Regression versus no regression in the autistic disorder: developmental trajectories.

Developmental regression is a complex phenomenon which occurs in 20-49% of the autistic population. Aim of the study was to assess possible differences in the development of regressed and non-regressed autistic preschoolers. We longitudinally studied 40 autistic children (18 regressed, 22 non-regressed) aged 2-6 years. The following developmental areas were considered fundamental in the first years of life, and were assessed at ages 2, 3, 4, 5, and 6: receptive and expressive language, communicative and request modalities, play activities, and mental age. Children who regressed showed lower mean performances than those who did not regress and, in the time intervals considered, non-regressed children improved their ratings in the above mentioned variables significantly more than regressed children.

An open-label study of controlled-release melatonin in treatment of sleep disorders in children with autism.

Long-term effectiveness of controlled-release melatonin in 25 children, aged 2.6-9.6 years with autism without other coexistent pathologies was evaluated openly. Sleep patterns were studied using Children's Sleep Habits Questionnaire (CSHQ) and sleep diaries at baseline, after 1-3-6 months melatonin treatment and 1 month after discontinuation. Sleep diary and CSHQ showed a more problematic sleep in autistic children compared with controls. During treatment sleep patterns of all children improved. After discontinuation 16 children returned to pre-treatment score, readministration of melatonin was again effective. Treatment gains were maintained at 12 and 24-month follow-ups. No adverse side effects were reported. In conclusion, controlled-release melatonin may provide an effective and well-tolerated treatment for autistic children with chronic sleep disorders.

HERG potassium channels are constitutively expressed in primary human acute myeloid leukemias and regulate cell proliferation of normal and leukemic hemopoietic progenitors.

An important target in the understanding of the pathogenesis of acute myeloid leukemias (AML) relies on deciphering the molecular features of normal and leukemic hemopoietic progenitors. In particular, the analysis of the mechanisms involved in the regulation of cell proliferation is decisive for the establishment of new targeted therapies. To gain further insight into this topic we report herein a novel approach by analyzing the role of HERG K(+) channels in the regulation of hemopoietic cell proliferation. These channels, encoded by the human ether-a-gò-gò-related gene (herg), belong to a family of K(+) channels, whose role in oncogenesis has been recently demonstrated. We report here that herg is switched off in normal peripheral blood mononuclear cells (PBMNC) as well as in circulating CD34(+) cells, however, it is rapidly turned on in the latter upon induction of the mitotic cycle. Moreover, hergappears to be constitutively activated in leukemic cell lines as well as in the majority of circulating blasts from primary AML. Evidence is also provided that HERG channel activity regulates cell proliferation in stimulated CD34(+) as well as in blast cells from AML patients. These results open new perspectives on the pathogenetic role of HERG K(+) channels in leukemias.

Gravitational unloading induces osteoclast-like differentiation of FLG 29.1 cells.

FLG 29.1 cells, cultured at 1xg, are able to switch on a differentiating process only when they are suitably induced by chemical factors. On the contrary, when FLG 29.1 cells are cultured in conditions of gravitational unloading, simulated by a Random Positioning Machine, the switching on of the differentiation process occurs in the absence of any added differentiating agent or any stimulating factor. The phenotypic characterization of the cells and quantitative measures of their bone resorption activity are consistent with a differentiation process through the osteoclastic pathway.

Interferon-gamma receptor 2 expression as the deciding factor in human T, B, and myeloid cell proliferation or death.

The heterodimeric interferon (IFN)-gamma receptor (IFN-gammaR) is formed of two chains. Here we show that the binding chain (IFN-gammaR1) was highly expressed on the membranes of T, B, and myeloid cells. Conversely, the transducing chain (IFN-gammaR2) was highly expressed on the surfaces of myeloid cells, moderately expressed on B cells, and poorly expressed on the surfaces of T cells. Differential cell membrane expression of IFN-gammaR2 determined the number of receptor complexes that transduced the IFN-gamma signal and resulted in a different response to IFN-gamma. After IFN-gamma stimulation, high IFN-gammaR2 membrane expression induced rapid activation of signal transducer and activator of transcription-1 (STAT-1) and high levels of interferon regulatory factor-1 (IRF-1), which then triggered the apoptotic program. By contrast, low cell membrane expression resulted in slow activation of STAT-1, lower levels of IRF-1, and induction of proliferation. Because the forced expression of IFN-gammaR2 on T cells switched their response to IFN-gamma from proliferative to apoptotic, we concluded that the surface expression of IFN-gammaR2 determines whether a cell stimulated by IFN-gamma undergoes proliferation or apoptosis.

