico/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Antagonistas de Aminoácidos Excitatórios/farmacologia , Aminoácidos Excitatórios/metabolismo , Ácido Glutâmico/metabolismo , Camundongos/embriologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Valores de Referência , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia
7.
Arch Biochem Biophys ; 319(1): 23-35, 1995 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-7771789

RESUMO

Sublethal concentrations of reactive oxygen intermediates including H2O2 can alter human T cell function and inhibit proliferative responses but relatively little is known about the effects of low levels of oxidant stress on signaling pathways. In the present study, we investigated whether the exposure of Jurkat T cells to micromolar concentrations of H2O2 might influence the activity of certain serine/threonine kinases and protein phosphatases important for T cell signaling as well as initiation of nuclear events. Jurkat cells treated with 100-200 microM H2O2 exhibited rapid increases in cytosolic protein kinase C (PKC) activity without detectable translocation of PKC to the membrane/particulate compartment. The stimulation of PKC activity by H2O2 was associated with an increase in the activation of kinases phosphorylating myelin basic protein (MBP), a substrate for mitogen-activated protein (MAP) kinase and RRLSSLRA (S6 peptide; a substrate for the approximately 90-kDa ribosomal S6 kinases). Optimal activation of MAP kinase in cells treated with H2O2 was preceded by increases in protein tyrosine phosphorylations and occurred at sublethal concentrations of H2O2 which did not markedly deplete intracellular ATP. Pretreatment of cells with the PKC inhibitors sangivamycin and H7 suppressed but did not block the stimulation of MAP kinase activity in response to H2O2 or phytohemagglutinin. The activities of both protein tyrosine phosphatase (PTP) and protein phosphatase 2A (PP2A) were reduced after H2O2 treatment of intact cells. Furthermore, kinetic studies showed that H2O2 was capable of suppressing the activities of PTP and PP2A before inducing optimal increases in MAP kinase activity. These results demonstrate that the exposure of T cells to sublethal levels of oxidant stress acutely stimulates the MAP kinase cascade and suggest that this activation may involve PKC-dependent and -independent pathways as well as inhibition of certain protein phosphatases.


Assuntos
Fosfoproteínas Fosfatases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Linfócitos T/enzimologia , Sequência de Aminoácidos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/toxicidade , Cinética , Dados de Sequência Molecular , Estresse Oxidativo , Peptídeos/química , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Fosfatase 2 , Proteínas Quinases S6 Ribossômicas , Transdução de Sinais , Especificidade por Substrato , Linfócitos T/efeitos dos fármacos , Tirosina/metabolismo
8.
Mech Ageing Dev ; 77(3): 197-211, 1995 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-7739267

RESUMO

The expression of alpha- and beta-isoforms of protein kinase C (PKC) was analyzed in the peripheral blood T and B cells from 11 elderly and young humans. Immunoblot analysis with isoenzyme specific antibodies showed that T cells from five of 11 elderly subjects exhibited selective reductions in PKC alpha which was < 60% of those in young subjects whereas the levels of PKC beta were comparable to T cells of young subjects. No age-related reductions of PKC alpha or beta were observed in B cells. Among individual elderly subjects, the reductions in T cell PKC alpha were not associated with lower levels of PKC beta thereby resulting in only approximately 60-70% reductions of combined PKC alpha plus PKC beta. In addition, the functional properties of PKC in stimulated T cells of elderly subjects with respect to activation/translocation were comparable to T cells of young subjects. These results suggest that selective alterations in PKC isoenzymes can occur in human T cells during aging which may not be readily apparent in standard enzymatic assays and may contribute to aberrancies in intracellular signal transduction.


