Organ-specific inhibition of metastatic colon carcinoma by CXCR3 antagonism.

Liver and lung metastases are the predominant cause of colorectal cancer (CRC)-related mortality. Recent research has indicated that CXCR3/chemokines interactions that orchestrate haematopoetic cell movement are implicated in the metastatic process of malignant tumours, including that of CRC cells to lymph nodes. To date, however, the contribution of CXCR3 to liver and lung metastasis in CRC has not been addressed. To determine whether CXCR3 receptors regulate malignancy-related properties of CRC cells, we have used CXCR3-expressing CRC cell lines of human (HT29 cells) and murine (C26 cells) origins that enable the development of liver and lung metastases when injected into immunodeficient and immunocompetent mice, respectively, and assessed the effect of CXCR3 blockade using AMG487, a small molecular weight antagonist. In vitro, activation of CXCR3 on human and mouse CRC cells by its cognate ligands induced migratory and growth responses, both activities being abrogated by AMG487. In vivo, systemic CXCR3 antagonism by preventive or curative treatments with AMG487 markedly inhibited the implantation and the growth of human and mouse CRC cells within lung without affecting that in the liver. In addition, we measured increased levels of CXCR3 and ligands expression within lung nodules compared with liver tumours. Altogether, our findings indicate that activation of CXCR3 receptors by its cognate ligands facilitates the implantation and the progression of CRC cells within lung tissues and that inhibition of this axis decreases pulmonary metastasis of CRC in two murine tumour models.

A comparison of four new antiepileptic medications.

In order to select a new medication for a patient with epilepsy, it would be helpful to have an idea of which drug might have the greatest overall chance for success. Since epilepsy is a chronic disorder, the long-term effectiveness and tolerability of the medications are very important. Here, we compared gabapentin, lamotrigine, topiramate and vigabatrin using Kaplan-Meier survival analysis to see how long patients chose to stay on each drug and if they stopped, why they stopped. The results seem to suggest the type of responses to be expected in a hospital seizure clinic.

Novel methods for studying new antiepileptic drug pharmacology.

A number of new antiepileptic drugs act by indirect mechanisms and thus produce effects that may not best be measured by traditional blood studies of the drugs and their metabolites. Study of the indirect action of these drugs on GABA-mediated inhibition by microdialysis and nuclear MR spectroscopy has proved more relevant. These new investigative techniques may also prove valuable as compounds affecting glutamate or other excitatory neurotransmitters are developed.

Human brain GABA levels rise rapidly after initiation of vigabatrin therapy.

OBJECTIVE: The purpose of this study was to measure changes in brain GABA after a single oral dose (50 mg/kg) of vigabatrin in patients with intractable epilepsy. BACKGROUND: Vigabatrin is a safe and effective antiepileptic medication designed to increase brain GABA by irreversibly inhibiting GABA-transaminase. Serial measurements showed that brain GABA levels increased from 1.0 (SEM, 0.07) to 2.4 mmol/kg (SEM, 0.09) in patients who were regularly taking vigabatrin (50 mg/kg/day divided into two doses). METHODS: In vivo measurements of GABA in human brain were made using 1H magnetic resonance spectroscopy. We used a 2.1-T NMR spectrometer and an 8-cm surface coil to measure a 13.5 cm3 volume in the occipital cortex. RESULTS: Brain GABA increased by more than 40% within 2 hours of administration of a single 50 mg/kg oral dose of vigabatrin from 0.95 (SEM, 0.07; n = 7) to 1.34 mmol/kg (SEM, 0.13). By the next day, brain GABA increased further to 1.44 mmol/kg (SEM, 0.08). Levels declined gradually to 1.16 mmol/kg (SEM, 0.14) by day 5 and 1.03 mmol/kg (SEM, 0.10) at day 8. The patients reported no side effects and were calm but not drowsy. CONCLUSIONS: A single oral dose of vigabatrin rapidly increased brain GABA without side effects. Once-a-day dosing should be as effective as divided doses.