Developmental profile and regression in a child with autism: a single case study.

The developmental profile of a child with autism during the first 3 years of life is presented. Clinical material obtained from different sources is discussed: home videos from birth to 3 years, and cognitive and communicative evaluations at 24, 34 and 38 months. The videos show how the child appeared to make progress up to 12 months, but from 12 to 18 months some abilities that had been previously acquired were lost, and a decrease in social interaction, communication and language was observed. From 18 to 38 months communicative and linguistic abilities remained unchanged, but social interactive behaviours continued to decrease. The particular profile identified is discussed as one of the possible pathways through which autism may develop.

Biased activation of human T lymphocytes due to low extracellular pH is antagonized by B7/CD28 costimulation.

As T cell response to tumor-associated antigens may be impaired by the acidic microenvironment typical of solid tumors, we assessed the effect of extracellular pH (pH(e)) on the activation and proliferation of human T lymphocytes and generation of the cytotoxic response. T lymphocytes stimulated with anti-CD3 mAb or PHA at low pH(e) were unable to secrete IL-2 and IFN-gamma and their ability to progress through the cell cycle was impaired. T lymphocytes also displayed up-regulation of IFN-gammaR2 chain and CTLA-4 expression, rendering them sensitive to negative regulatory signals. Agonistic mAb against CD28, but not against CD2, completely restored cytokine production and cell cycle progression, but down-regulated IFN-gammaR2 and CTLA-4 expression. The anti-CD28mAb rescued the CTL response of allogeneic anti-tumor cultures generated at low pH(e). Following anti-CD28 mAb treatment, T cells synthesized cyclooxygenase-2 (Cox-2) protein, which is involved in the early phases of T cell activation. This rescue of T cell activation was independent of the inducible 6-phosphofructo-2-kinase (iPFK-2) pathway, which stimulates proliferation in hypoxic and acidic conditions. The restoration of proliferative and cytotoxic T cell responses by CD28-triggering provides insight into the mechanisms by which B7 enhances the T cell anti-tumor response in vivo.

In vitro activity of vinorelbine on human leukemia cells.

Vinorelbine (VNR) is a semi-synthetic Vinca rosea alkaloid that has been employed both as a single agent and in combination, and has shown significant antitumor activity. As little is known about VNR activity on human leukemia, we studied its in vitro cytotoxic effect on human leukemia cell lines (FLG 29.1, HL60, K562, Balm 4, CEM and Daudi) and on fresh leukemia cells from 28 patients: 2 acute myeloid leukemia (AML); 3 chronic myeloid leukemia in blastic phase (CML-BP); 5 acute lymphoblastic leukemia (ALL); 18 B-chronic lymphatic leukemia (B-CLL), employing the colorimetric INT assay and determining the IC50. We observed that VNR exerts its cytotoxic activity on leukemic cell lines in a dose-dependent fashion. The lymphoid cell lines appear more sensitive than the myeloid ones to the VNR-dependent growth inhibition. A similar pattern was noticed for leukemia cells in primary cultures. VNR is not effective on CML-BP cells, shows variable activity on the AML and ALL cells and is very effective against B-CLL cells. VNR inhibited the growth of fresh B-CLL cells from 15 of 18 patients, the IC50 doses ranging from 4 ng/ml to 83 microg/ml (doses coinciding with the plasma levels obtained in clinics). These observations strongly suggest that VNR could be useful in clinics for the treatment of B-CLL.

Regulation of interferon-gamma receptor (INF-gammaR) chains: a peculiar way to rule the life and death of human lymphocytes.

Interferon-gamma (IFN-gamma) is a lymphokine produced by activated T lymphocytes and NK cells, that plays an important role in host defense mechanisms by exerting pleiotropic activities on a wide range of cell types. Cellular responses to IFN-gamma are mediated by its heterodimeric cell surface receptor (IFN-gammaR), which activates downstream signal transduction cascades, ultimately leading to the regulation of gene expression. Several observations suggest that the signals resulting from the binding of IFN-gamma to its receptor depend on the number of surface receptors transducing the IFN-gamma signal. This review summarizes recent advances in the understanding of the fine regulation of the response of human lymphocytes to IFN-gamma through an interplay between surface expression of IFN-gammaR and a variety of environmental factors that combine to control their fate.

Interleukin 12-activated lymphocytes influence tumor genetic programs.

