Search

Unlabelled: Two siblings, a 12-year-old girl and a 7-year-old boy, had recurrent episodes of haemolytic-uraemic syndrome/thrombotic thrombocytopenic purpura, manifested mainly by thrombocytopenia and micro-angiopathic haemolytic anaemia. During 11 years of follow up the girl responded only to steroids, whereas many other therapeutic modalities were ineffective. Following treatment with low dose danazol relapses became fewer and of diminished severity and completely subsided after 6 months. The boy started his illness with signs of haemolytic uraemic syndrome and later developed neurological manifestations. During a 6 year follow up he responded only to plasma exchange. Although chronic thrombocytopenia persisted during the past 3 years, the boy's clinical condition improved.Conclusion: A family with two children with recurrent episodes of thrombotic thrombocytopenic purpura is described. In one child danazol could have had a beneficial effect. [ABSTRACT FROM AUTHOR]

Background: Subjects carrying mutations in the GBA gene, whether in one or two alleles pose a risk to develop Parkinson's disease (PD). This type of GBA-related PD has usually a severe course, and patients tend to develop cognitive deficits faster. Herein we describe 8 cases of GBA-PD patients with a markedly benign course (bGBA-PD), and compared them to the other regular GBA-PD group (rGBA-PD) and to mutation negative patients (MNP)., Methods: All patients who performed next generation testing were enrolled. We defined bGBA-PD as patients with disease duration of at least 5 years, with Hoehn and Yahr (HY) ≤3 and MoCA score of ≥24. Following a selection of patients with a disease duration of 3 years or longer up to 15 years or shorter, we compared the bGBA-PD group to the rGBA-PD and MNP in terms of demographic and clinical features., Results: 166 patients (58 males) diagnosed with PD were genotyped. Twenty-five patients were GBA-PD, of whom 7 were defined as bGBA-PD. Seventeen patients were LRRK2-PD and 123 patients were MNP. The rate of Rapide Eye Movement Behavioral Disorder (RBD) was highest in the rGBA-PD group and lowest in the bGBA-PD group (100 % vs. 28.6 %) (p < 0.001). The bGBA-PD group scored significantly higher in the Montreal Cognitive Assessment (MoCA) than rGBA-PD and MNP (27.6 ± 2.0 vs. 19.8 ± 3.9 vs. 23.5 ± 3.7, respectively; p = 0.006). There were no significant differences in MDS-UPDRS part-3 among the groups. There was no specific GBA mutation which was more commonly associated with bGBA-PD., Conclusion: While GBA-PD has a severe course of disease, a subgroup of which shows a benign course with a relatively preserved cognitive function even years after onset. We might suggest that these cases have other pathomechanisms leading to PD, including an incidental GBA carriership., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Tissue engineering is considered a promising approach to treating advanced degenerative maculopathies such as nonexudative age-related macular degeneration (AMD), the leading cause of blindness worldwide. The retina consists of several hierarchical tissue layers, each of which is supported by a layer underneath. Each of these layers has a different morphology and requires distinct conditions for proper assembly. In fact, a prerequisite step for the assembly of each of these layers is the organization of the layer underneath. Advanced retinal degeneration includes degeneration of the other retina layers, including the choroid, the retinal pigmented epithelium (RPE), and the photoreceptors. Here, we report a step-by-step fabrication process of a three-layer retina-like structure. The process included the 3D printing of a choroid-like structure in an extracellular matrix (ECM) hydrogel, followed by deposition of the RPE monolayer. After the formation of the blood vessel-RPE interface, the photoreceptor cells were deposited to interact with the RPE layer. At the end of the fabrication process, each layer was characterized for its morphology and expression of specific markers, and the integration of the three-layer retina was evaluated. We envision that such a retina-like structure may be able to attenuate the deterioration of a degenerated retina and improve engraftment and regeneration. This retinal implant may potentially be suitable for a spectrum of macular degenerative diseases for which there are currently no cures and may save millions from complete blindness.

Introduction: Post-traumatic stress disorder (PTSD) is characterized by changes in both brain activity and microstructural integrity. Cumulative evidence demonstrates that hyperbaric oxygen therapy (HBOT) induces neuroplasticity and case-series studies indicate its potentially positive effects on PTSD. The aim of the study was to evaluate HBOT's effect in veterans with treatment resistant PTSD., Methods: Veterans with treatment resistant PTSD were 1:1 randomized to HBOT or control groups. All other brain pathologies served as exclusion criteria. Outcome measures included clinician-administered PTSD scale-V (CAPS-V) questionnaires, brief symptom inventory (BSI), BECK depression inventory (BDI), brain microstructural integrity evaluated by MRI diffuse tensor imaging sequence (DTI), and brain function was evaluated by an n-back task using functional MRI (fMRI). The treatment group underwent sixty daily hyperbaric sessions. No interventions were performed in the control group., Results: Thirty-five veterans were randomized to HBOT (N = 18) or control (n = 17) and 29 completed the protocol. Following HBOT, there was a significant improvement in CAPS-V scores and no change in the control (F = 30.57, P<0.0001, Net effect size = 1.64). Significant improvements were also demonstrated in BSI and BDI scores (F = 5.72, P = 0.024 Net effect size = 0.89, and F = 7.65, P = 0.01, Net effect size = 1.03). Improved brain activity was seen in fMRI in the left dorsolateral prefrontal, middle temporal gyri, both thalami, left hippocampus and left insula. The DTI showed significant increases in fractional anisotropy in the fronto-limbic white-matter, genu of the corpus callosum and fornix., Conclusions: HBOT improved symptoms, brain microstructure and functionality in veterans with treatment resistant PTSD., Competing Interests: AH and ES works for AVIV scientific. SE is a shareholder in AVIV scientific.

A family of monomodified bovine serum albumin (BSA) linked to methotrexate (MTX) through a variety of spacers was prepared. All analogues were found to be prodrugs having low MTX-inhibitory potencies toward dihydrofolate reductase in a cell-free system. The optimal conjugates regenerated their antiproliferative efficacies following entrance into cancerous glioma cell lines and were significantly superior to MTX in an insensitive glioma cell line. A BSA-MTX conjugate linked through a simple ethylene chain spacer, containing a single peptide bond located 8.7 Å distal to the protein back bone, and apart from the covalently linked MTX by about 12 Å, was most effective. The inclusion of an additional disulfide bond in the spacer neither enhanced nor reduced the killing potency of this analogue. Disrupting the native structure of the carrier protein in the conjugates significantly reduced their antiproliferative activity. In conclusion, we have engineered BSA-MTX prodrug analogues which undergo intracellular reactivation and facilitate antiproliferative activities following their entrance into glioma cells.

