Structural and biologic characterization of pegylated recombinant IFN-alpha2b.

The type I interferon-alpha (IFN-alpha) family is a family of natural small proteins that have clinically important anti-infective and antitumor activity. We have developed a semisynthetic protein-polymer conjugate of IFN-alpha2b (Intron A) by attaching a 12,000-Da monomethoxypolyethylene glycol (PEG-12000) polymer to the protein. PEG conjugation is thought to increase the serum half-life and thereby prolong patient exposure to IFN-alpha2b without altering the biologic potency to the protein. Matrix-assisted laser desorption ionization/mass spectrometry (MALDI-MS), high-performance size exclusion chromatography (HPSEC), circular dichroism (CD) analysis and tryptic digestion peptide analysis of PEG Intron demonstrated that the IFN-alpha2b protein was approximately 95% monopegylated and that the primary, the secondary, and the tertiary structures were unaltered. Pegylation did not affect the epitope recognition of antibodies used for Intron A quantitation. An extensive analysis of the pegylated positional isomers revealed that approximately 50% of PEG Intron was monopegylated on the His(34) residue of the IFN-alpha2b protein. The highest antiviral activity of the pegylated positional isomers for PEG Intron was associated with the His(34) pegylated isomer. The specific activity for PEG Intron in an antiviral cytopathic protection assay was 28%, relative to Intron A. However, the potency of PEG Intron, defined as bioactivity independent of protein concentration, was comparable to Intron A at both the molecular and cellular levels in a battery of in vitro assays. Equivalent units of PEG Intron and Intron A were indistinguishable for the induction of several key IFN-induced genes, including 2',5'-oligoadenylate synthetase (2',5'-OAS) and protein kinase R (PKR), in Molt 4 cells. The antiviral dose-response curves revealed that there were no significant differences between PEG Intron and Intron A. This demonstrated that the introduction of more IFN-alpha2b protein associated with equivalent unit dosing of PEG Intron did not create any antagonism or agonism in the antiviral assay. In assays for the immune response, PEG Intron and Intron A displayed comparable potency for both natural-killer (NK) and lymphokine-activated killer (LAK) cell cytolytic activity and for the induction of class I major histocompatibility protein. These results demonstrate that PEG Intron maintains an in vitro biologic potency profile for both antiviral and immunotherapeutic activity that is highly comparable to that of Intron A.

A review of the effects of moderate alcohol intake on the treatment of anxiety and mood disorders.

BACKGROUND: There is no published review to help the clinician clarify the potential role of moderate ethanol consumption in patients being treated for anxiety and mood disorders. Product labels and textbook chapters routinely warn the individual against the consumption of ethanol when using prescription psychotropic drugs. A general understanding is that the reason for this recommendation is the potential for adverse synergistic effects or sedation and decreased psychomotor performance. What is overlooked by this emphasis on safety is the effect of alcohol use both on the underlying psychiatric disorder being treated and on the effectiveness of drug therapy. METHOD: We review the available literature on the interactions of ethanol with neurotransmitters and psychotropic medications and explore the clinical consequences of these interactions. RESULTS: Ethanol might affect anxiety and mood disorders by different mechanisms. Principal among these are the effects of ethanol on multiple neurotransmitter systems, which adapt in different ways to the acute and/or chronic presence of ethanol. Perturbations in the balance of CNS neurotransmitter systems may modify the acute clinical course of primary mood disorders and undermine the therapeutic response to psychotropic medications. Ethanol also modifies the clearance and disposition of psychotropic metabolites and interferes with their clinical effectiveness. Neurotransmitter responses may additionally be manifested clinically by rebound phenomena, akin to a subsyndromal withdrawal, which affect sleep and precipitate anxiety and mood symptoms. Recent alcohol use also may alter the subjective interpretation of the patient's "internal milieu," causing confusion and eliciting reactive psychopathology. CONCLUSION: While much research remains to be done, there is abundant evidence that patients with mood and anxiety disorders should abstain from even moderate ethanol use, as this adversely affects their clinical course and response to treatment.

