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A series of relatively small studies collectively suggest that zonisamide may be effective in the treatment of infantile spasms. Using a large single‐center cohort of children with infantile spasms, we set out to evaluate the efficacy and safety of zonisamide. We retrospectively identified all patients with infantile spasms who were treated with zonisamide at our center. For each patient, we recorded dates of birth, infantile spasms onset, response (if any), and most recent follow‐up. To quantify zonisamide exposure, we recorded daily dosage and patient weight at each sequential encounter so as to allow calculation of peak and weighted‐average weight‐based dosage. We identified 87 children who were treated with zonisamide, of whom 78 had previously been treated with hormonal therapy or vigabatrin. Peak and weighted‐average zonisamide dosage were 7.1 (interquartile range 3.6, 10.2) and 5.4 (interquartile range 3.0, 8.9) mg/kg/day, respectively. Whereas five (6%) patients exhibited resolution of epileptic spasms, only two (2%) patients exhibited video‐EEG confirmed resolution of both epileptic spasms and hypsarrhythmia (electroclinical response). Importantly, both electroclinical responders had not previously been treated with hormonal therapy or vigabatrin; in contrast, none of the 78 children with prior failure of hormonal therapy or vigabatrin subsequently responded to zonisamide. Zonisamide was well tolerated, and there were no deaths. This study suggests that zonisamide exhibits favorable tolerability but very limited efficacy among patients who do not respond to first‐line therapy.

To investigate spatial correlation between interictal HFOs and neuroimaging abnormalities, and to determine if complete removal of prospectively identified interictal HFOs correlates with post-surgical seizure-freedom.Interictal fast ripples (FRs: 250-500 Hz) in 19 consecutive children with pharmacoresistant focal epilepsy who underwent extra-operative electrocorticography (ECoG) recording were prospectively analyzed. The interictal FRs were sampled at 2000 Hz and were visually identified during 10 min of slow wave sleep. Interictal FRs, MRI and FDG-PET were delineated on patient-specific reconstructed three-dimensional brain MRI.Interictal FRs were observed in all patients except one. Thirteen out of 18 patients (72%) exhibited FRs beyond the extent of neuroimaging abnormalities. Fifteen of 19 children underwent resective surgery, and survival analysis with log-rank test demonstrated that complete resection of cortical sites showing interictal FRs correlated with longer post-operative seizure-freedom (p 0.01). Complete resection of seizure onset zones (SOZ) also correlated with longer post-operative seizure-freedom (p = 0.01), yet complete resection of neuroimaging abnormalities did not (p = 0.43).Prospective visual analysis of interictal FRs was feasible, and it seemed to accurately localize epileptogenic zones.Topological extent of epileptogenic region may exceed what is discernible by multimodal neuroimaging.

Objective Delta-gamma phase-amplitude coupling in EEG is useful for localizing epileptic sources and evaluate severity in children with infantile spasms. We (1) develop an automated EEG preprocessing pipeline to clean data using artifact subspace reconstruction (ASR) and independent component (IC) analysis (ICA) and (2) evaluate delta-gamma modulation index (MI) as a method to distinguish children with epileptic spasms (cases) from normal controls during sleep and awake. Methods Using 400 scalp EEG datasets (200 sleep, 200 awake) from 100 subjects, we calculated MI after applying high-pass and line-noise filters (Clean 0), and after ASR followed by either conservative (Clean 1) or stringent (Clean 2) artifactual IC rejection. Classification of cases and controls using MI was evaluated with Receiver Operating Characteristics (ROC) to obtain area under curve (AUC). Results The artifact rejection algorithm reduced raw signal variance by 29-45% and 38-60% for Clean 1 and Clean 2, respectively. MI derived from sleep data, with or without preprocessing, robustly classified the groups (all AUC > 0.98). In contrast, group classification using MI derived from awake data was successful only after Clean 2 (AUC = 0.85). Conclusions We have developed an automated EEG preprocessing pipeline to perform artifact rejection and quantify delta-gamma modulation index.

OBJECTIVE Favorable neurodevelopmental outcomes in epileptic spasms (ES) are tied to early diagnosis and prompt treatment, but uncertainty in the identification of the disease can delay this process. Therefore, we investigated five categories of computational electroencephalographic (EEG) measures as markers of ES. METHODS We measured 1) amplitude, 2) power spectra, 3) Shannon entropy and permutation entropy, 4) long-range temporal correlations, via detrended fluctuation analysis (DFA) and 5) functional connectivity using cross-correlation and phase lag index (PLI). EEG data were analyzed from ES patients (n = 40 patients) and healthy controls (n = 20 subjects), with multiple blinded measurements during wakefulness and sleep for each patient. RESULTS In ES patients, EEG amplitude was significantly higher in all electrodes when compared to controls. Shannon and permutation entropy were lower in ES patients than control subjects. The DFA intercept values in ES patients were significantly higher than control subjects, while DFA exponent values were not significantly different between the groups. EEG functional connectivity networks in ES patients were significantly stronger than controls when based on both cross-correlation and PLI. Significance for all statistical tests was p < 0.05, adjusted for multiple comparisons using the Benjamini-Hochberg procedure as appropriate. Finally, using logistic regression, a multi-attribute classifier was derived that accurately distinguished cases from controls (area under curve of 0.96). CONCLUSIONS Computational EEG features successfully distinguish ES patients from controls in a large, blinded study. SIGNIFICANCE These objective EEG markers, in combination with other clinical factors, may speed the diagnosis and treatment of the disease, thereby improving long-term outcomes.