Human serum albumin (HSA) is efficiently taken up by cancer cells as a source of carbon and energy. In this study, we prepared a monomodified derivative of HSA covalently linked to an EDTA derivative and investigated its efficacy to shuttle weakly anti-proliferative EDTA associating ligands such as vanadium, into a cancer cell line. HSA-S-MAL-(CH 2 ) 2 -NH-CO-EDTA was found to associate both with the vanadium anion (+5) and the vanadium cation (+4) with more than thrice the associating affinity of those ligands toward EDTA. Both conjugates internalized into glioma tumor cell line via caveolae-mediated endocytosis pathway and showed potent anti-proliferative capacities. IC 50 values were in the range of 0.2 to 0.3 µM, potentiating the anti-proliferative efficacies of vanadium (+4) and vanadium (+5) twenty to thirty fold, respectively. HSA-EDTA-VO ++ in particular is a cancer permeable prodrug conjugate. The associated vanadium (+4) is not released, nor is it active anti-proliferatively prior to its engagement with the cancerous cells. The bound vanadium (+4) dissociates from the conjugate under acidic conditions with half maximal value at pH 5.8. In conclusion, the anti-proliferative activity feature of vanadium can be amplified and directed toward a cancer cell line. This is accomplished using a specially designed HSA-EDTA-shuttling vehicle, enabling vanadium to be anti-proliferatively active at the low micromolar range of concentration.

Bleomycin is an anticancer antibiotic effective against a range of human malignancies. Yet its usefulness is limited by serious side effects. In this study, we converted bleomycin into a prodrug by covalently linking 2-sulfo, 9 fluorenylmethoxycarbonyl (FMS) to the primary amino side chain of bleomycin. FMS-bleomycin lost its efficacy to bind transition metal ions and therefore was converted into an inactive derivative. Upon incubation in vitro under physiological conditions, the FMS-moiety undergoes spontaneous hydrolysis, generating native bleomycin possessing full anti-bacterial potency. FMS hydrolysis and reactivation takes place with a t 1/2 value of 17 ± 1 h. In silico simulation predicts a narrow therapeutic window in human patients of seven hours, starting 40 min after administration. In mice, close agreement was obtained between the experimental and the simulated pharmacokinetic profiles for FMS-bleomycin. FMS-bleomycin is thus shown to be a classical prodrug: it is inactive at the time of administration and the non-modified (active) bleomycin is released with a desirable pharmacokinetic profile following administration, suggesting it may have therapeutic value in the clinic., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Accurate and timely diagnoses of acute appendicitis (AA) during pregnancy avoids maternal and fetal morbidity and mortality. We present our experience of using an initial transabdominal ultrasound (US) performed at presentation to diagnose AA in pregnant patients as well as the value of a delayed repeat study in those who remain equivocal. We explore the sensitivity and specificity of this algorithm as well as the maternal and fetal safety of this approach., Methods: Of the 225 patients identified within the study period who underwent laparoscopic appendectomy, 216 met the inclusion criteria and were retrospectively analyzed. If the US performed on presentation revealed AA, surgery was performed. Patients with a non-diagnostic US were admitted with surgery performed if there was clinical and/or biochemical deterioration. Patients who remained equivocal underwent a repeat delayed study. The results of the initial versus delayed studies were compared. Maternal and fetal complications were recorded and contrasted., Results: Of the 216 patients included, 164 (75.9%) had AA, 14 (6.5%) had complicated AA and 38 (17.6%) had a normal appendix. Initial US was diagnostic for 125/216 (57.9%) of patients and 19/34 (55.8%) of patients who underwent a delayed repeat study. The remaining patients underwent empirical surgery. The pooled sensitivity and specificity of US for the cohort was 79.2% and 92.1%, respectively. There was no difference in proxies of maternal or fetal safety between the groups., Conclusion: US is a useful tool for diagnosing AA in pregnancy. In this cohort, performing a delayed repeat US during a period of observation in those patients who remained otherwise equivocal increased the diagnostic yield of the US. Delaying surgery in this specific group of patients does not affect maternal or fetal safety.

Aims: Lead perforation is a rare, well-known complication of cardiac implantable electronic device (CIED) implants, whose management is mostly not evidence-based. Main management strategies include conservative approach based on clinical and lead function follow-up vs. routine invasive lead revision approach. This study compared the complications of both strategies by composite endpoint, including recurrent perforation-related symptoms, recurrent pericardial effusion (PEf), lead dysfunction, and device infection during 12 month follow-up., Methods and Results: Multicentre retrospective analysis, inquiring data from imaging studies, device interrogation, pericardiocentesis, and clinical charts of patients with suspected perforating leads between 2007 and 2014 in five hospitals. All cases were reviewed by electrophysiologist and defined as definite perforations by suggestive symptoms along with lead perforation on imaging, bloody PEf on pericardiocentesis shortly after implant, or right ventricular (RV) lead non-capture along with diaphragmatic stimulation upon bipolar pacing. Clinical outcomes associated with both management approaches were compared, with respect to the composite endpoint. The study included 48 definitive perforation cases: 22 managed conservatively and 26 via lead revision. Conservative management was associated with an increased composite endpoint compared with lead revision (8/22 vs. 1/26; P = 0.007). The dominant complication among the conservative cohort was appearance of cardiac tamponade during follow-up; 5/6 occurring in cases which presented with no or only mild PEf and were treated by antiplatelets/coagulants during or shortly after CIED implantation., Conclusion: A conservative management of CIED lead perforation is associated with increased complications compared with early lead revision. Lead revision may be the preferred management particularly in patients receiving antiplatelets/coagulants., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

In this study, we examined the possibility of introducing methotrexate (MTX) to the carboxylate rather than to the ε-amino side chains of proteins. We found that MTX-amino compounds covalently linked to the carboxylate moieties of macromolecules, undergo unusual peptide-bond cleavage, with the release of the MTX amino derivatives from the conjugates. This event takes place at an accelerated rate under acidic conditions, and at a slower rate at physiological pH values. The glutamate portion of MTX is responsible for this behavior, with little or no contribution of the p-aminobenzoate-pteridine ring that is linked to the α-amino side chain of the glutamate. Carboxylate-linked Fmoc-Glu-γ-CONH-(CH2)6-NH2 undergoes hydrolysis in a nearly indistinguishable fashion. A free α carboxylate moiety is essential for this effect. Carboxylate linked Fmoc-glutamic-amide-γ-CONH-(CH2)6-NH2 undergoes no hydrolysis under acidic conditions. Based on these findings, we engineered a cysteine specific MTX containing reagent. Its linkage to bovine serum albumin (BSA) yielded a conjugate with profound antiproliferative efficacy in a MTX-sensitive glioma cell line. In conclusion, carboxylate linked MTX-amino derivatives in particular, and carboxylate linked R-α-GLU-γ amino compounds in general are equipped with'built-in chemical machinery' that releases them under mild acidic conditions.