Assuntos
Envelhecimento/metabolismo , Linfócitos B/enzimologia , Células Sanguíneas/enzimologia , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Linfócitos T/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Transporte Biológico , Cálcio/fisiologia , Feminino , Humanos , Immunoblotting , Masculino , Fosfolipídeos/fisiologia
9.
Mech Ageing Dev ; 71(1-2): 31-46, 1993 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-8309282

RESUMO

Age-related changes in the functional properties of human B cells have been reported by several groups, but little is known about the early biochemical events and signaling pathways that might be altered during aging. The present investigation examined whether differences in the activation of protein tyrosine kinases (PTK) and in the expression of protein kinase C (PKC) enzymatic activity could be identified in B cells from 16 elderly subjects (mean 77 years) compared to B cells from 15 young subjects (mean 33 years). B cells from young subjects stimulated with the surface immunoglobulin (sIg) crosslinkers anti-IgM or Staphylococcus aureus Cowan I (SAC) demonstrated rapid increases in PTK mediated de novo tyrosine phosphorylation of endogenous proteins. In comparison, stimulated B cells from elderly subjects were reduced 22-46% in tyrosine phosphorylations. Quantitation of the enzymatic levels and activation/translocation of PKC activity among resting and sIg stimulated B cells showed that B cells from approximately 50% of elderly subjects had significant reductions compared to B cells from young subjects. Further analyses of the expression of PTK and PKC enzymatic activity by stimulated B cells from elderly subjects demonstrated that aging was associated with greater heterogeneity in PKC expression and that defects in PKC enzymatic activity could coexist with relatively normal PTK activity. Thus, these data suggest that aging can alter the expression of PTK/PKC enzymatic activity in human B cells and that these age-related alterations might perturb the balance between PKC-dependent and -independent signaling pathways.


Assuntos
Envelhecimento/sangue , Linfócitos B/enzimologia , Proteína Quinase C/sangue , Proteínas Quinases/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Ativação Enzimática , Feminino , Humanos , Fosforilação , Proteína Quinase C/biossíntese , Proteínas Quinases/biossíntese , Receptores de Antígenos de Linfócitos B/sangue
10.
Cell Immunol ; 142(2): 398-415, 1992 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1320464

RESUMO

The ability of cyclic AMP (cAMP) to modulate human B cell proliferative responses and the possible role of cAMP-dependent kinases (PKA) in cAMP modulation of proliferative responses were investigated. The addition of dibutyl cAMP (Bt2 cAMP) or the cAMP-elevating agent forskolin to B cells stimulated by crosslinking surface immunoglobulins (sIg) resulted in a concentration-dependent inhibition of proliferative responses. By contrast, Bt2 cAMP or forskolin enhanced the proliferative responses of B cells after direct stimulation by phorbol myristate acetate (PMA) and the calcium ionophore ionomycin. The inhibition and enhancement of B cell proliferative responses by Bt2 cAMP were observed at different incubation intervals and were not due to temporal shifts of optimal responses. Also, Bt2 cAMP caused only small changes in B cell RNA synthesis compared to modulation of proliferative responses. Exposure of B cells to Bt2 cAMP rapidly activated PKA. Blocking Bt2 cAMP activation of PKA with the kinase inhibitor HA1004 prevented Bt2 cAMP enhancement of B cell responses after direct stimulation by PMA and ionomycin. In reciprocal experiments, the kinase inhibitor H7 resulted in some inhibition of PKC activation but did not inhibit Bt2 cAMP activation of PKA or Bt2 cAMP enhancement of proliferative responses. Other experiments demonstrated that B cells treated with Bt2 cAMP had selective increases in the de novo phosphorylations of two endogenous substrates which reflected PKA activation. Furthermore, concentrations of HA1004 or H8 which inhibited Bt2 cAMP enhancement of proliferative responses also inhibited PKA phosphorylations of these substrates whereas H7 did not. Thus, elevations of cAMP can enhance or inhibit human B cell proliferative responses to different stimuli and the activation of PKA is important for cAMP enhancement of certain responses.


Assuntos
Linfócitos B/efeitos dos fármacos , Bucladesina/farmacologia , Divisão Celular/efeitos dos fármacos , Proteínas Quinases/efeitos dos fármacos , Sulfonamidas , Sequência de Aminoácidos , Anticorpos Anti-Idiotípicos , Bucladesina/antagonistas & inibidores , Sinergismo Farmacológico , Humanos , Ionomicina/farmacologia , Isoquinolinas/farmacologia , Dados de Sequência Molecular , Ésteres de Forbol/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases
11.
Vet Pathol ; 29(3): 216-22, 1992 May.
Artigo em Inglês | MEDLINE | ID: mdl-1621333