ObjectiveFavorable neurodevelopmental outcomes in epileptic spasms (ES) are tied to early diagnosis and prompt treatment, but uncertainty in the identification of the disease can delay this process. Therefore, we investigated five computational electroencephalographic (EEG) measures as markers of ES.MethodsWe measured 1) amplitude, 2) power spectra, 3) entropy, 4) long-range temporal correlations, via detrended fluctuation analysis (DFA) and 5) functional connectivity of EEG data from ES patients (n=40 patients) and healthy controls (n=20 subjects), with multiple blinded measurements during wakefulness and sleep for each patient.ResultsIn ES patients, EEG amplitude was significantly higher in all electrodes. Shannon and permutation entropy were lower in ES patients than control subjects, while DFA intercept values in ES patients were significantly higher than control subjects. DFA exponent values were not significantly different between the groups. EEG functional connectivity networks in ES patients were significantly stronger than controls. Using logistic regression, a multi-attribute classifier was derived that accurately distinguished cases from controls (area under curve of 0.96).ConclusionsComputational EEG features successfully distinguish ES patients from controls in a large, blinded study.SignificanceThese objective EEG markers, in combination with other clinical factors, may speed the diagnosis and treatment of the disease, thereby improving long-term outcomes.HighlightsObjective computational EEG features may aid diagnosis of epileptic spasms (ES)ES EEG has increased delta and theta power and decreased entropy relative to controlsStronger functional connectivity networks differentiate ES patients from controls

Purpose Epileptic spasms are often preceded by focal (or multifocal) seizures. Based on a series of case reports suggesting that carbamazepine and oxcarbazepine may induce epileptic spasms, we set out to rigorously evaluate the potential association between exposure to voltage-gated sodium channel blockade and latency to epileptic spasms. Methods We identified 50 cases (children with focal seizures and evolution to epileptic spasms) and 50 controls (children with focal seizures without evolution to epileptic spasms). For each patient, we reviewed all sequential neurology encounters between onset of epilepsy and emergence of epileptic spasms. For each encounter we recorded seizure-frequency and all anti-seizure therapy exposures. Using multivariable Cox proportional hazards regression, we evaluated the association between voltage-gated sodium channel exposure (carbamazepine, oxcarbazepine, lacosamide, or phenytoin) and latency to epileptic spasms onset, with adjustment for etiology and seizure-frequency. Results Latency to epileptic spasms onset was independently associated with exposure to sodium channel blockade (hazard ratio = 2.4; 95% CI 1.1–5.2; P = 0.03) and high-risk etiology (hazard ratio = 2.8; 95% CI 1.5–5.1; P = 0.001). With assessment for interaction between sodium channel blockade and etiology, we identified an estimated 7-fold increased risk of epileptic spasms with the combination of sodium channel blockade and high-risk etiology (hazard ratio = 7.0, 95% CI 2.5–19.8; P Conclusion This study suggests that voltage-gated sodium channel blockade may induce epileptic spasms among children at risk on the basis of etiology. Further study is warranted to replicate these findings, ascertain possible drug- and dose-specific risks, and identify potential mechanisms of harm.

Background There is ongoing debate regarding the comparative effectiveness of adrenocorticotropic hormone and prednisolone in the treatment of infantile spasms. With a large cohort and extended follow-up, we set out to evaluate a protocol in which adrenocorticotropic hormone is reserved for prednisolone nonresponders. Methods The following standardized hormonal therapy protocol was adopted. Patients initially receive prednisolone (8 mg/kg/day [maximum 60 mg/day], divided in three daily doses for 14 days). Prednisolone responders taper it over 14 days, whereas prednisolone nonresponders immediately transition to natural adrenocorticotropic hormone (150 U/m2/day, divided in two daily doses for 14 days). We evaluated short-term response, defined as video-electroenecphaloagraphy-confirmed resolution of both epileptic spasms and hypsarrhythmia on day 14, without relapse for 28 additional days. We then evaluated long-term relapse and calculated the rates of sustained response at six, 12, and 18 months. Results We identified 102 children with infantile spasms who were treated with prednisolone. Prior exposure to hormonal therapy and vigabatrin was observed among 12% and 35% of patients, respectively. Sixty (59%) patients responded to prednisolone, and 13 (33%) prednisolone nonresponders then responded to adrenocorticotropic hormone. Cumulative response to prednisolone and adrenocorticotropic hormone (if needed) was higher among treatment-naive patients (84%) than among patients with prior exposure to first-line treatment (51%), with P Conclusion Short-term response to prednisolone was favorable and higher among treatment-naive patients. These data suggest that prednisolone is a reasonable approach to initial therapy and that adrenocorticotropic hormone exhibits substantial efficacy after prednisolone failure.