Role of the Lck Src homology 2 and 3 domains in protein tyrosine phosphorylation.

Many protein tyrosine phosphorylation events that occur as a result of T cell receptor (TCR) stimulation are enhanced when CD4 is co-cross-linked with the TCR, and this increased phosphorylation is thought to be a mechanism by which T cell functions are augmented by CD4. Such enhanced tyrosine phosphorylation was originally attributed to the kinase activity of the CD4-associated tyrosine kinase Lck. However, it has been shown that CD4-associated Lck lacking the catalytic domain can enhance T cell functions, suggesting that the noncatalytic domains of Lck are also important in CD4 signaling. Using T cells expressing various CD4-Lck chimeric molecules, we assessed the role of different Lck domains in early T cell signaling. Following TCR-CD4 co-cross-linking, cells expressing a CD4-Lck full-length chimera showed enhanced tyrosine phosphorylation of many cellular proteins in a CD4-dependent manner. Surprisingly, cells expressing a CD4-Lck chimera lacking the catalytic domain (termed CD4-N32) also showed enhanced phosphorylation. This enhancement of phosphorylation required both the Src homology 2 (SH2) and SH3 domains of Lck. Lck has been postulated to dimerize through the SH2 and SH3 domains. In this way CD4-N32 may interact with endogenous Lck, and although it lacks intrinsic kinase activity, it may be capable of enhancing phosphorylation through the associated full-length Lck. Consistent with this model, when CD4-Lck chimeric molecules were expressed in J. CaM1.6 cells lacking endogenous Lck, CD4-N32 failed to enhance tyrosine phosphorylation. Moreover, a Lck SH2 and SH3 domain fragment expressed as a glutathione S-transferase fusion protein associated with Lck when incubated with activated Jurkat T cell lysates, suggesting that the SH2 and SH3 domains of Lck can associate with endogenous full-length Lck upon activation. Thus, our data suggest that dimerization is an important mechanism of Lck function in T cell activation.

Direct interaction between protein kinase C theta (PKC theta) and 14-3-3 tau in T cells: 14-3-3 overexpression results in inhibition of PKC theta translocation and function.

Recent studies have documented direct interactions between 14-3-3 proteins and several oncogene and proto-oncogene products involved in signal transduction pathways. Studies on the effects of 14-3-3 proteins on protein kinase C (PKC) activity in vitro have reported conflicting results, and previous attempts to demonstrate a direct association between PKC and 14-3-3 were unsuccessful. Here, we examined potential physical and functional interactions between PKC theta, a Ca(2+)-independent PKC enzyme which is expressed selectively in T lymphocytes, and the 14-3-3 tau isoform in vitro and in intact T cells. PKC theta and 14-3-3 tau coimmunoprecipitated from Jurkat T cells, and recombinant 14-3-3 tau interacted directly with purified PKC theta in vitro. Transient overexpression of 14-3-3 tau suppressed stimulation of the interleukin 2 (IL-2) promoter mediated by cotransfected wild-type or constitutively active PKC theta, as well as by endogenous PKC in ionomycin- and/or phorbol ester-stimulated cells. This did not represent a general inhibition of activation events, since PKC-independent (but Ca(2+)-dependent) activation of an IL-4 promoter element was not inhibited by 14-3-3 tau under similar conditions. Overexpression of wild-type 14-3-3 tau also inhibited phorbol ester-induced PKC theta translocation from the cytosol to the membrane in Jurkat cells, while a membrane-targeted form of 14-3-3 tau caused increased localization of PKC theta in the particulate fraction in unstimulated cells. Membrane-targeted 14-3-3 tau was more effective than wild-type 14-3-3 tau in suppressing PKC theta-dependent IL-2 promoter activity, suggesting that 14-3-3 tau inhibits the function of PKC theta not only by preventing its translocation to the membrane but also by associating with it. The interaction between 14-3-3 and PKC theta may represent an important general mechanism for regulating PKC-dependent signals and, more specifically, PKC theta-mediated functions during T-cell activation.

Vav: function and regulation in hematopoietic cell signaling.