Despite aggressive therapy, existing treatments offer poor prognosis for glioblastoma multiforme patients, in part due to poor penetration of most drugs across the blood-brain barrier (BBB). We propose a minimal-invasive combined treatment approach consisting of local BBB disruption in the tumor in parallel to systemic drug administration. Local BBB disruption is obtained by convection-enhanced delivery of a novel BBB disruption agent, enabling efficient/targeted delivery of the systemically administered drug by the tumors own vasculature. Various human serum albumin (HSA) analogs were synthesized and screened for BBB disruption efficacy in custom in vitro systems. The candidate analogs were then delivered into naïve rat brains by convection-enhanced delivery and screened for maximal BBB disruption and minimal brain toxicity. These studies found a noncationized/neutralized analog, ethylamine (EA)-HSA, to be the optimal BBB-opening agent. Immunocytochemical studies suggested that BBB disruption by EA-HSA may be explained by alterations in occludin expression. Finally, an efficacy study in rats bearing intracranial gliomas was performed. The rats were treated by convection-enhanced delivery of EA-HSA in parallel to systemic administration of Methotrexate, showing significant antineoplastic effects of the combined approached reflected in suppressed tumor growth and significantly (~x3) prolonged survival.

We studied the effects of chronic angiotensin 1-7 (Ang 1-7) treatment in an experimental model of the metabolic syndrome, i.e., rats given high-fructose/low-magnesium diet (HFrD). Rats were fed on HFrD for 24 weeks with and without Ang 1-7 (576 µg/kg/day, s.c., Alzet pumps). After 6 months, Ang 1-7-treated animals had lower body weight (-9.5%), total fat mass (detected by magnetic resonance imaging), and serum triglycerides (-51%), improved glucose tolerance, and better insulin sensitivity. Similar metabolic effects were also evident, albeit in the absence of weight loss, in rats first exposed to HFrD for 5 months and then subjected to short-term (4 weeks) treatment with Ang 1-7. Six months of Ang 1-7 treatment were associated with lower plasma renin activity (-40%) and serum aldosterone (-48%), less hepatosteatatitis, and a reduction in epididymal adipocyte volume. The marked attenuation of macrophage infiltration in white adipose tissue (WAT) was associated with reduced levels of the pP65 protein in the epididymal fat tissue, suggesting less activation of the nuclear factor-κB (NFκB) pathway in Ang 1-7-treated rats. WAT from Ang 1-7-treated rats showed reduced NADPH-stimulated superoxide production. In single muscle fibers (myofibers) harvested and grown ex vivo for 10 days, myofibers from HFrD rats gave rise to 20% less myogenic cells than the Ang 1-7-treated rats. Fully developed adipocytes were present in most HFrD myofiber cultures but entirely absent in cultures from Ang 1-7-treated rats. In summary, Ang 1-7 had an ameliorating effect on insulin resistance, hypertriglyceridemia, fatty liver, obesity, adipositis, and myogenic and adipogenic differentiation in muscle tissue in the HFrD rats.

Most chemotherapeutic agents are blood-brain barrier (BBB) impermeants. HIV-1-derived TAT protein variants contain a transmembrane domain, which may enable them to cross the BBB and reach the brain. Here we synthesized CAYGRKKRRQRRR, a peptide containing a cysteine moiety attached to the N terminus of the transmembrane domain (C-TAT peptide), and studied its effects in an in vitro BBB model, which we found to reflect penetration by a receptor-independent pathway. Incubation of the brain capillary endothelial cell monolayer with 0.3-0.6 μmol/ml of this C-TAT peptide, for a period of 1-2 h, destabilizes brain capillary endothelial cell monolayer and introduces the ability of impermeant therapeutic agents including high molecular weight proteins to penetrate it substantially. The cysteinyl moiety at position 1 of the C-TAT peptide contributes largely to the destabilizing potency and the penetration efficacy of impermeant substances. The destabilizing effect was reversed using heparin. In summary, experimental conditions allowing a significant increase in entry of impermeant low and high molecular weight substances from the luminal (blood) to the abluminal side (brain) were found in an in vitro BBB model reflecting in vivo protein penetrability by a receptor-independent pathway.

We found that human serum albumin (HSA) contains a single binding domain for derivatives of long-chain fatty acid (LCFA)-like molecules in which the carboxylate is replaced by sulfonate. Accordingly, we have synthesized 16-sulfo-hexadecanoic acid-N-hydroxysuccinimide ester [HO(3)S-(CH(2))(15)-CONHS], an agent that reacts selectively with the amino side chains of peptides and proteins. A macromolecule containing a single 16-sulfohexadecanoate moiety associating with albumin with a K(a) value of 0.83 ± 0.08 × 10(6) M(-1), a sufficient affinity to extend the actions in vivo of such short-lived peptides and proteins. Subcutaneous administration of insulin-NHCO-(CH(2))(15)-SO(3)(-) into mice facilitated a glucose-lowering effect 4.3 times in duration and 6.6 times in area under the curve (AUC) as compared to an in vitro equipotent amount of Zn(2+)-free insulin. Similarly, subcutaneous and intravenous administration of exendin-4-NHCO-(CH(2))(15)-SO(3)(-) to mice yielded prolonged and stable reduction in glucose level, 5-9-fold longer than that of exendin-4. Also, a single subcutaneous administration of human interferon-α2-[NH-CO-(CH(2))(15)-SO(3)(-)](3) to mice yielded circulating antiviral activity over a period of 40 h. In conclusion, a simple, hydrophilic reagent has been engineered, synthesized, and studied. Its linkage to peptides and proteins in a monomodified fashion yielded hydrophilic, prolonged acting derivatives, due to their acquired ability to associate with serum albumin after administration.