Several small case series provide conflicting impressions of the efficacy of felbamate for treatment of epileptic spasms. Using a large single-center cohort of children with epileptic spasms, we retrospectively evaluated the efficacy and safety of felbamate. We identified all patients with video-EEG confirmed epileptic spasms who were treated with felbamate at our center. We quantified felbamate exposure by calculating peak and weighted-average weight-based dose. Clinical response was defined as resolution of epileptic spasms for at least 28 days, beginning not more than 3 months after felbamate initiation. Electroclinical response was defined as clinical response accompanied by overnight video-EEG demonstrating freedom from epileptic spasms and hypsarrhythmia. Among a cohort of 476 infants, we identified 62 children who were treated with felbamate, of whom 58 had previously failed treatment with hormonal therapy or vigabatrin. Median peak and weighted-average felbamate dosages were 47 and 40 mg/kg/day, respectively. Five (8%) children were classified as clinical responders and two (3%) children were classified as electroclinical responders. Among 17 patients with latency from epileptic spasms onset to felbamate initiation of less than 12 months, we observed 4 (24%) clinical responders. This study suggests that felbamate may be efficacious for treatment of epileptic spasms and that further rigorous study is warranted.

We present the case of a child with long-standing, super-refractory status epilepticus (SRSE) who manifested prompt and complete resolution of SRSE upon exposure to pure cannabidiol. SRSE emerged in the context of remote suspected encephalitis with previously well-controlled epilepsy. We discuss the extent to which response may be specifically attributed to cannabidiol, with consideration and discussion of multiple potential drug–drug interactions. Based on this case, we propose that adjunctive cannabidiol be considered in the treatment of SRSE., Highlights • Adjunctive cannabidiol may be effective in the treatment of super refractory status epilepticus

Objective: To develop a novel software method (AR2) for reducing muscle contamination of ictal scalp electroencephalogram (EEG), and validate this method on the basis of its performance in comparison to a commercially available software method (AR1) to accurately depict seizure-onset location. Methods: A blinded investigation used 23 EEG recordings of seizures from 8 patients. Each recording was uninterpretable with digital filtering because of muscle artifact and processed using AR1 and AR2 and reviewed by 26 EEG specialists. EEG readers assessed seizure-onset time, lateralization, and region, and specified confidence for each determination. The two methods were validated on the basis of the number of readers able to render assignments, confidence, the intra-class correlation (ICC), and agreement with other clinical findings. Results: Among the 23 seizures, two-thirds of the readers were able to delineate seizure-onset time in 10 of 23 using AR1, and 15 of 23 using AR2 (p Conclusions: EEG artifact reduction methods for localizing seizure-onset does not result in high rates of interpretability, reader confidence, and inter-reader agreement. However, the assignments by groups of readers are often congruent with other clinical data. Utilization of the AR2 software method may improve the validity of ictal EEG artifact reduction.

SummaryObjective There is scant evidence to guide the management of infantile spasms after successful response to initial therapies. There is significant risk of relapse, largely because effective pharmacologic treatments cannot be continued long term because of concern for significant adverse events. Zonisamide (ZNS) and topiramate (TPM) are commonly used to prevent relapse, and the purpose of this study was to specifically evaluate the efficacy of ZNS and TPM as agents for secondary prevention of infantile spasms. Methods Patients with video–electroencephalography (EEG) confirmed resolution of infantile spasms were retrospectively identified. Relevant clinical data were systematically collected, including lead time from onset of spasms to successful treatment response, etiology of infantile spasms, number of treatment failures prior to response, timing of relapse, and detailed exposure data for ZNS and TPM. Results We identified 106 patients with response to hormonal therapy (n = 58), vigabatrin (n = 25), or surgery (n = 23). To prevent relapse of infantile spasms, 37 patients received ZNS, 34 received TPM, 3 received both ZNS and TPM, and 38 patients received neither ZNS nor TPM. There were 44 relapses, occurring a median of 6.9 (3.2–10.8) months after initial response. Time to relapse was not affected by treatment with ZNS or TPM. Relapse was less likely among patients who were older (hazard ratio 0.97 [per month], p = 0.036) and those who responded to surgical resection (hazard ratio = 0.28, p = 0.017). Of note, we identified a relatively refractory cohort with multiple treatment failures and long lead time to initial response. Significance In this refractory cohort, neither ZNS nor TPM was successful in preventing relapse of infantile spasms, despite relatively high dosages. At this time, aside from surgical resection in eligible candidates, there is no known treatment that is efficacious in the prevention of relapse of infantile spasms.