T-lymphocytes (LYs) from normal and IFN-gamma knockout mice were activated by anti-CD3 and anti-CD28 antibodies and cultured in inserts in the presence of interleukin (IL)-12 (IL-12-activated LYs) or not (activated LYs). Their ability to modulate the genetic programs of two tumor lines growing at the bottom of transwells was evaluated. cDNA gene expression array, reverse transcription-PCR, and protein expression showed that LPS, transcription termination factor 1, transforming growth factor, and fibroblast growth factor genes were up-modulated by factors other than IFN-gamma released by activated LYS: The high levels of IFN-gamma released by normal IL-12-activated LYs up-modulated the expression of STAT1, IRF-1, LMP2, LMP7, monokine induced by IFN-gamma, monocyte chemoattractant protein 1, and angiopoietin 2 genes but down-modulated the expression of vascular endothelial growth factor. PA28, IFN-inducible protein 10, inducible NO synthetase, and macrophage-inhibitory protein 2 genes were up-modulated by factors released only by IL-12-activated LYs apart from IFN-gamma. The opposite modulations of vascular endothelial growth factor expression and of angiopoietin 2, monokine induced by IFN-gamma, IFN-inducible protein 10, and inducible NO synthetase by IL-12-activated LYs fit in well with the inhibition of angiogenesis that characterizes the antitumor activity of IL-12. T-LYs thus modify a tumor's behavior so that it becomes a party to its own inhibition.

HERG K+ channels activation during beta(1) integrin-mediated adhesion to fibronectin induces an up-regulation of alpha(v)beta(3) integrin in the preosteoclastic leukemia cell line FLG 29.1.

Integrin receptors have been demonstrated to mediate either "inside-to-out" and "outside-to-in" signals, and by this way are capable of regulating many cellular functions, such as cell growth and differentiation, cell migration, and activation. Among the various integrin-centered signaling pathways discovered so far, we demonstrated that the modulation of the electrical potential of the plasma membrane (V(REST)) is an early integrin-mediated signal, which is related to neurite emission in neuroblastoma cells. This modulation is sustained by the activation of HERG K(+) channels, encoded by the ether-à-go-go-related gene (herg). The involvement of integrin-mediated signaling is being discovered in the hemopoietic system: in particular, osteoclasts are generated as well as induced to differentiate by interaction of osteoclast progenitors with the stromal cells, through the involvement of integrin receptors. We studied the effects of cell interaction with the extracellular matrix protein fibronectin (FN) in a human leukemic preosteoclastic cell line (FLG 29.1 cells), which has been demonstrated to express HERG currents. We report here that FLG 29.1 cells indeed adhere to purified FN through integrin receptors, and that this adhesion induces an osteoclast phenotype in these cells, as evidenced by the appearance of tartrate-resistant acid phosphatase, as well as by the increased expression of CD51/alpha(v)beta(3) integrin and calcitonin receptor. An early activation of HERG current (I(HERG)), without any increase in herg RNA or modifications of HERG protein was also observed in FN-adhering cells. This activation is apparently sustained by the beta(1) integrin subunit activation, through the involvement of a pertussis-toxin sensitive G(i) protein, and appears to be a determinant signal for the up-regulation of alpha(v)beta(3) integrin, as well as for the increased expression of calcitonin receptor.

Multispectral imaging autofluorescence microscopy for the analysis of lymph-node tissues.

Although histochemical and immunohistochemical methods are the standard procedures in diagnosis of lymphoproliferative disorders, useful improvements in evidencing histopathologic manifestations can be obtained with the introduction of tissue autofluorescence analyses. We used microspectrofluorometry and a Multispectral Imaging Autofluorescence Microscopy (MIAM) technique to analyze lymph-node biopsies from patients with lymphoadenopathy of different origins. Images of tissue autofluorescence were obtained by excitation at 365 nm of lymph-node sections and sequential detection with interference filters (50 nm bandwidth) peaked at 450, 550 and 658 nm. Monochrome images were combined together in a single red-green-blue color image. Most of the fluorescence was observed within the blue spectral band because of large contributions from extracellular collagen and elastin fibers as well as from reduced form of intracellular nicotinamide adenine dinucleotide (phosphate). Autofluorescence imaging shows morphological differences between neoplastic and non-neoplastic tissues. The reactive hyperplasia samples show the typical lymph-node organization with weak fluorescent follicles separated by high fluorescent connective trabeculae. In the neoplastic lymph nodes the loss of follicle organization is observed. Consequently, MIAM permits to discriminate between non-neoplastic and neoplastic tissues on the basis of their autofluorescence pattern. Multispectral imaging of tissue autofluorescence may present some advantages with respect to standard histochemical microscopy since it (1) does not require any chemical manipulation of samples; (2) gives real-time results performing the analysis immediately upon specimen resection; and (3) supplies a representation of the biological structure organization linked to endogenous fluorophores.