Several important pharmacological features can be integrated into injected drugs to enhance their therapeutic efficacy following administration. Short-lived peptide/protein drugs should be converted into long-lived species in vivo to avoid multiple injections. Circulating levels of anticancer agents need to be maintained within a narrow therapeutic range for prolonged period. Water-insoluble drugs must be turned into soluble species and blood-brain barrier-impermeable agents need to be modified to cross it following peripheral administrations. The derivatization requiring for achieving those desirable pharmacological features typically result in biologically/pharmacologically inactive products, unless those derivatizations can be carried out in a reversible fashion.

Cystine disulfide bond is a common feature in numerous biologically active peptides and proteins and accordingly its replacement by various surrogates presents a potential route to obtain analogs with improved pharmacokinetic characteristics. The purpose of the present study was to assess whether an azo-bridge can serve as such a surrogate. In view of the marked clinical significance of somatostatin and the brain natriuretic peptide (BNP) we choose these peptides as a model. Three cyclic-azo somatostatin analogs and three cyclic-azo BNP analogs were effectively prepared in solution through azo bond formation between p-amino phenylalanine and His or Tyr residues that were positioned in the peptide sequences in place of the native Cys residues. The peptides binding affinities to the sst₂ and ANP-receptor (NPR-A) expressed on rat acinar pancreating carcinoma AR4-2J cell membranes and HeLa cells, respectively, were examined. The somatostatin analogs displayed good to moderate affinities to the rat sst₂ in the nM range with best results obtained with peptide 2, that is, IC₅₀ = 8.1 nM. Molecular dynamics simulations on these peptides suggests on a correlation between the observed binding potencies and the degree of conformational space overlapping with that of somatostatin. The BNP analogs exhibited binding affinities to the NPR-A in the nM range with best results obtained with BNP-1, that is, IC₅₀ = 60 nM., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objective: To investigate pregnancy and labour outcomes in grand and great grand multiparous women., Study Design: A retrospective population-based study was conducted between the years 1988 and 2007. Parturients were classified into three groups: multiparous; 2-5 deliveries, grand multiparous; 6-9 deliveries, and great grand multiparous; 10+ deliveries. Stratified analyses included multiple logistic regression models., Results: A significant linear association was found between parity and adverse maternal and perinatal outcomes such as malpresentation, labour dystocia, caesarean delivery, postpartum haemorrhage, maternal anaemia (HB<10), congenital malformations and perinatal mortality. Using multivariable logistic regression models, grand multiparity was found an independent risk factor for labour dystocia, first stage (OR=1.5, P<0.001), and perinatal mortality (OR=2.0, P<0.001). Great grand multiparity was found an independent risk factor for labour dystocia, first stage (OR=2.6, P<0.001), labour dystocia, second stage (OR=2.1, P<0.001), and perinatal mortality (OR=2.5, P<0.001)., Conclusion: Women with high birth order are at increased risk for adverse obstetric outcomes. The risk is higher for great grand multiparous women compared to grand multiparous women. Grand and great grand multiparity are independent risk factors for labour dystocia and perinatal mortality.

Many peptides with the potential of therapeutic action for brain disorders are not in clinical use because they are unable to cross the blood-brain barrier (BBB) following peripheral administration. We have developed two potential strategies for the delivery of peptides to the brain and demonstrated their feasibility with enkephalins. In the first approach, designated induced reversible lipophilization, Leu/Met Enkephalins were converted to 9-fluorenylmethoxycarbonyl (Fmoc) derived lipophilic prodrug analogues, which undergo slow, spontaneous hydrolysis under physiological conditions, generating the native agonists. In contrast to Enkephalin, Fmoc-Met-Enkephalin was found to facilitate an analgesic effect following intraperitoneal administration in mice. Fmoc-Leu-Enkephalin was not analgesic. In the second approach, Enkephalin was linked to BBB transport vectors through an Fmoc based linker spacer, forming conjugates that slowly release Enkephalin under physiological conditions. A pronounced antinociceptive response was thus obtained following intraperitoneal administration of either cationized-human serum albumin-Fmoc-Enkephalin or polyethylene glycol(5)-Fmoc-Enkephalin. Derivatives of Enkephalin covalently linked to the same BBB-transport vectors through a stable (nonreversible) chemical bond were not analgesic. In summary, we have demonstrated that lipophilicity can be conferred to hydrophilic peptides to a degree permitting the permeation of the BBB by passive diffusion, without the drawback of agonist inactivation, which is often caused by irreversible derivatization. Similarly, in the second strategy, the conjugation to BBB-permeable vectors overcomes the obstacle of peptide inactivation by releasing the active form in the central nervous system.

Pegylation is a powerful technology to prolong the action of proteins in vivo, but it is impractical for low-molecular-weight (LMW) drugs, which are usually inactivated upon such modification. Here, we have applied a recently developed strategy of reversible pegylation to gentamicin, a LMW antibiotic. Variable length polyethyleneglycol (PEG-SH) chains were covalently linked to gentamicin using two heterobifunctional agents, each containing a spontaneously hydrolyzable bond. The inactive derivatives regained full antibacterial potency upon incubation under physiological conditions in vitro, and following systemic administration to rats, they released native active gentamicin with half-lives 7- to 15-fold greater than those of systemically administered nonderivatized gentamicin. In conclusion, reversibly pegylated prodrug derivatives of gentamicin were found to be capable of releasing gentamicin for prolonged periods in vivo. Most importantly, the major drawback of conventional pegylation, namely, the loss of pharmacological potency following irreversible derivatization, has been overcome.