Guinea pig peritoneal exudate cells were harvested 3 to 4 days after the intraperitoneal injection of Marcol oil. The washed cells were exposed to various concentrations of sodium periodate in phosphate-buffered saline (PBS) at pH 7.4 for 10 min at +4 degrees C. The cells were then used in the in vitro migration assay, and migration was consistently inhibited at concentrations from 10(-3) to 10(-5) M. The viability of the macrophages was not affected by this treatment. Sodium borohydride (10(-3) to 10(-5) M) in PBS for 10 min at pH 7.4 reversed the periodate effect. Experiments with purified macrophages showed that sodium periodate has a direct effect on macrophage function rather than an indirect effect via the potentiation of migration inhibition factor. In support of this, the in vitro spreading of macrophages on glass substrate for 1 h has been shown to be inhibited. This spreading inhibition can also be reversed by treatment with sodium borohydride. These results provide a new approach to understanding the biological significance and role of macrophage migration inhibition.

Uterine sarcomas are rare, highly malignant tumours comprising < 1 % of all gynaecologic malignancies. To evaluate clinical presentation, histolopathologic pattern and outcome of uterine sarcomas presenting to a tertiary referral centre over an 8 year period (2004–2012). All histologically proven uterine sarcomas were retrospectively analysed. Clinical presentation, histology, treatment and outcome were analysed. Mean age was 42 years. Predominant histopathology was endometrial stromal sarcoma (n = 13); 9 were low grade, carcinosarcoma (n = 8) and leiomyosarcoma (n = 2). Fourteen patients had Stage I disease, 3 Stage II, 4 Stage III and 2 were Stage IV at presentation. Patients with disease confined to uterus received no adjuvant treatment (61 %). Of these, 11 were endometrial stromal sarcoma (7 were low grade) and 3 were carcinosarcomas. Four patients received adjuvant EBRT following hysterectomy (17 %). Two patients who presented with metastases received palliative chemotherapy. Mean follow-up period was 46 months (0–86 months). Eleven patients (47 %) developed disease recurrence. Seven (30 %) had local recurrence, while 4 (17 %) developed pulmonary metastases. A total of eight patients died and all deaths were within 1 year of recurrence. The only prognostic factor that correlated with survival was the stage of disease at diagnosis.

The void swelling behavior of heavy ion irradiated D9 steel has been investigated using variable low energy positron beam. The normalized defect-sensitive S-parameter shows up a large increase in the depth region corresponding to the maximum radiation damage as a function of irradiation temperature. From the variation of S-parameter as a function of irradiation temperature, the peak swelling temperature has been deduced and the results are discussed.

Inflammatory bowel disease has been shown to affect children's health-related quality of life (HRQOL) through the use of lengthy questionnaires. We examined whether a pediatric patient's HRQOL, measured by a rapid visual analog scale ("feeling thermometer"), correlates with the perceptions of the HRQOL as determined by the patient's pediatric gastroenterologist and parent(s). Additionally, we attempted to determine whether the HRQOL correlates with the patient's disease activity as determined by validated activity indices.A cross-sectional study of pediatric patients (ages 7-21 years) who were diagnosed as having Crohn disease, ulcerative colitis, or indeterminate colitis was conducted from January 2011 to May 2011. Each participant (patient, parent(s), and treating pediatric gastroenterologist) completed feeling thermometers to determine the symptom burden as well as therapeutic burden of the patient. The parent(s) and doctor were blinded to the patient's results. Pediatric Ulcerative Colitis Activity Index or a Short Pediatric Crohn Disease Activity Index (S-PCDAI) was calculated. Correlations between the participant's perceived burdens as well as their calculated disease activity were determined.Sixty-seven children and their families participated, resulting in 101 visits. Patients had a mean age of 15.0 years, and there were 38 boys. There was a strong significant correlation between the patient's perceived symptom burden and that of the parent's (ρ 0.59, P0.001) and physician (ρ 0.48, P0.001). Similarly, there was a strong significant correlation between patient's perceived treatment burden and that of the parent treatment burden (ρ 0.49, P0.001) and, to a lesser degree, the physician (ρ 0.29, P0.003). The correlation coefficient was strongest between the physician's perception of the patient's symptom burden against the standard disease activity indices Pediatric Ulcerative Colitis Activity Index (ρ 0.69, P0.001) and Short Pediatric Crohn Disease Activity Index (ρ 0.65, P0.001).The patient's HRQOL was highly correlated to both the physician's and parent's perceptions as well as their disease activity. The feeling thermometer is a quick, easy-to-use, visual analog scale that can be implemented in everyday practice to measure a pediatric patient's HRQOL.