Correlates of protracted homelessness in a sample of dually diagnosed psychiatric inpatients.

The authors assessed sociodemographic, drug use, and diagnostic correlates of protracted homelessness in a sample of 147 dually diagnosed patients who required admission to the hospital. When 58 patients with protracted homelessness, defined as continuous undomiciled status for over a year, were compared with 74 patients without protracted homelessness, significant differences were found with regard to diagnosis, employment status, criminality, Brief Psychiatric Rating Scale score on admission, and history of injection drug use. The results of a multiple logistic regression analysis confirmed that a history of injection drug use, current unemployment, and a diagnosis of schizophrenia were positively associated with a history of protracted homelessness. No significant relationships were obtained between protracted homelessness and demographics or chronicity of mental illness.

A modified therapeutic community for the dually diagnosed. Greenhouse Program at Bellevue Hospital.

Techniques used in therapeutic communities may be applicable to patients dually diagnosed with mental illness and a psychoactive substance use disorder (PSUD). This study was designed to evaluate the demographics, course, and outcome for 100 patients treated in one such residential program. One hundred indigent male patients admitted to a drug-free therapeutic community for the dually diagnosed were studied on admission and over the course of their treatment, and subjects were monitored throughout their stays on the basis of observed urine toxicology tests and a clinical assessment of drug or alcohol use. The mean age of the patients was 33.8 years, and the average length of stay was 121.0 days. Thirty-three of the patients completed the full 6-month program and moved on to another stable living environment. Only 12 patients had urine toxicologies positive for illicit drugs or alcohol while in the program. These findings support the possibility of applying the residential drug-free therapeutic community to dually diagnosed patients.

Crack cocaine and schizophrenia as risk factors for PPD reactivity in the dually diagnosed.

We studied factors contributing to an increased risk of PPD positive status among 147 inpatients dually diagnosed for mental illness and substance abuse in a large urban hospital. Ninety-three percent (N = 137) were tested for PPD on admission. The rate of positive PPDs was 30.7%. Significant correlates of PPD positive status were the diagnosis of schizophrenia/psychosis NOS (p < .05), and crack cocaine use in the 30 days prior to admission (p < .01). A multiple logistic regression revealed a relative risk of 3.53 (p < .005) for PPD positive status for the crack using group and a relative risk of 2.16 (p < .06) for PPD positive status for the schizophrenic group. Reasons for why patients whose primary drug of abuse is crack cocaine and those whose diagnosis is schizophrenia/psychosis NOS may be at an increased risk for exposure to tuberculosis are discussed as are the implications for public health.

A review of the effects of moderate alcohol intake on psychiatric and sleep disorders.

In this chapter we discuss the effects of moderate ethanol consumption on the treatment of psychiatric and sleep disorders. A review of the literature on the interactions of ethanol with neurotransmitters and psychotropic medications suggests that although ethanol affects the clinical course of psychiatric and sleep disorders by different mechanisms, it does so principally through perturbations it causes in the balance of central nervous system neurotransmitter systems, which may modify the clinical course of primary psychiatric and sleep disorders and undermine the therapeutic response to psychotropic medications. Neurotransmitter responses may also be manifested clinically by rebound phenomena, akin to a subsyndromal withdrawal, which affect sleep and precipitate anxiety and mood symptoms. In addition, ethanol also modifies the clearance and disposition of a variety of psychotropic metabolites and interferes with their clinical effectiveness. We recommend that most psychiatric patients, and all patients with sleep disorders, should abstain from even moderate ethanol use, as this may adversely affect their clinical course and response to treatment.

Assessing an adult patient's suicide risk. What primary care physicians need to know.

There is no pathognomonic sign or symptom that predicts suicide. However, suicide threats, hopelessness, and other risk factors may signal a patient's intent for self-harm. Through careful history taking and examination, the physician can assess the patient's risk profile for suicide and construct an appropriate treatment plan, which may include hospitalization, drug therapy, and/or referral to a psychiatrist.

Physical examination of substance abusers. How to gather evidence of concealed problems.