Natriuretic peptides (NP), including atrial natriuretic peptide (ANP), induce potent natriuresis and vasodilation and thereby generate hypotension in vivo. Despite intensive efforts, clinical application of NP as an antihypertensive agent is limited because of their short biological half-life and poor bioavailability. Recently, we have developed a strategy that facilitates slow release of peptides from PEG-peptide inactive conjugates, based on reversible pegylation. Peptides prepared by this approach undergo slow, spontaneous chemical hydrolysis at physiological conditions, releasing the native active peptide/protein drug from the inactive conjugates over prolonged periods. A PEG chain of 30 kDa was linked covalently to the alpha-amino side chain of the hormone via a MAL-Fmoc-NHS spacer, yielding PEG 30-Fmoc-ANP, a prodrug that releases the native hormone upon incubation at physiological conditions. Bolus administration of native ANP to Wistar rats receiving adrenaline yields a short, transitory effect in lowering blood pressure (BP), reaching a maximum at 2 min, and then returning to control values after 12 to 25 min. In contrast, administration of PEG 30-Fmoc-ANP lowered BP following a lag period of 50 min, and maintained low BP for a period exceeding 60 min. Saline or PEG 30-Fmoc-Alanine were not effective in lowering BP in Wistar rats. These results show that the novel compound, PEG 30-Fmoc-ANP, is a reversible pegylated prodrug derivative that facilitates a prolonged BP lowering effect in rats and may be considered as a candidate for development into an antihypertensive drug.

The covalent linkage of peptides or protein drugs to human serum albumin (HSA) greatly prolongs their lifetime in vivo but is pharmacologically irrelevant when it irreversibly inactivates them. We retain drug bioactivity by synthesizing a heterobifunctional reagent (MAL-Fmoc-OSu, 9-hydroxymethyl-2-(amino-3-maleimidopropionate)-fluorene-N-hydroxysuccinimide) that generates HSA-Fmoc-insulin on covalent conjugation to the amino group of insulin and the Cys-34 side chain of HSA. HSA-Fmoc-insulin is water-soluble and, upon incubation in aqueous buffers reflecting normal human serum conditions, slowly, spontaneously, and homogeneously hydrolyzes to release unmodified insulin with a t 1/2 of 25 +/- 2 h. A single subcutaneous or intraperitoneal administration of HSA-Fmoc-insulin to diabetic rodents lowers circulating glucose levels for about 4 times longer than an equipotent dose of Zn2+-free insulin. Following subcutaneous administration, onset of the glucose-lowering effect is delayed 0.5-1 h and persists for 12 h. Thus, we present a prototype insulin formulation possessing three desirable parameters: high aqueous solubility, delayed action following subcutaneous administration, and prolonged therapeutic effect.

Administration of peptide YY(3-36) (PYY(3-36)) to fasting humans or mice shortly before re-feeding effectively reduced their food intake, but PYY(3-36) exhibited a functional half-life of only approximately 3 h. Attachment of poly(ethylene glycol) to proteins and peptides (PEGylation) prolongs their half-life in vivo, but completely inactivated PYY(3-36). We developed a reversibly PEGylated PYY(3-36) derivative by coupling it to a 40 kDa PEG through a spontaneously cleavable linker. The resulting conjugate (PEG(40)-FMS-PYY(3-36)) gradually released unmodified PYY(3-36) in vivo, exhibiting an eightfold increase in its functional half-life, to approximately 24h. This long-acting PYY(3-36) pro-drug may serve as an effective means for controlling food intake in humans.

Many peptide and protein drugs have a short circulatory half-life in vivo. The covalent attachment of polyethylene glycol (PEG) chains (PEGylation) can overcome this deficiency, but pegylated peptides and proteins are often inactive. In this study, we present a novel PEG-IFNalpha2 conjugate, PEG(40)-FMS-IFNalpha2, capable of regenerating native interferon alpha2 (IFNalpha2) at a slow rate under physiological conditions. A 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) containing bifunctional reagent, MAL-FMS-NHS, has been synthesized, enabling the linkage of a 40 kDa PEG-SH to IFNalpha2 through a slowly hydrolyzable bond. By use of a BIAcore binding assay, the in vitro rate of regeneration of native interferon was estimated to have a half-life of 65 h. Following subcutaneous administration to rats and monitoring circulating antiviral activity, active IFNalpha2 levels peaked at 50 h, with substantial levels still being detected 200 h after administration. This value contrasts with a half-life of about 1 h measured for unmodified interferon. The concentration of active IFNalpha2 scaled linearly with the quantity injected. Comparing subcutaneous to intravenous administration of PEG(40)-FMS-IFNalpha2, we found that the long circulatory lifetime of IFNalpha2 was affected both by the slow rate of absorption of the PEGylated protein from the subcutaneous volume and by the slow rate of discharge from the PEG in circulation. A numerical simulation of the results was in good agreement with the results observed in vivo. The pharmacokinetic profile of this novel IFNalpha2 conjugate combines a prolonged maintenance in vivo with the regeneration of active-native IFNalpha2, ensuring ready access to peripheral tissues and thus an overall advantage over currently used formulations.

Polyethylene glycol (PEG)-conjugated therapeutic peptides/proteins have been shown to exhibit clinical properties superior to those of their corresponding unmodified parent molecules. However, the desirable pharmacological features gained by protein PEGylation become irrelevant if conjugates are inactivated or cannot reach their target tissues. Here we describe the design and synthesis of MAL-FMS-OSU. This bifunctional agent enables PEG chains to be linked to peptides and proteins through a slowly hydrolysable chemical bond. PEG-FMS-peptide/protein conjugates thus formed undergo spontaneous hydrolysis at a slow rate upon incubation at pH 8.5, 37 degrees C with a t(1/2) value of 8-14 +/- 2 h, generating the unmodified parent molecule. The validity of this approach was studied with exendin-4 and human growth hormone. A single subcutaneous administration of PEG(40,000)-FMS-exendin-4 facilitated a prolonged and stable reduction in glucose levels in mice (t(1/2) = 30 +/- 2 h) and exceeded the effect obtained by the same dose of the native hormone by 7-8 times.

FMS(3)-insulin (2-sulfo-9-fluorenylmethoxycarbonyl)(3)-insulin is a water soluble, inactive-reactivated derivative of insulin with protracted action in vivo. In this study we find that FMS(3)-insulin preserves insulin's capacity to crystallize when associated with Zn(2+) ions or with basic protamine. Zinc or protamine suspended preparations of FMS(3)-insulin manifest substantially prolonged, blood glucose-lowering pharmacokinetic profiles in STZ-treated rats (STZ-rats). A dose of up to 1mg suspended FMS(3)-insulin/STZ rat can be subcutaneously administered with no hypoglycemic episodes at any time point after administration. This dose yielded glucose-lowering profiles with t(1/2) values at the range of 50-70h, turning catabolic STZ-rats into anabolic ones over a period of 2-3 days. The obtained glucose-lowering patterns exceeded 7-8 times in duration those produced by nonhypoglycemic doses of NPH-insulin. In summary, subcutaneous administration of suspended insulin prodrugs, such as FMS(3)-insulin, can bring about prolonged, nonhypoglycemic glucose-lowering profiles, unattainable with insulin preparations, which are known to be active at the time of administration.