Immunization of BALB/c mice with EBV-CLL-1 cells, derived from Epstein-Barr virus transformed B lymphocytes from a chronic lymphocytic leukemia (CLL) patient, yielded 2 monoclonal antibodies (IgG1 Kappa and IgG2a Kappa) against a membrane antigen on a subset of normal B lymphocytes and non-Hodgkin's lymphomas. Immunofluorescence revealed strong reactivity of the antibodies with EBV-CLL-1 cells and with most lymphocytes in tonsil follicles, in the intestinal wall, around splenic arterioles and near Hassall's corpuscles in the neonatal thymus as well as with a small proportion of lymphocytes in some large reactive lymph node follicles, weak reactivity with 1/5 of peripheral blood B lymphocytes (PBL), and no reactivity with platelets, granulocytes and non-lymphoid tissues. PBL from 3 CLL patients showed weak staining of only larger cells. Intense fluorescence was observed in several non-Hodgkin's lymphomas of various histological types and in Burkitt's lymphoma lines but not in the 3 T lymphoblastoid and 12 nonlymphoid tumor lines examined. The antibodies precipitated Mr 22,000 and 33,000 bands from surface labeled RAJI or EBV-CLL-1 cells and cross-competed in a binding inhibition assay. The antibodies had approximately 6 million binding sites per EBV-CLL-1 or RAJI cell but were not cytotoxic. This high antigen-density and limited expression in normal cells may permit their use for immunocytological diagnosis and targeting cytotoxic agents and radionuclides against appropriate lymphoma cells.

C57 B1/6 mice were immunized with bovine serum albumin (BSA) and Freund’s complete and incomplete adjuvants in various concentrations. Spleen cells from these animals were subsequently stimulated with concanavalin A (Con A), purified protein derivative or BSA, and lymphokine responses were measured in one-stage migration assays. Con A consistently produced macrophage migration inhibition factor (MIF) responses in nonimmunized animals and those immunized with complete adjuvant. This was switched to migration stimulation factor (MStF) responses by prior immunization with incomplete adjuvant. Immunization with complete adjuvant and with BSA alone was followed by MIF responses to antigenic stimulation whereas incomplete adjuvant promoted MStF responses. The MStF responses to both mitogenic and antigenic stimulation were inhibited by the addition of affinity purified interleukin-1 to the spleen cells. Interleukin-1 also inhibited MStF responses and potentiated MIF responses to Con A stimulation by human mononuclear cells whereas interleukin-2 did not influence these responses

Background Oxidative stress by reactive oxygen species (ROS) and nitrosative stress by reactive nitrogen species (RNS) are proven initiators and promoters in carcinogenesis. Elevated ROS/RNS with lowered antioxidants occur in patients with squamous cell carcinoma of oral cavity. Ours is the first study to evaluate the effect of curative resection on both oxidative and nitrosative stress in such patients. Methods This study was conducted on 24 cancer patients and with age- and sex-matched healthy controls. Lipid peroxidation products, nitric oxide (NO) products and ceruloplasmin (CPL) in plasma were measured before and after surgery. Similarly enzymatic antioxidants in erythrocytes and non-enzymatic antioxidants in plasma were measured. Results Statistically significant elevation of lipid peroxidation, NO products and CPL and depletion of antioxidants were found in cancer patients compared with controls. After curative surgical resection there was a statistically significant fall in oxidants and CPL coupled with a rise in antioxidants. Conclusions Our results suggest that oxidative/nitrosative stress could play a significant role in oral cavity cancer (OCC) and that curative resection is effective in alleviating this oxidative/nitrosative burden. Significant mitigation of oxidative/nitrosative stress could indicate the completeness of resection since tumor forms the major source of oxidants. J. Surg. Oncol. 2007;96: 194–199. © 2007 Wiley-Liss, Inc.

We recently described a novel form of cell division termed neosis, which appears to be the mode of escape of cells from senescence and is involved in the neoplastic transformation and progression of tumors (Cancer Biol & Therap 2004;3:207-18). Neosis is a parasexual somatic reduction division and is characterized by (1) DNA damage-induced senescence/mitotic crisis and polyploidization, (2) followed by production of aneuploid daughter cells via nuclear budding, (3) asymmetric cytokinesis and cellularization conferring extended, but, limited mitotic life span to the offspring, and (4) is repeated several times during tumor growth. The immediate neotic progeny are termed the Raju cells, which seem to transiently display stem cell properties. The Raju cells immediately undergo symmetric mitotic division and become mature tumor cells. Exposure of tumor cells to genotoxic agents yields neosis-derived Raju cell progenies that are resistant to genotoxins, thus contributing to the recurrence of drug-resistant tumor growth. Similar events have been described in the literature under different names through several decades, but have been neglected due to the lack of appreciation of the significance of this process in cancer biology. Here we review and interpret the literature in the light of our observations and the recent advances in self-renewal in cancer. Neosis paradigm of self-renewal of cancer growth is consistent with the telomere attrition, aging and origin of cancer cells after reactivation of telomerase, and constitutes an alternative to the cancer stem cell hypothesis. We summarize the arguments favoring Raju cells and not cancer stem cells, as the source of self-renewal in cancer and present a comprehensive hypothesis of carcinogenesis, encompassing various aspects of cancer biology including senescence, tumor suppressor genes, oncogenes, cell cycle checkpoints, genomic instability, polyploidy and aneuploidy, natural selection, apoptosis, endoapoptosis, development of resistance to radiotherapy and chemotherapy leading tumor progression into malignancy.