RESUMO

Twenty-two congenitally athymic nude (rnu/rnu) rats were transplanted with large granular lymphocyte leukemia derived from F344 rats and then compared with ten similar rats inoculated with a suspension of normal F344 rat spleen cells. The normal spleen cells and tumor cells from a spontaneous, naturally occurring leukemia did not grow or cause clinical disease in any of the rats. All rats inoculated with a serially passaged leukemia cell inoculum had local growth at the inoculation site that spread widely and resulted in progressive tumor growth. Death occurred between 16 and 38 days after inoculation. The 22 rats that received passaged tumor cells developed leukemia and splenomegaly. Spleens were diffusely infiltrated by tumor cells and had severe depletion of lymphocytes in the white pulp. Leukemic rats were thrombocytopenic and had hemolytic anemia characterized by increased osmotic fragility, red cell width, and many nucleated erythrocytes. The disease syndrome appears similar to that of F344 rats transplanted with the same inoculum. Because the host rats lacked T cells, it is concluded that the hemolytic anemia and thrombocytopenia that develop in transplanted rats are independent of T cell function.


Assuntos
Leucemia de Células T/veterinária , Ratos Nus , Doenças dos Roedores/patologia , Anemia Hemolítica/etiologia , Anemia Hemolítica/veterinária , Animais , Contagem de Células/veterinária , Leucemia de Células T/complicações , Leucemia de Células T/patologia , Contagem de Leucócitos/veterinária , Pulmão/patologia , Masculino , Mesentério/patologia , Transplante de Neoplasias/veterinária , Omento/patologia , Ratos , Doenças dos Roedores/etiologia , Baço/patologia , Trombocitopenia/etiologia , Trombocitopenia/veterinária
12.
Lymphokine Cytokine Res ; 10(6): 463-73, 1991 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1804309

RESUMO

Protein phosphorylation is considered an early cellular mechanism of signal transduction by surface immunoglobulins (sIg) and other receptors of B cells. Using intact human peripheral blood B cells of young subjects labeled with orthophosphate, increased phosphorylation levels of serine/threonine and tyrosine substrates were demonstrated on indicator phosphoproteins corresponding to the CD20 isoforms and microtubule-associated protein 2 kinase after cross-linking sIg and costimulation with phorbol diesters. By contrast, stimulated B cells from certain elderly subjects displayed substantial alterations in the phosphorylation patterns of serine/threonine or tyrosine indicator phosphoproteins. Also, age-related impairments in sIg stimulated mobilization of cytosolic protein kinase C (PKC) enzymatic activity and in cytosolic calcium [Ca2+]i responses of B cells were observed with the altered phosphorylation reactions. Comparison of the substrate phosphorylation profiles to the proliferative responses of stimulated B cells from individual elderly subjects suggested a model of signal transduction in which differing stimuli have different dependencies on phosphorylation reactions. Diminished proliferative responses after sIg ligation coincided with decreased phosphorylations of either tyrosine or serine/threonine indicator substrates. However, the decreased proliferative responses of B cells from elderly subjects with substantial reductions of tyrosine phosphorylation after sIg ligation were enhanced by the direct stimulation of serine/threonine kinase activity with phorbol diesters or CD40 ligation. Experiments with kinase inhibitors evaluated the relative dependency of different B cell stimuli on tyrosine and serine/threonine phosphorylation reactions. The proliferative responses of normal B cells to sIg ligation were quite sensitive to the tyrosine kinase inhibitor genistein whereas those observed following costimulations with phorbol diesters or CD40 ligation were more resistant. However, treatment of B cells with H7, an inhibitor of PKC activity, led to a more uniform reduction of B-cell responses after different stimuli. Results from RNase protection assays of c-myc expression also suggested that different B-cell stimuli might utilize distinct intracellular signaling pathways. Both the type of stimuli and mode of sIg ligation were important in determining the stimulated levels of c-myc mRNA expression. Thus, the current findings suggest that age-related defects are present in human B cell signaling pathways as reflected by tyrosine and serine/threonine phosphorylation reactions. Also, these age-related defects can coexist with altered mobilization of PKC enzymatic activity and with alterations in [Ca2+]i and proliferative responses.