Exendin-4, a glucagon-like peptide-1 agonist, is a relatively short-lived species in the circulatory system in vivo. We have linked three moieties of 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) to the three amino functions of exendin-4. FMS(3)-exendin-4 thus obtained has about 0.1% the glucose-lowering potency of the native peptide. Upon in vitro incubation at physiological conditions, the FMS moieties undergo hydrolysis generating the parent fully active, exendin-4. A single subcutaneous administration of FMS(3)-exendin-4 to healthy and type II diabetic mice has induced a glucose-lowering profile that exceeds in length several times that obtained by the native peptide. The reduction of blood glucose level began 1h after administration and was maximal 3-4h later. The blood glucose level returned to pre-administered values with t(1/2) of 12+/-0.7, 26+/-2, and 44+/-3h with doses of 1, 10, and 100 micro g/mouse, respectively. In sum, we have synthesized and characterized FMS(3)-exendin-4, a prodrug derivative of the native insulinotropic peptide, and found it to facilitate a prolonged and stable, glucose-lowering action in healthy and diabetic mice.

Tetramethylrosamine (TMR) is excluded from P-glycoprotein (MDR1)-enriched cell lines, but it stains efficiently MDR1-poor parent lines. Application of the TMR resistance assay to cells obtained from chronic myelogenous leukaemia (CML) patients revealed, in all individuals, a significant resistance compared with healthy donors (P < 0.001). Cells from the same patients at later phases exhibited a further increase in TMR resistance. Doxorubicin was excluded from all cell samples obtained from CML patients at presentation. The resistance to TMR and doxorubicin was energy-dependent, and was not modulated by inhibitors of MDR1 and multidrug-resistance protein-1 (MRP1). Transcription of mRNAs suspected as relevant to multidrug resistance was assessed using comparative reverse transcription polymerase chain reaction. All cells from the CML patients transcribed high levels of MRP3, MRP4 and MRP5 compared with healthy donors. Low levels of MDR1, MRP1, MRP2, MRP6, lung resistance-related protein and anthracycline resistance-associated protein were equally transcribed in cells from healthy donors and CML patients. These results indicated that neither MDR1 nor MRP1 mediate the resistance in these cells. Our results shed light on a resistance mechanism operative in CML patients, which, together with the resistance to apoptosis, is responsible for the lack of response of CML patients to induction-type protocols used to treat acute myeloid leukaemia patients.

Objective: To study the qualities of laser myringotomy (LM) as a treatment for middle ear ventilation problems., Design: Prospective study and follow-up of consecutive cases of adults, children, and infants. Patients were observed for up to 2 years., Setting: Children underwent LM, with or without adenoidectomy, under general anesthesia in the operating room. Adults and infants underwent LM under topical anesthesia, as an outpatient procedure., Patients: All consecutive patients with either secretory otitis media (SOM) (adults and children) or acute otitis media (AOM) (infants) who agreed to participate were included without selection., Intervention: Myringotomy was performed using new laser equipment, enabling a 0.1-second ablation with changeable diameter., Outcome Measures: Close follow-up, with microscopic examination of all ears. Findings were noted on the medical charts., Results: Among all age groups, 136 ears were followed up. Perforation lasted a mean 22 days in adults, 17 days in children, and 11 days in infants. Patient age was found to be a significant determining factor for duration of perforation (P =.002). Laser myringotomy in the anterior and inferior areas lasted longer than posterior LM (P<.001). In patients with SOM, during the time the LM was patent, all ears were ventilated. In children, 38% of SOM cases resolved after a single LM treatment. All infants with AOM recovered promptly without antibiotic treatment., Conclusions: Laser myringotomy is a convenient, quick procedure that can be performed in the medical office with the use of topical anesthesia and is suitable for patients with AOM or for those who need short-term ventilation for SOM. It was found to be a safe alternative to ventilation tubes in these patients. In AOM, it was used instead of antibiotics and gave prompt relief from symptoms and cure of the AOM.

Polypeptide drugs are generally short-lived species in circulation. In this study, we have covalently linked seven moieties of 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) to the amino groups of human interferon-alpha2. The derivative thus obtained (FMS(7)-IFN-alpha2) has approximately 4% the biological potency and 33 +/- 4% the receptor binding capacity of the native cytokine. Upon incubation, FMS(7)-IFN-alpha2 undergoes time-dependent spontaneous hydrolysis, generating active interferon with t(1/2) values of 24 +/- 2 h at pH 8.5 and 98 +/- 10 h at pH 7.4. When native IFN-alpha2 is intravenously administered to mice, circulating antiviral activity is maintained for a short duration and then declines with t(1/2) = 4 +/- 0.5 h, reaching undetectable values at approximately 18 h after administration. With intravenously administered FMS(7)-IFN-alpha2, there is a lag period of 2 h, followed by a progressive elevation in circulating antiviral-active protein, which peaked at 20 h and declined with t(1/2) = 35 +/- 4 h. FMS(7)-IFN-alpha2 is resistant to alpha-chymotrypsin digest and to proteolytic inactivation by human serum proteases in vitro. We have thus introduced here an inactive IFN-alpha2 derivative, which is resistant to in situ inactivation and has the capability of slowly reverting to the native active protein at physiological conditions in vivo and in vitro. Having these attributes, FMS(7)-IFN-alpha2 maintains prolonged circulating antiviral activity in mice, exceeding 7-8 times the activity of intravenously administered native cytokine.

Atypical hemolytic uremic syndrome (HUS) is a heterogeneous group of disorders, the pathogenesis of which is unclear. Plasma transfusions and plasmapheresis are widely used modes of therapy for adults with this life-threatening syndrome. There is very limited experience in using plasmapheresis therapy in children and infants with atypical HUS. Plasmapheresis, which is considered a relatively safe procedure in adults and older children, may be hazardous in neonates and very young infants and can result in severe complications. We report a 2-month-old infant with idiopathic atypical HUS, who was successfully treated with a 1-month course of plasmapheresis during the acute phase of the disease. Appropriate preparations as well as several adjustments were made in order to meet the special needs of this very young infant who, to the best of our knowledge, is the youngest reported patient with atypical HUS to undergo plasmapheresis. Plasmapheresis therapy of the infant was not associated with any complications of the procedure and resulted in marked clinical improvement. We conclude that plasmapheresis in neonates and in very small infants is technically feasible, can be performed without major complications, and may be of benefit in individual cases.