The scaling behavior of hardness with ligament diameter and vacancy defect concentration in nanoporous Au (np-Au) has been investigated using a combination of Vickers Hardness, Scanning electron microscopy, and positron lifetime measurements. It is shown that for np-Au, the hardness scales with the ligament diameter with an exponent of −0.3, that is, at variance with the conventional Hall-Petch exponent of −0.5 for bulk systems, as seen in the controlled experiments on cold worked Au with varying grain size. The hardness of np-Au correlates with the vacancy concentration CV within the ligaments, as estimated from positron lifetime experiments, and scales as CV1/2, pointing to the interaction of dislocations with vacancies. The distinctive Hall-Petch exponent of −0.3 seen for np-Au, with ligament diameters in the range of 5–150 nm, is rationalized by invoking the constrained motion of dislocations along the ligaments.

Positron lifetime experiments, coupled with scanning electron microscopy studies, have been used to follow the coarsening of the ligaments of nanoporous Au (np-Au), prepared by electrochemical dealloying of Ag75Au25. Positron lifetime measurements in the as-prepared np-Au indicate two lifetime components, identified with annihilation at vacancies within the ligaments and at the ligament-pore interface. The variation of these lifetime parameters with annealing temperature indicates distinct changes that correlate the migration of vacancies to the growth of ligaments. The lifetime component, corresponding to the annihilation at the ligament-pore interface, shows signature of ligament surface instability.

Using a number of drugs that increase cellular cAMP levels, alterations in the amount of cell surface fibronectin and other transformation parameters were studied in Chinese hamster ovary (CHO) cells. The drugs include db-cAMP, different methylxanthines (theophylline, aminophylline, methyl isobutyl xanthine (MIX), caffeine and theobromine), papaverine and cholera toxin. Methylxanthines that have a methyl group at the seventh position lack reverse transforming potential; those that lack a methyl group at the seventh position induced reverse transformation in CHO cells, causing an increase in surface fibronectin, cell substratum adhesive strength and anchorage dependence for growth. Further, as methyl xanthines are substituted in other positions different from the seventh position, the more efficient they become in restoring normal phenotypic properties; the later agents also induced low saturation density via a cytostatic state causing accumulation of cells in the S and G2 phases of the cycle in contrast to the G1 arrest of normal cells at low saturation density. db-cAMP and cholera toxin induced cell elongation but like caffeine and theobromine, did not induce surface fibronectin. The non-methylxanthine phosphodiesterase inhibitor papaverine induced neither cell elongation nor surface fibronectin but produced a cytostatic effect similar to aminophylline and MIX. These studies suggest that the reverse transformation properties fall into two groups: ( a ) Differentiation-related properties including cell morphology, parallel alignment and surface matrix fibronectin, etc.; ( b ) cell cycle-related properties — low saturation density, cell arrest at G1 phase and anchorage-dependent growth. Phosphodiesterase inhibitors reversibly eliminate indefinite division potential of CHO cells by inducing a cytostatic situation and not by inducing a G1-specific arrest.

Several types of normal diploid cells, established ‘normal’ cells, and transformed cells of human and rodent origin have been studied with reference to cell surface fibronectin distribution and their anchorage-independent growth behavior. All cell types that showed intercellular and fibrillar surface fibronectin were anchorage-dependent for growth. Lack of surface fibrillar fibronectin in spontaneously or virus-transformed cells and cells of neoplastic origin was accompanied by anchorage-independent growth potential. The degree of three-dimensional organization of cells under anchorage-independent growth conditions was dependent on the amount of intercellular fibronectin. The significance of these observations to in vivo tumor growth is discussed.

Purified human plasma fibronectin assembles into liposome-like vesicles, called fibrosomes, when layered on agar-coated substrata. Fibrosome assembly is caused by a non-proteinaceous, non-dialysable soluble component that elutes out of the gelled agar; fractionation studies indicated that the sulfated galactans are the active ligands in agar. Fibrosome formation is relatively independent of Ca++, Mg++ ions, and pH, and is not affected by the presence of EDTA, β-mercaptoethanol or dithiothrietol but is sensitive to trypsin. Gel electrophoresis of the recovered fibrosomes showed the typical doublet bands characteristic of plasma fibronectin. Fine structure of fibrosomes showed membrane-like vesicles with no bilayer arrangement, suggesting that under certain circumstances fibronectin can be induced to become amphiphilic which may facilitate hydrophobic interactions with the cell membrane.