Incubation of murine bone marrow cells in hypoxia ensures the maintenance of marrow-repopulating ability together with the expansion of committed progenitors.

We developed previously a hypoxic culture system in which progenitors endowed with marrow-repopulating ability (MRA), unlike committed progenitors, were selected and maintained better than in air. We report here an improvement to this system targeted at combining the maintenance of progenitors sustaining MRA with the numerical expansion of multipotent and committed progenitors. Murine bone marrow cells were incubated at 1% oxygen in liquid medium supplemented with stem cell factor, granulocyte colony-stimulating factor, interleukin-6 and interleukin-3. In day 8 hypoxic cultures, the numbers of high proliferative potential and granulocyte/macrophage colony-forming cells (HPP-CFC and CFU-GM) were increased with respect to time zero. Colonies generated by HPP-CFC derived from hypoxic cultures exhibited a high replating ability, whereas colonies generated by HPP-CFC derived from control cultures exhibited a low replating ability. MRA was fully maintained in hypoxia and markedly reduced in air. Thus, severe hypoxia is able to ensure a full maintenance of progenitors sustaining MRA, together with a significant expansion of in vitro-detectable clonogenic progenitors, including those endowed with replating ability. This system could contribute to the improvement of current techniques for the in vitro treatment of human haematopoietic cell populations before transplantation.

A "stealth effect": adenocarcinoma cells engineered to express TRAIL elude tumor-specific and allogeneic T cell reactions.

BALB/c mammary adenocarcinoma cells engineered to express TNF-related apoptosis-inducing ligand (TRAIL)/APO-2 ligand (APO-2L) on their membrane (TSA-TRAIL) grow with kinetics similar to that of parental cells (TSA-pc) in vitro and in nu/nu mice. In contrast, TSA-TRAIL cells grow faster than TSA-pc in normal BALB/c mice. In DBA/2 mice, which differ from BALB/c mice at minor histocompatibility Ags, they also grow faster and display a higher percentage of tumor takes than TSA-pc. In fully histoincompatible C57BL/6 (B6) mice, TSA-TRAIL cells form evident tumors that are slowly rejected by most mice, but outgrow in a few. In contrast, TSA-pc cells are rejected at once by B6 mice. Since TRAIL/APO-2L induces apoptosis by interacting with a variety of specific receptors, this rapid growth in both syngeneic and allogeneic mice may be the result of an immunosuppressive mechanism. The following evidence supports this hypothesis: 1) TSA-TRAIL cells overcome the strong immunity against TSA-pc cells elicited in BALB/c mice by preimmunization with TSA cells engineered to release IL-4; 2) their rejection by B6 mice does not prime a CTL-mediated memory; 3) thymidine uptake by T lymphocytes unstimulated or stimulated by allogeneic cells is inhibited when TSA-TRAIL cells are added as third party cells; 4) CTL kill TSA-pc but not TSA-TRAIL cells in 48-h assays; and 5) activated lymphocytes interacting with TSA-TRAIL cells in vivo and in vitro undergo apoptosis.

Nitric oxide suppresses human T lymphocyte proliferation through IFN-gamma-dependent and IFN-gamma-independent induction of apoptosis.

Human normal and malignant T cells cease to proliferate, down-modulate Bcl-2 expression, and undergo apoptosis when cultured in the presence of NO-donor compounds (sodium nitroprusside and NOC12) for 48 h. At 72 h, cells that evade apoptosis start to proliferate again, overexpress both chains of the IFN-gammaR, and thus become susceptible to apoptosis in the presence of IFN-gamma. By contrast, in the presence of IFN-gamma, no apoptosis, but an increase of proliferation was displayed by control cultures of T cells not exposed to NO and not overexpressing IFN-gammaR chains. The NO-induced cell surface overexpression of IFN-gammaR chains did not affect the transduction of IFN-gamma-mediated signals, as shown by the expression of the transcription factor IFN regulatory factor 1 (IRF-1). However, transduction of these signals was quantitatively modified, because IFN-gamma induces enhanced levels of caspase-1 effector death in NO-treated cells. These findings identify NO as one of the environmental factors that critically govern the response of T cells to IFN-gamma. By inducing the overexpression of IFN-gammaR chains, NO decides whether IFN-gamma promotes cell proliferation or the induction of apoptosis.