How can physicians make appropriate treatment decisions in substance abusers who have serious medical illness, especially when patients may not offer the information needed? Drs Westreich and Rosenthal summarize common physical findings that suggest abuse of specific substances. Recognizing these signs allows physicians to reliably diagnose chemical use, leads to improve outcomes, and lends weight to recommendations for substance-abuse therapy.

Delirium and acute psychosis. Mental states calling for clear diagnostic thinking and careful management.

Management of a delirious or acutely psychotic patient calls for rapid but well-considered decision making by the treating physician. Clear diagnostic thinking is vitally important in making a presumptive diagnosis and forming a treatment plan. Appropriate treatment usually returns patients to a less agitated state and allows for definitive treatment of any underlying illness.

Concurrent cocaine withdrawal is associated with reduced severity of alcohol withdrawal.

The purpose of this study was to implement an empirical assessment of the clinical response to standard alcohol detoxification during withdrawal from both alcohol and cocaine. One hundred forty-nine males consecutively admitted in acute alcohol withdrawal to a hospital-based detoxification unit were studied. All subjects completed a 4-day chlordiazepoxide detoxification. Patients who used drugs other than cocaine were excluded. Fifty-five subjects withdrawing only from alcohol and 94 subjects withdrawing from both alcohol and cocaine, as evidenced by positive urinalysis and history, were studied. Both groups reported similar amounts of daily alcohol intake and had a similar age of onset of alcohol dependence. Parental alcoholism was equally frequent in both groups. Statistically, several variables were directly related to severity of alcohol withdrawal, including associated cocaine abuse, age, abnormal laboratory values, and duration of homelessness. As measured by the Alcohol Withdrawal Scale (AWS), alcohol withdrawal was less severe among cocaine users, not only at intake but throughout the 4-day detoxification. Singly addicted alcoholics were older and had longer drinking histories, more prior detoxifications, and more abnormal laboratory values than cocaine users. A multiple regression analysis demonstrated a significant relationship between cocaine and severity of alcohol withdrawal. Cocaine users more frequently requested reductions in chlordiazepoxide dosages than singly addicted alcoholics, complaining of dysphoria, sedation, and weakness. The severity of alcohol withdrawal was associated with recent cocaine use, age, laboratory abnormalities, and duration of homelessness. Concurrent cocaine withdrawal in the sample was associated with reduced severity of alcohol withdrawal. Possible neurobiological mechanisms, as well as study limitations affecting interpretation of the findings, are discussed. Tailored detoxification as opposed to standard detoxification regimens may be more appropriate for the clinical management of combined alcohol-cocaine withdrawal.

Patient knowledge about electroconvulsive therapy: effect of an informational video.

We wished to ascertain whether the addition of an informational video to the informed consent procedure for electroconvulsive therapy (ECT) results in improved patient knowledge about ECT. Eighteen ECT patients were randomized to consent using the usual written document or using the written document and an informational video. The two groups were similar when compared on demographic variables and scores on the Brief Psychiatric Rating Scale (BPRS) and Mini-Mental State Examination (MMSE). Each subject, just after signing the informed consent document, was administered an 8-question ECT knowledge questionnaire. The addition of an informational video to the consent process for ECT did not result in improved knowledge about ECT. Poor knowledge about ECT might be accounted for by unsuccessful communication from the doctors or cognitive impairment and apathy on the part of the patients. One benefit of the video was increased interest from family members in ECT and the consent process.

Differences between men and women in dual-diagnosis treatment.

The authors reviewed the charts of all women and a randomly selected sample of men over a 6-month period on two addiction treatment units at Bellevue Hospital Center in New York. The men were more likely to be admitted with schizophrenia and to have used substances of abuse other than alcohol, and the women were more likely to be admitted with affective disorders. Also, the women on the dual-diagnosis ward were more likely to be domiciled (i.e., not homeless), and the women on both units were significantly more likely to report having been crime victims. These findings suggest that dually diagnosed women need a substantially different treatment paradigm from men.

Perceived social support and treatment retention on an inpatient addiction treatment unit.