Vav, a 95 kDa proto-oncogene product expressed specifically in hematopoietic cells, was originally isolated as a transforming human oncogene. Vav contains an array of functional domains that are involved in interactions with other proteins and, possibly, with lipids. These include, among others, a putative guanine nucleotide exchange domain, a cysteine-rich region similar to the phorbol ester/diacylglycerol-binding domain of protein kinase C, a pleckstrin-homology domain, and Src-homology 2 and 3 (SH2 and SH3, respectively) domains. The presence of these domains, the transforming activity of the vav oncogene, and the rapid increase in tyrosine phosphorylation of Vav induced by triggering of diverse receptors indicate that it plays an important role in hematopoietic cell signaling pathways. Such a role is supported by recent studies using "knockout" mice and transiently transfected T cells, in which Vav deletion or overexpression, respectively, had marked effects on lymphocyte development or activation. The presence of a putative guanine nucleotide exchange domain, the prototype of which is found in the dbl oncogene product, implies that Vav functions as a guanine nucleotide exchange factor (GEF) for one (or more) members of the Ras-like family of small GTP-binding proteins. In support of such a role, Vav preparations were found in some (but not other) studies to mediate in vitro-specific GEF activity for Ras. Additional studies are required to identify the physiological regulators and targets of Vav, and its exact role in hematopoietic cell development and signaling.

CD4 and signal transduction.

The CD4 molecule plays an important role in the development of CD4+T lymphocytes and it also acts as a coreceptor to enhance responses mediated via the TCR. It is now established that CD4 functions both as an adhesion molecule favoring the T cell: APC interaction and as a signaling molecule. The coreceptor function mediated via CD4 depends on its association with Lck, a src-family tyrosine kinase. Lck, while interacting via its unique NH2-terminal domain with CD4, also interacts via its SH2 and SH3 domains with other intracellular signaling proteins. Although the Lck association with CD4 is essential for CD4 coreceptor activity, the tyrosine kinase activity of CD4-associated Lck appears to be dispensable for CD4 function. Given the necessity of Lck kinase activity for T lymphocyte development and for mature T cell functions, perhaps Lck may function at different stages during T cell activation and at some stages the kinase activity of Lck may not be necessary. This raises an intriguing possibility that CD4-associated Lck may function more as an adapter protein than a kinase and may help to recruit other signaling proteins into the TCR/CD3 complex. However, determination of the precise role of Lck in CD4 coreceptor activity and the domains of Lck that are necessary for CD4-dependent and CD4-independent functions awaits further experiments.

Cognitive functioning as a contraindication to organ transplant surgery: Dilemmas encountered in medical decision making.

A case is presented to illustrate some of the difficulties encountered when providing psychological consultation to evaluate the readiness of patients for pediatric heart-lung transplantation. The outcome of complex medical decision making can often hinge on information provided by the psychological consultant who is attempting to simultaneously serve the needs of the patient as well as the transplant team. Ethical dilemmas frequently arise when medical decision making is driven by limited health care resources and cost constraints. The utility of cognitive functioning as a variable in pediatric transplant decision making is discussed. Recommendations are made for further work in this area on both conceptual and empirical grounds to guide the integration of psychological information into transplant decision making as health care delivery continues to evolve in the future.

The CD4-associated tyrosine kinase p56lck is required for lymphocyte chemoattractant factor-induced T lymphocyte migration.