Assuntos
Envelhecimento/fisiologia , Aminoácidos/metabolismo , Linfócitos B/fisiologia , Cálcio/metabolismo , Citosol/metabolismo , Transdução de Sinais/fisiologia , Divisão Celular/fisiologia , Células Cultivadas , Humanos , Fosforilação , Proteína Quinase C/sangue , Proteína Quinase C/fisiologia , Proteínas Tirosina Quinases/fisiologia , Ribonucleases/antagonistas & inibidores , Serina/metabolismo , Treonina/metabolismo , Tirosina/metabolismo
13.
Leuk Res ; 15(6): 427-33, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-1861529

RESUMO

A unique oncofetal protein (OFP) previously identified in rat fetal tissue and rat and human solid tumors, is now shown to be present in rat and human leukemia cells by use of a monoclonal antibody-based assay. Using a highly specific anti-rat OFP monoclonal antibody OFP has been unquivocally immunolocalized to the cytoplasm of the rat leukemia cells. The factor is rapidly released to the circulation as 50 and 55 kD species which share the immunological determinants. When leukemia cells are transplanted to normal rats, OFP increases in the circulation in a biphasic manner which may be due to immune clearance since circulating anti-OFP antibodies have been demonstrated. Induction of differentiation in the human HL-60 leukemia cell line by 13-cis-retinoic acid caused a down regulation of OFP synthesis, both intra- and extra-cellular levels dropping to essentially zero. Induction of differentiation with dibutyryl cyclic AMP caused a cessation of secretion of OFP, with a marked increase in its intracellular concentration, a condition resembling the retention in fetal cells. Leukemia cells add to a growing list of tumors previously shown to produce OFP, suggesting that OFP is intimately involved in some facet of tumorigenesis.


Assuntos
Antígenos de Neoplasias/genética , Leucemia Experimental/genética , Leucemia Mieloide/genética , Animais , Antígenos de Neoplasias/sangue , Antígenos de Neoplasias/metabolismo , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo/fisiologia , Imunofluorescência , Regulação Leucêmica da Expressão Gênica/fisiologia , Humanos , Leucemia Experimental/metabolismo , Leucemia Experimental/patologia , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Peso Molecular , Transplante de Neoplasias , Ratos , Ratos Endogâmicos F344 , Tretinoína/farmacologia , Células Tumorais Cultivadas
14.
Vet Pathol ; 27(6): 404-10, 1990 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2278128

RESUMO

A spontaneous large granular lymphocyte (LGL) leukemia was serially transplanted in 92 male F344 rats kept under standard laboratory conditions. Serial transplantation into groups of four rats each resulted in a rapid reduction in the latent period of the disease. After 23 serial transplantations, F344 rats in groups that were injected intraperitoneally with 10(7) cells died between 12 and 16 days after transplantation. At necropsy, "transplanted" rats had enlarged mesenteric lymph nodes, thymus, and spleen. Neoplastic cells were detected in the spleen on day 3 and in peripheral blood on day 6. Extreme leukocytosis with leukemia was present on day 9. Severe hemolytic anemia coincided with a sharp increase in osmotic fragility on day 12. Splenic lymphoid depletion was observed histologically and confirmed by differential cell counts of isolated spleen cells. Analysis for surface markers of splenic lymphocytes by monoclonal antibodies and flow cytometry indicated that cells with T helper/inducer phenotypes were disproportionately decreased, while the number of T suppressor cells did not significantly change. The T helper/T suppressor lymphocyte ratio (normal = 2.09 +/- 0.35) was decreased on day 9 (0.76 +/- 0.10) and day 12 (0.25 +/- 0.04). Hemolytic anemia was not related to a decrease in the number of T suppressor cells. The passaged leukemia cell model should provide investigators with an easily maintained neoplasm of short latency with which to study pathogenesis of leukemia-related disorders.