The authors measured perceived social support among patients and their families as a predictor of retention in an inpatient addiction rehabilitation program. After detoxification from all substances of abuse, 66 sequentially admitted inpatients gave demographic and diagnostic information and completed scales of perceived social support from the program and their own families. Scales were completed at 7, 14, and 21 days. A total of 46 subjects completed the 21-day program, and 20 did not. Homeless status, initial weak perceived social support from family, and a relatively shorter history of crack-cocaine use were correlated with completion of the program. Patients with stronger connections to shelter and family were less likely to complete an inpatient addiction rehabilitation program. Patients who reported more years of crack-cocaine use were also less likely to complete the program. The authors discuss implications for treatment.

HIV risk factors in dually diagnosed patients.

The authors examined correlates of HIV seropositivity in a sample of dually diagnosed inpatients. The subjects were 147 consecutively admitted patients to a specialized dual-diagnosis unit in a municipal hospital who were given a structured interview and HIV testing. The HIV seroprevalence was 19%, with women having a nearly fourfold increased risk of being HIV seropositive, as compared with men. Cocaine as drug of choice was also highly significant as a risk factor for HIV infection, independent of gender. This finding suggests that targeted prevention and education programs need to be developed for the dually diagnosed patient.

Isolation and characterization of a monomethioninesulfoxide variant of interferon alpha-2b.

PURPOSE: To isolate and characterize a monomethioninesulfoxide variant of the commercially available therapeutic protein interferon alpha-2b. METHODS: The methionine (Met)-oxidized variant was isolated by reverse-phase high performance liquid chromatography and characterized by SDS-PAGE, peptide mapping and mass spectrometric analysis of the trypsin/V8-generated peptide fragments. The biological and immunological activities of the isolated variant were also evaluated. RESULTS: The rHuIFN alpha-2b variant was found to contain a Met sulfoxide residue at position 111 of the rHuIFN alpha-2b molecule. The far-UV CD spectra showed a slight loss of alpha-helical content and an increase in the beta-sheet contribution. The CD spectra indicate that both chromatographic conditions and Met oxidation contribute to the observed secondary structure changes. Both interferon alpha-2b main component and its methionine-oxidized variant showed different reactivity to monoclonal antibodies employed in immunoassays for the protein. CONCLUSIONS: A monomethioninesulfoxide rHuIFN alpha-2b variant was found to be present in the rHuIFN alpha-2b bulk drug substance in solution. The Met(111) residue was identified as Met sulfoxide by comparative tryptic/V8 mapping and mass spectrometric analysis. Nevertheless, the oxidation of the Met(111) residue did not seem to have a detectable effect on the biological activity of the molecule.

Generating p53-specific cytotoxic T lymphocytes by recombinant adenoviral vector-based vaccination in mice, but not man.

Mutations and aberrant expression of the p53 tumor suppressor protein are the most frequent molecular alterations in human malignancy. Peptides derived from the wild-type (wt) p53 protein and presented by major histocompatibility complex (MHC) molecules for T lymphocyte recognition are believed to serve as universal tumor-associated antigens for cancer immunotherapy. We studied the immunogeneicity of a recombinant replication-defective adenoviral vector encoding human full-length wt p53 (rAd/hup53) in human leukocyte antigen (HLA)-A2K(b)-transgenic (Tg) mice and man. The generation of p53 epitope-specific cytotoxic T lymphocytes (CTLs) in p53-proficient and p53-deficient A2K(b)-Tg mice was affected by self-tolerance and a selective inability of rAd/hup53 to induce p53.264-272 peptide-reactive effector cells. To extend this study into a pilot clinical trial, six advanced-stage cancer patients received sequential injections of rAd/hup53. The treatment was well tolerated. To date, no evidence for objective tumor responses was observed. An amplification of humoral and cellular anti-adenoviral immune responses was demonstrated in all patients following rAd/hup53 vaccination. However, p53-reactive antibodies and HLA-A*0201 (A2.1)-restricted CTLs specific for wt p53 epitopes were not generated. Tailoring p53-based cancer immunotherapy thus requires the interference with p53-specific self-tolerance and the induction of the entire repertoire of p53-reactive T lymphocytes.