Lymphocyte chemoattractant factor (LCF) is a polypeptide cytokine which induces both cell motility and activation of T lymphocytes. These LCF-induced events demonstrate an absolute requirement for the cell surface expression of CD4. Because many CD4-mediated T lymphocyte activation events have been demonstrated to require the association of the src-related tyrosine kinase p56lck with the cytoplasmic domain of CD4, we examined the role of p56lck in LCF-induced lymphocyte migration in a murine T cell hybridoma line expressing transfected human CD4. LCF induces the catalytic activity of CD4 associated p56lck at chemoattractant concentrations of cytokine. Hybridoma cells that express CD4 with cytoplasmic point mutations which uncouple the CD4-lck association lack both lck enzymatic activity and chemotactic responses to LCF. The enzymatic activity of lck however does not appear to be required for CD4-mediated migratory signal. First, the protein tyrosine kinase inhibitor herbimycin A blocked LCF-induced p56lck activation but had no effect on the LCF-induced motile response. Second, T cell hybridomas expressing a chimeric receptor combining the extracellular domain of human CD4 and murine p56lck which lacked the kinase domain had a normal LCF-induced motile response. We conclude from these observations that CD4-lck coupling is essential for LCF-induced T lymphocyte migration but the motile response is independent of the enzymatic activity of CD4-associated p56lck.

Adhesion receptors in lymphocyte activation.

The past several years have seen significant progress in understanding the role of T lymphocyte coreceptors in adhesion and activation. New insights have been gained in several areas: the avidity regulation of beta 1 and beta 2 integrins and their role in signal transduction; the regulation of CD8 avidity; the role of Lck in CD4 coreceptor activity; and the novel role for CD2 adhesion in the T cell antigen response.

Tyrosine kinase activity of CD4-associated p56lck may not be required for CD4-dependent T-cell activation.

The lymphoid-specific tyrosine kinase p56lck (Lck) is critical for the development and activation of T lymphocytes, and Lck kinase activity has been implicated in both T-cell antigen receptor/CD3- and CD4-mediated signaling. CD4-dependent T-cell activation has been demonstrated to be dependent upon the association of CD4 with Lck. To examine the role of the kinase activity of Lck in CD4-dependent T-cell activation, we have generated several kinase-deficient mutants of Lck. When transfected into CD4+ murine T-cell hybridoma cells, these mutants cause approximately 90% diminution in CD4-associated Lck kinase activity. Specifically, upon CD4 crosslinking there is decreased Lck autophosphorylation and decreased phosphorylation of an exogenous substrate. When CD4 is crosslinked to the T-cell antigen receptor-CD3 complex, decreased phosphorylation of associated substrates is also observed. In spite of this striking inhibition of Lck kinase function, cells expressing the kinase-deficient mutants demonstrate normal or enhanced CD4-dependent antigen responsiveness. These data demonstrate that the level of Lck kinase activity does not correlate with its CD4-associated function and suggest that the kinase activity of Lck may not be required for CD4-mediated signaling.

Disruption of the CD4-p56lck complex is required for rapid internalization of CD4.

CD4 is a cell surface glycoprotein expressed by a subset of T lymphocytes and functions to enhance T-cell activation. CD4 is noncovalently associated via the cytoplasmic domain with the protein-tyrosine kinase p56lck, a member of the src protein-tyrosine kinase family. Upon activation of protein kinase C by phorbol ester, CD4 is phosphorylated on cytoplasmic serine residues and internalized from the cell surface, and disruption of the CD4-p56lck complex occurs. The exact relationship between these events is likely to be functionally significant, as cytoplasmic-domain serine phosphorylation and internalization have been shown to regulate the function of receptors that possess intrinsic protein-tyrosine kinase activity. Here we demonstrate that p56lck slows the rate of phorbol 12-myristate 13-acetate-induced internalization of CD4 in a manner that depends on a physical association between p56lck and CD4. This decreased rate is due at least in part to a requirement for disruption of the CD4-p56lck complex prior to internalization of CD4. Furthermore, disruption of the CD4-p56lck complex appears to depend on the integrity of the cytoplasmic-domain serine at position 408, probably due to a requirement for phosphorylation.

p56lck association with CD4 is required for the interaction between CD4 and the TCR/CD3 complex and for optimal antigen stimulation.