Assuntos
Modelos Animais de Doenças , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Ratos Endogâmicos F344 , Anemia Hemolítica/etiologia , Animais , Eritrócitos/patologia , Contagem de Leucócitos , Fígado/patologia , Linfócitos , Masculino , Mesentério/patologia , Transplante de Neoplasias , Tamanho do Órgão , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicações , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiologia , Ratos , Baço/patologia , Linfócitos T/patologia , Células Tumorais Cultivadas
15.
Vet Pathol ; 27(6): 397-403, 1990 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-2148994

RESUMO

A spontaneous large granular lymphocyte leukemia from a F344 rat was transplanted to 36 syngeneic recipients to study the interactions among leukemia, T lymphocytes, and the development of immunemediated hemolytic anemia. Six rats were euthanatized at biweekly intervals, and spleen weight, total spleen cellularity, and differential spleen cell counts were correlated with hemograms and osmotic fragility. Sequential changes in splenic architecture were correlated with hematologic parameters. Monoclonal antibodies defining all T lymphocytes (W3/13), T helper-inducer cells (W3/25), and T suppressor cells (OX-8) were used to identify T cells in immunocytochemical techniques on spleen sections, as well as in fluorescence activated cell sorter analysis of spleen cell suspensions. The onset of hemolytic anemic at 7 weeks after transplantation coincided with the first detection of tumor cells in the spleen and peripheral blood. Tumor cells first accumulated in the marginal zones, and then they infiltrated the red pulp sinusoids. Although the leukemia caused dispersion of the splenic lymphoid tissue, there was no significant lymphopenia, and the relative number of helper (W3/25+) and suppressor (OX-8+) lymphocytes did not change. Because the induction of anemia was a relatively early event in splenic involvement, we concluded that anemia was unrelated to disruption of lymphoid architecture; furthermore, it does not appear to be caused by changes in the numbers of regulatory T lymphocytes.


Assuntos
Anemia Hemolítica/etiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Baço/patologia , Linfócitos T , Animais , Contagem de Células , Separação Celular , Citometria de Fluxo , Imuno-Histoquímica , Macrófagos , Masculino , Transplante de Neoplasias , Tamanho do Órgão , Fragilidade Osmótica , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicações , Ratos , Ratos Endogâmicos F344 , Linfócitos T Auxiliares-Indutores , Linfócitos T Reguladores
16.
J Clin Lab Immunol ; 31(1): 27-31, 1990 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1966981

RESUMO

The effect of large granular lymphocyte leukemia on B lymphocyte function was studied by determining the number of plaques formed in an in vitro hemolytic plaque assay. Leukemia cells inhibited plaque formation by normal splenic lymphocytes in a logarithmic, dose-dependent manner. At the highest leukemia cell concentrations, spleen cell suspensions made 50% fewer plaques. Plaque forming responses were very sensitive to duration of preincubation time in all assays. The number of plaques formed decreased markedly if incubated 2 hr before the assay was performed. Incubation of the cells at 56 degrees C for 8 min did not alter the inhibitory activity but pretreatment with 0.01% trypsin did. Supernatant fluids from leukemia cell suspensions did not inhibit plaque formation. These data suggest that diffuse infiltration of lymphoid tissues by leukemia cells may interfere with some normal lymphocyte functions. Although leukemia cells inhibited splenic B lymphocyte function, leukemic rats did not have hypogammaglobulinemia.


Assuntos
Subpopulações de Linfócitos B/imunologia , Técnica de Placa Hemolítica , Leucemia Linfocítica Crônica de Células B/imunologia , Células-Tronco Neoplásicas/fisiologia , Animais , Subpopulações de Linfócitos B/patologia , Membrana Celular/fisiologia , Relação Dose-Resposta Imunológica , Leucemia Linfocítica Crônica de Células B/complicações , Masculino , Ratos , Ratos Endogâmicos F344 , Baço/imunologia , Esplenomegalia/etiologia , Células Tumorais Cultivadas/imunologia , Células Tumorais Cultivadas/patologia
17.
Leuk Lymphoma ; 1(3-4): 227-33, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-27463990

RESUMO

The pattern of disease relapse in nine patients with multiple myeloma who had received double hemi body irradiation therapy is analysed. This analysis highlights the localisation of these relapses to skeletal areas excluded from initial radiation fields. This phenomenon was also documented in a patient who received systemic irradiation as part of a conditioning regimen per allogeneic bone marrow transplantation.

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