Activity of vinorelbine on B-chronic lymphocytic leukemia cells in vitro.

Vinorelbine (VNR) is a new semi-synthetic Vinca rosea alkaloid that has been employed both in combination and as a single agent, showing a significant antitumour activity. Since little is known about VNR in human leukemia, we studied the in vitro cytotoxic effect of VNR on peripheral blood lymphocytes from 18 patients affected by B-chronic lymphocytic leukemia (CLL), employing the INT assay. VNR inhibited fresh B-CLL cells from 15/18 patients in primary cultures, the ID50 doses ranging from 4 ng/ml to 83 micrograms/ml. These data strongly suggest that VNR could be effective in the treatment of B-CLL.

A simple, one-step clonal assay allows the sequential detection of committed (CFU-GM-like) progenitors and several subsets of primitive (HPP-CFC) murine progenitors.

Murine bone marrow (BM) cells were cultured in semisolid medium containing interleukin 3 (IL-3) and high doses of G-CSF. Colonies were counted twice, at day 7 and day 14, and the number of granulocyte/macrophage colony-forming units (CFU-GM) accurately estimated by the subtraction of day-14 from day-7 colonies, based on the principle that colonies detectable at day 7 and persisting beyond day 14 are generated by significantly more immature progenitors. The frequency of colonies relative to their size was determined and used to define subsets of high proliferative potential colony-forming cells (HPP-CFC). Two main groups of HPP-CFC were considered: those generating colonies of 0.6-1.8 mm of diameter or larger than 1.8 mm. The characterization of these groups showed that they correspond to different functional subsets of HPP-CFC. The replating ability of colonies was estimated. The percentage of clonogenic progenitors in the S phase of cell cycle was measured by cytosine arabinoside suicide assay. The sensitivity of colonies to 5-fluorouracil (5-FU) in vitro was determined and their survival after an in vivo treatment with 5-FU compared with that of colony-forming units in spleen (CFU-S). This technique allowed identification of: A) CFU-GM; B) relatively mature HPP-CFC, probably corresponding to CFU-S day12; C) more primitive HPP-CFC, relatively resistant to 5-FU in vivo and closely corresponding to CFU-S day 14, and D) very primitive HPP-CFC, resistant to 5-FU in vitro. This simple, rapid, and versatile method allows the detection of a broad range of hematopoietic progenitors in murine BM, from committed progenitors to largely quiescent, primitive stem cells, as well as the evaluation of the progenitors' self-renewal and proliferative potential.

Functional analysis of T lymphocytes infiltrating the dermis and epidermis of post-burn hypertrophic scar tissues.

The cytokine profile of T cell clones (TCC) from the dermis and epidermis of burn patients with hypertrophic scars (HS) in active (AHS) and remission phases (RHS) was determined in this study. We found that AHS tissues are heavily infiltrated by Type 0-Type 1 polarized CD3+ lymphocytes producing high IFN-gamma and low IL-4 levels. Analysis of their surface marker phenotype showed that the high IFN-gamma production was shared equally between the CD4+ TCRalpha/beta and CD8+ TCRalpha/beta clones. The profile of TCC from RHS tissues revealed pronounced infiltration of Type 0-Type 1 polarized lymphocytes with an even more evident Type 1 profile. However, the levels of IFN-gamma produced by RHS-derived TCC were 4-6 times lower than those produced by AHS-derived TCC. These data show that high levels of IFN-gamma produced by Type 0-Type 1 lymphocytes infiltrating HS are a feature of AHS, whereas reduction of this ability to produce high levels of IFN-gamma, though without a shift towards a Type 0-Type 2 phenotype through an increase in IL-4, is characteristic of RHS.

Disturbance of imagination and symbolization in pervasive developmental disorders: preliminary study utilizing the Rorschach Inkblot Test.

Two clinical cases of children of 6 and 7 years are presented with their respective Rorschach records. The first case had a diagnosis of autism, the second of Pervasive Developmental Disorder Not Otherwise Specified. The modes of elaboration and responses to the ambiguous stimuli of the Rorschach test were compared with the clinical symptoms of the two subjects, which are centered on the two opposite poles of absence of imagination in the first case and distortion of the imaginative processes in the second.