By fluorescence resonance energy transfer, we have previously demonstrated that upon anti-CD3 mAb-mediated activation of a murine T cell hybridoma expressing human CD4, CD4 moves into close association with the TCR/CD3 complex. It was shown that this association between CD4 and the TCR/CD3 complex was dependent upon the presence of an intact CD4 cytoplasmic domain. We have now expressed, in a murine T cell hybridoma, mutated forms of CD4 containing cysteine to serine point mutations at positions 420, 422, or 430. The mutations at positions 420 and 422, but not 430, abolish association with p56lck. By using fluorescence resonance energy transfer, we demonstrate that mutations of CD4 which fail to interact with p56lck are unable to associate with the TCR/CD3 complex under conditions in which wild-type CD4 and the 430 mutant CD4 do associate with the TCR/CD3 complex. In addition, these mutants have a diminished response to CD4-dependent stimuli. We conclude that the association between CD4 and the TCR/CD3 complex during T cell activation plays an important role in CD4-dependent responsiveness and this association requires the interaction of CD4 with p56lck. These results also suggest that a substrate for p56lck may be expressed in the TCR/CD3 complex.

Definitive diagnosis of breast implant rupture by ultrasonography.

Silicone breast implantation is entering its fourth decade. Our ability to monitor the integrity of "old" prostheses is questioned. Clinical and mammographic examinations are reliable indicators of implant rupture only if there has been gel migration away from the implant pocket. Ultrasonography is presented as a reliable, sensitive method of evaluation of implant integrity. It should be considered the definitive study of prosthesis integrity. When sonography is added to mammographic and clinical examination, the preoperative evaluation of symptomatic augmented breasts is complete. Ultrasonography may be considered with mammography in the routine breast examination of all previously augmented patients.

Heterogeneity of helper/inducer T lymphocytes. IV. Stimulation of resting and activated B cells by Th1 and Th2 clones.

To test the hypothesis that resting and previously activated B lymphocytes differ in their proliferative and differentiative responses to various Th cell-derived stimuli, we have examined the interactions of purified small (resting) and large (activated) murine B cells with rabbit Ig-specific Th1 and Th2 clones in the presence of the Ag analogue, rabbit anti-mouse Ig antibody. Small numbers of Th2 cells induce strong Ag-dependent proliferation of and Ig secretion by both resting and activated B lymphocytes. In contrast, Th1 clones stimulate lower responses of activated B cells and fail to stimulate small resting B cells. An interaction with Th1 clones does make small B cells responsive to the Th2-derived cytokine, IL-4, indicating that Th1 clones are capable of delivering some but not all the stimuli necessary for the induction of humoral immunity. Finally, in order to compare the responses of small and large B cells to cognate interactions and secreted cytokines, we used an autoreactive I-Ak-specific Th2 line. This line induces proliferation of and Ig secretion by I-Ak expressing but not H-2d resting and activated B cells as a result of cognate interactions. However, when the H-2d B cells are bystanders in the presence of cytokine secretion by this Th2 line, or are directly exposed to Th2-derived cytokines, both small and large B cells are induced to proliferate but only the large B cells secrete antibody. These results indicate that the magnitude and nature of antibody responses depend on three principal factors: the cytokines produced by Th cells, the state of activation of the responding B lymphocytes, and whether the B cells are recipients of cognate help or are bystanders at the site of T cell stimulation. Our findings also confirm the view that cognate T-B interactions are most efficient for initiating B cell responses and may allow B cells to subsequently respond to a variety of T cell-derived cytokines.

Serial biparietal diameter and femur length measurements in twin gestations.

Biparietal diameter and femur length measurements from 60 twin pairs were used to construct fetal twin growth tables. From 18 through 38 weeks of gestation the biparietal diameter measurements were accurate within +/- 1.9 weeks, and the femur length within +/- 2.1 weeks in the prediction of gestational age. Since this data was obtained from a predominantly white middle class population it may prove more reliable in similar groups for predicting the adequacy of fetal growth and gestational age than prior data obtained using indigent populations.

Percutaneous drainage of a psoas abscess in a young child.

Percutaneous drainage of abdominal and retroperitoneal abscesses has become a widely practiced alternative to surgery in selected patients. Although such techniques and results have been widely reported in adults, these series do not include reports of percutaneous drainage of a psoas abscess in a young child to emphasize that interventional radiologic techniques can be effective even in very young children.