While the in vitro adhesion and spreading behaviour of fibroblastic cells have been extensively documented, similar studies on the cells of immunological importance are still lacking. We report here the in vitro adhesion and spreading behaviour of normal human peripheral blood mononuclear cells (PBMC) isolated by the Ficoll-Hypaque technique. Since the presence of serum retarded cell adhesion by 25%, serum-free RPMI-1640 medium was used in these studies. Based on the light and scanning electron microscope studies, glass-immobilized PBMC were classified on the basis of their spreading behaviour as follows: (A) Cells with non-deformable nucleus: I, Cytoplasm non-spread; II, Cytoplasm partially spread; III, Cytoplasm well spread. (B) Cells with deformable nucleus: IV, Nucleus partially spread; cytoplasm well spread; V, Nucleus and cytoplasm well spread. These different cell types occur in the normal human peripheral blood in the relative frequency of 24 ± 3: 27 ± 4: 13 ± 2: 20 ± 5: 16 ± 2 percent. Cell types I–III show the characteristics of lymphocytes and the cells of type V were identified as monocytes, while the nature of cell type IV is not known at present. The immobilized PBMC contained T and B cells as confirmed by sheep erythrocyte (SE) and mouse erythrocyte (ME) rosette formation and surface immunoglobulin. The spreading behaviour of these cells appears to be limited by factors such as available surface membrane area, number and density of adhesion sites, other membrane receptors and the deformability of cell and nuclear membranes. Further characterization of these cell types would aid as a behavioural marker for the subpopulations of lymphocytes and monocytes in health and disease.

Summary Lung-specific, cellular hypersensitivity was studied in patients with chronic pulmonary diseases. To do this, a leucocyte migration inhibition assay was performed using patient and control leucocytes incubated in the presence and absence of a soluble lung extract. Control antigens consisted of liver and kidney extracts. The immunological reactivity of these control antigens was tested by measuring migration, subsequent to incubation of leucocytes from patients with hepatic or renal disease with the liver and kidney extracts, respectively. As a second in vitro test of cell-mediated immunity, a lymphocyte cytotoxicity assay was performed, using as target cells normal chromium-labelled human lung cells. Leucocyte migration inhibition was produced by the lung extract in four out of six patients with chronic bronchitis, five out of seven patients with tuberculosis, one out of five patients with sarcoidosis and in the single patient studied with rheumatoid lung disease. Inhibition did not occur with the leucocytes from control subjects. Neither was migration inhibited when leucocytes from the patients with lung disease were incubated with the control antigens. The reactivity of these control antigens was confirmed by the fact that leucocyte inhibition occurred in two out of three patients with hepatic disease, studied using the liver extract, and in two out of three patients with renal disease, studied with the kidney extract. The cytotoxic potential of anti-lung lymphocytes was demonstrated by the fact that significant target cell cytotoxicity occurred using lymphocytes from two out of four patients with chronic bronchitis, four out of five patients with tuberculosis and in two out of four patients with sarcoidosis.

Polyionenes have been shown recently (A. Rembaum, Appl. Polym. Symp. No. 22, 299, 1973) to produce the following biological effects: (1) bactericidal action, (2) formation of insoluble complexes with DNA and heparin, (3) neuromuscular blocking action, (4) cell aggregation and lysis, and (5) cell adhesion. In the present study, polyionenes of various structures (mainly I3,3, I6,10) were used as molecular probes to gain an understanding of the cell surface phenomena of adhesion on glass- and polyionenes-treated surfaces. Since tumor cells show different surface cell properties, including an increase in the anodic mobility, they bind preferentially to polyionene-treated surfaces. Normal human diploid WI-38 cells were found to adhere at a lower rate than SV-transformed WI-38 cells. However, cell spreading was accelerated in both cases. A study of the interaction of polyionenes in solution in vitro and in vivo and polyionenes covalently bound to polymeric microspheres with leukemic murine EL 4 cells and normal thymocytes showed specific cytotoxity towards the leukemic cells.

Laminin is a complex extracellular matrix molecule consisting of one A-subunit (M r 400KD) and 3 B-subunits (M r 220KD) and is found in the basement membrane. Even though it is now apparent that different cell types are synthesizing laminin-like molecules, the role of these molecules in different systems is not well understood. We have characterized laminin and raised specific antiserum in rabbits. The distribution of laminin was studied by indirect immunofluorescence in different cells such as PFHR-9, WI-38, MRC-5, CHO, 3T3, WI38VA132RA, RAW264-7 and Ki3T3. All normal and transformed cells display a high amount of intracellular submembranous network-like component cross-reacting with antilaminin serum (anti-Lm) and not with anti-fibronectin (anti-Fn) serum as seen by immunofluorescence in permeabilized cells. Preabsorption of anti-Lm with increasing amounts of laminin progressively decreased the staining of the submembranous network. Anti-Lm sera from four other laboratories also showed similar staining pattern. The structural and non-secretory nature of this submembranous staining was confirmed by (a) inhibiting protein synthesis in 0.5% serum and 4 μg/ml puromycin and (b) by immunoelectron microscopy of permeabilized cells. Immunoprecipitation of 3 H-leucine labelled cellular proteins with anti-laminin sera showed proteins of M r 220-210 KD in SDS-PAGE fluorography. These studies suggest that an antigen(s) crossreacting with anti-Lm sera is localized in the membrane associated cytoskeletal region where spectrin/fodrin family of proteins have been localized.