Several ligands, when complexed with vanadium, potentiate its insulinomimetic activity both in vivo and in vitro. We have recently found that L-Glu-gamma-monohydroxamate (HXM) and L-Asp(beta)HXM were especially potent in this regard. In the present study, we used vanadium-enriched adipose cells and cell-free experimental systems to determine the features of L-Glu(gamma)HXM and L-Asp(beta)HXM that turn these ligands into optimal-synergizing vanadium chelators. We found that L-Glu(gamma)HXM and L-Asp(beta)(HXM) possess the following characteristics: 1) They associate with vanadium(+5) at pH 7.2 within a narrow range of an apparent formation constant of 1.3 to 1.9 x 10(2) M(-1); 2) they have nearly the same binding affinity for the vanadyl(+4) cation and the vanadate(+5) anion at physiological pH values; and 3) they form intense ultraviolet absorbing complexes upon associating with vanadium(+4) at 1 and 3 M stoichiometry, respectively, at pH 3.0. Vanadium ligands lacking any of these three defined criteria synergize less effectively with vanadium to activate glucose metabolism.

Vanadate mimics the metabolic actions of insulin. In diabetic rodents, vanadate also sensitizes peripheral tissues to insulin. We have analyzed whether this latter effect is brought about by a mechanism other than the known insulinomimetic actions of vanadium in vitro. We report that the levels of glucose 6-phosphate (G-6-P) in adipose, liver, and muscle of streptozotocin-treated (STZ)-hyperglycemic rats are 77, 50, and 58% of those in healthy control rats, respectively. Normoglycemia was induced by vanadium or insulin therapy or by phlorizin. Vanadate fully restored G-6-P in all three insulin-responsive peripheral tissues. Insulin did not restore G-6-P in muscle, and phlorizin was ineffective in adipose and muscle. Incubation of diabetic adipose explants with glucose and vanadate in vitro increased lipogenic capacity three- to fourfold (half-maximally effective dose = 11 +/- 1 microM vanadate). Lipogenic capacity was elevated when a threshold level of approximately 7.5 +/- 0.3 nmol G-6-P/g tissue was reached. In summary, 1) chronic hyperglycemia largely reduces intracellular G-6-P in all three insulin-responsive tissues; 2) vanadate therapy restores this deficiency, but insulin therapy does not restore G-6-P in muscle tissue; 3) induction of normoglycemia per se (i.e., by phlorizin) restores G-6-P in liver only; and 4) glucose and vanadate together elevate G-6-P in adipose explants in vitro and significantly restore lipogenic capacity above the threshold of G-6-P level. We propose that hyperglycemia-associated decrease in peripheral G-6-P is a major factor responsible for peripheral resistance to insulin. The mechanism by which vanadate increases peripheral tissue capacity to metabolize glucose and to respond to the hormone involves elevation of this hexose phosphate metabolite and the cellular consequences of this elevated level of G-6-P.

The activating effect of vanadate on glycogenesis and on glycogen synthase (uridine diphosphate-glucose-glycogen glucosyl transferase) activity was studied in rat adipocytes and compared with that of insulin. Using several approaches and specific blockers, we found that vanadate and insulin resemble each other, in the activation of glycogen synthase, in several aspects: both require nonarrested protein phosphatase 1 activity; they are equally suppressed by conditions that elevate cAMP-levels; and both depend on the activation of phosphatidylinositol-3 kinase. The basic differences between them are as follows: 1) vanadate promotes glycogenesis through the activation of a cytosolic protein tyrosine kinase, in an insulin-receptor-independent manner; 2) vanadate elevates glucose-6-phosphate (G-6-P) to a higher level than insulin; 3) vanadate-activated glycogenesis is accompanied by an increase in the cellular content of immunoreactive glycogen synthase, an effect less noticeable with insulin; 4) adipose glucose-6-phosphatase is inhibited by vanadate (dose for 50% inhibition, IC50 = 7 +/- 0.7 microM) but not by insulin. We have concluded that insulin and vanadate activate glycogenesis through a phosphatidylinositol-3 kinase and dephosphorylation-dependent mechanism. Vanadate, however, uses a receptor-independent pathway and is superior to insulin in elevating the level of G-6-P, a key metabolite for activating glycogen synthase. This is attributed to the combined effect of vanadate in enhancing glucose entry and in inhibiting dephosphorylation of endogenously formed G-6-P. The latter effect is not exerted by insulin.

We have established an experimental system in rats in which the lipogenic capacity of adipose tissue was decreased in vivo by prolonged fasting, and restored in vitro by glucose together with insulin or vanadate. Incubation of fasted adipose explants for 5 h at 37 C with 2 mM glucose alone did not elevate lipogenic capacity. However, glucose with insulin (17 nM) or vanadate (100 microM), led, respectively, to 2.2- and 8- to 10-fold elevation. Actinomycin D (50 microM) completely blocked this increase, while low concentrations (ED50 = 4.0 +/- 0.4 microM) of vanadate potentiated it. Neither insulin nor vanadate elevated fasted adipose explants' lipogenic capacity in the absence of glucose, or in the presence of the nonmetabolizable glucose analog 3-O-methylglucose. Upon replacing glucose with 2-deoxyglucose (1 mM), a glucose analog that undergoes phosphorylation to 2-deoxyglucose-6-phosphate with no further metabolism, vanadate was nearly as potent as with glucose in elevating lipogenic capacity. Vanadate was superior to insulin in increasing glucose-6-phosphate level in fasted-adipose explants. Adipose glucose-6-phosphatase activity was inhibited by vanadate (IC50 = 7.0 +/- 0.4 microM). We have concluded that glucose-6-phosphate is the key metabolite of glucose involved in the transcriptionally regulated elevation of lipogenic capacity of fasted adipose explants. Vanadate has a more profound effect than insulin, as it elevates glucose-6-phosphate to higher levels and the subsequent increase in lipogenic capacity is four to five times greater than that induced by insulin. The mechanism involved is the combined action of vanadate in enhancing glucose entry and in inhibiting dephosphorylation of endogenously formed glucose-6-phosphate. The latter effect is not exerted by insulin.