We have studied the effect of serum, plasma and plasma fibronectin on the spreading behavior of several normal and neoplastic human cells using a quantitative spreading assay. Normal human cells are rich in surface fibronectin and are able to spread in the absence of serum or exogenous fibronectin. Virus-transformed cells and cells of neoplastic origin have reduced or no recognizable surface fibronectin and are dependent on serum or plasma for spreading. In agreement with previous studies on rodent cells, fibronectin appears to be the major factor responsible for this effect. Neoplastic cells can spread on fibronectin-coated substrata or can recruit fibronectin directly from the medium. The capacity of transformed cells to recruit fibronectin from the medium correlates with the presence of intercellular fibronectin as seen by the immunofluorescence technique on monolayer cultures. Our results are in keeping with the concept that neoplastic cells may be dependent on extracellular fibronectin for adherence in vivo and that this dependency may vary between different cell types.

The nature and significance of spontaneous association between unsensitized human T lymphocytes and sheep erythrocytes has Seen studied in relation to a possible cytotoxic reaction. Human lymphocytes and 51Cr labelled sheep erythrocytes when mixed in ratios of 5 or 10:l released 70–100% of 51Cr into the supernatants. This suggests that E rosette formation may be the first step in a spontaneous T cell mediated cytotoxic reaction to sheep erythrocytes.

It is proposed that patching, capping and endocytosis, and cell locomotion are manifestations of a single process whereby the cell discards foreign materials. Capping results from the binding to the cell surface of particulate (or molecular) objects which cannot function as immovable substratum. This might be described as unsuccessful or abortive cell adhesion in that the particles adhere to the cell rather than the cell adhering to the substratum. Lateral particle movements on the cell surface membrane are effected by the submembranous microfilament-microtubule system, resulting in capping without displacement of the cell. Successful adhesion of the cell to a substratum renders capping and endocytosis impossible and the cell attempts to discard the substratum by mechanisms analogous to capping. The cell achieves this by lateral movement and detachment of the trailing edge. The concept of abortive adhesion leading to capping has been amplified by the development of molecular models of normal and neoplastic cell adhesion in vitro in the presence and absence of serum. In these models, the normal cells have molecule A (adhesion sites) on their surface; they can spread on the substratum in the absence of serum. In the presence of serum, the A molecules on the normal cell surface bind with B molecules in serum, which may be substratum-bound or free in suspension. Binding of free B molecules with cell surface A molecules results in blockage of adhesion sites; these are cleared via capping. New adhesion sites ( A molecules) are produced at the active edges of the cell. Binding of cell surface A molecules with the substratum bound B molecules results in cell adhesion. Transformed cells do not have A molecules on their surface; they cannot spread in the absence of serum. The transformed cells may recruit A molecules from the serum to attain deformability and spreading. These models also relate to capping of gold or resin particles, cell locomotion and regulation of cell division, and lectin-induced agglutination of transformed cells.

Alterations in the distribution of surface fibronectin during reverse transformation of chinese hamster ovary (CHO) cells induced by dibutyryl adenosine cyclic monophosphate (dbcAMP) and theophylline was examined by indirect immunofluorescence. dbcAMP-induced reverse transformation was not followed by any significant increase in surface fibronectin up to 48 hrs after treatment. Reverse transformation induced by theophylliner by itself or in combination with dbcAMP is followed several hours later by a phenomenal increase in fibrillar surface fibronectin, which is largely persistent even in the presence of cytochalasin-B or colcemid but is sensitive to the presence of cycloheximide. It appears that reverse transformation consists at least of two steps: (a) morphological reversion to normal phenotype and (b) modulation of cell membrane properties or components favouring retention of fibronectin in the cell surface.

Human mononuclear cells from some individuals produce macrophage migration inhibition factor (MIF) when stimulated with Con A while those of others produce migration stimulation factor (MStF). T cells were responsible for these different responses but T4 cells produced MIF and T8 cells produced MStF regardless of the global response which was not explained by the individual T4:T8 ratios. Admixing the T-cell subpopulations in vitro revealed that MIF responses switched to MStF responses between T4:T8 ratios of 75:25 and 50:50 with MStF responders switching at higher ratios than MIF responders. Pulse exposure to supernatants from Con A-stimulated T4-enriched cells significantly reduced migration indices resulting from stimulation of fresh cells, promoting MIF responses regardless of the responder status of the supernatant donor. In contrast, supernatants from T8-enriched cells, when obtained from MStF responders, significantly increased migration indices while there was no effect when the supernatants were obtained from MIF responders. These results suggest that soluble factors from T8 cells are primarily responsible for determining whether an individual mounts a MIF or MStF response to Con A stimulation.

Ionene polymers are polyammonium salts with positive nitrogens in the backbone, resulting from the polycondensation of diamines with dihalides or from the polycondensation of halo-amines. They combine with a variety of molecules of biological importance. The nature of binding and the resulting biological activity depends, to a considerable extent, on the structure and charge density of the polymer.