Vanadium salts mimic most metabolic effects of insulin in vitro. We report here that vanadyl sulfate (VOSO4) and sodium vanadate (NaVO3) stimulate net K+ uptake in isolated perfused rat liver. Stimulation was evident at low concentrations of vanadyl ions (range 1-20 microM) and occurred within minutes following the addition of VOSO4. By comparison with VOSO4, insulin had less of a stimulatory effect on K+ uptake. Ouabain prevented the activating effect of VOSO4 on K+ uptake. Following a VOSO4 challenge, measured intracellular Na+ concentration ([Na+]i) fell (control, 17.1 +/- 1.2; VOSO4-treated, 13.0 +/- 1.1 mmol.g-1 wet weight, P = 0.027). The results indicate that active K+ uptake via the Na+/K+-ATPase was stimulated by vanadyl ions. An indirect mechanism due to changes in [Na+]i can be excluded. The tyrosine kinase inhibitor genistein was found to inhibit stimulation of K+ by vanadyl and vanadate ions which are known inhibitors of phosphotyrosine phosphatases. We conclude that stimulation of active K+ influx involves a tyrosine kinase. Possible mechanisms include phosphorylation at tyrosine residues and direct activation of the Na+/K+-ATPase, or phosphorylation of other proteins that regulate the activity or number of pumps in the cells.

We report here on a novel procedure for measuring glycogenolysis in rat adipocytes. In this procedure, cells are incubated for 30 min at 37 degrees C with insulin or vanadate, and with [U-14C]glucose to label the glycogen pool with radioactive glucose. The cells are washed and preincubated for an additional 1 h, before being assayed. The extent of glycogenolysis is determined by the decrease in radioactivity in precipitated glycogen, which was quite substantial under experimental conditions facilitating glycogenolysis. From the assay, we determined the following. (a) Glycogenolysis is activated in rat adipocytes in response to lipolytic hormones (i.e. catecholamines and adrenocorticotropic hormone). (b) Other agents and conditions elevating intracellular adenosine 3',5'-monophosphate levels (i.e. cholera toxin, dibutyryladenosine 3',5'-monophosphate, and isobutylmethylxanthine) also activate glycogenolysis. (c) Glycogenolysis (as opposed to lipolysis) is activated at concentrations of adrenocorticotropic hormone or isoproterenol 7-11-fold lower and at adenosine 3',5'-monophosphate concentrations 7-fold lower. (d) Calyculin A, a specific inhibitor of protein phosphatase 1, activates glycogenolysis as well. Calyculin A also activates lipolysis at an equimolar potency. (e) Insulin does not antagonize glycogenolysis in rat adipocytes. In conclusion, the assay allowed us to compare glycogenolysis to lipolysis within the same cell, and to find that the sensitivity to hormones and adenosine 3',5'-monophosphate was about 1 order of magnitude higher for glycogenolysis than for lipolysis. A more striking finding was the inability of insulin to antagonize glycogenolysis in the rat adipose cell, an effect which occurs readily in liver and muscle cells via protein phosphatase 1-activating machinery. This rules out a role for adipose protein phosphatase 1 activation in the mechanism by which insulin antagonizes lipolysis and supports the contention that the insulin effect in lowering adenosine 3',5'-monophosphate levels is the central mechanism by which insulin antagonizes lipolysis.

Vanadate, which mimics the biological effects of insulin, also inhibits lipolysis in rat adipocytes. Here we demonstrate that the antilipolytic effect of vanadate differs from that of insulin at least by the five following criteria: 1) vanadate inhibits lipolysis mediated by high (supraphysiological) concentrations of catecholamines; 2) vanadate antagonizes (Bu)2cAMP-mediated lipolysis; 3) vanadate antagonizes isobutylmethylxanthine-dependent lipolysis, 4) vanadate inhibits lipolysis mediated by okadaic acid; and 5) wortmannin, which blocks the antilipolytic effect of insulin, fails to block vanadate-mediated antilipolysis. Vanadate does activate phosphoinositol 3-kinase, and wortmannin blocks this activation. Our working hypothesis assumes that all of the insulin-like effects of vanadate, including antilipolysis, are initiated by the inhibition of protein phosphotyrosine phosphatases (PTPases). Among documented PTPase inhibitors we found that VOSO4 (oxidation state +4), several organic vanadyl compounds (+4), zinc (Zn2+), tungstate (W), and molybdate (Mo) also had antilipolytic activity. The order of potency was vanadyl acetylacetonate > or = VOSO4 > or = NaVO3 > or = vanadyl-dipicolinate > Zn2+ >> W > Mo, and it correlated better with the inhibition of adipose membranal-PTPases in cell-free experiments. We have concluded that the antilipolytic effect of vanadate is 1) mechanistically distinct from that of insulin, 2) independent of phosphoinositol 3-kinase activation, and 3) independent of the lipolytic cascade. We also strongly suggest that the antilipolytic effect of vanadate emanates from inhibiting adipose membranal, rather than cytosolic PTPases, and present preliminary data showing distinct differences in catalysis between these two PTPase categories. Overall, the study indicates that antilipolysis can be manifested via alternative, insulin-independent, signal-transducing pathways.

Vanadium and its compounds exhibit a wide variety of insulin-like effects. In this review, these effects are discussed with respect to the treatment of type I and type II diabetes in animal models, in vitro actions, antineoplastic role, treatment of IDDM and NIDDM patients, toxicity, and the possible mechanism(s) involved. Newly established CytPTK plays a major role in the bioresponses of vanadium. It has a molecular weight of approximately 53 kDa and is active in the presence of Co2+ rather than Mn2+. Among the protein-tyrosine kinase blockers, staurosporine is found to be a potent inhibitor of CytPTK but a poor inhibitor of InsRTK. Vanadium inhibits PTPase activity, and this in turn enhances the activity of protein tyrosine kinases. Our data show that inhibition of PTPase and protein tyrosine kinase activation has a major role in the therapeutic efficacy of vanadium in treating diabetes mellitus.

This article is based on clinical experience and offers principles for "tailoring" differential intervention packages for treating girls in distress. Profiles of ten types of girls are described and illustrated. Eight major features of intervention packages are discussed. Recommendation for matching intervention packages to the girls are presented.