Production of a Sindbis/Eastern Equine Encephalitis chimeric virus inactivated cell culture antigen.

Eastern Equine Encephalitis virus (EEEV) is a medically important pathogen that can cause severe encephalitis in humans, with mortality rates ranging from 30 to 80%. Unfortunately there are no antivirals or licensed vaccines available for human use, and laboratory diagnosis is essential to differentiate EEEV infection from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the EEEV immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). However, EEEV is classified as a HHS select agent and requires biosafety level (BSL) three containment, limiting EEEV antigen production in non-select agent and BSL-2 laboratories. A recombinant Sindbis virus (SINV)/EEEV has been constructed for use under BSL-2 conditions and is not regulated as a select agent. Cell culture production of inactivated EEEV antigen from SINV/EEEV for use in the EEEV MAC-ELISA is reported here. Cell culture conditions and inactivation procedures were analyzed for SINV/EEEV using a recently developed antigen production algorithm, with the MAC-ELISA as the performance indicator.

Ragweed pollen proteolytic enzymes: possible roles in allergies and asthma.

Extracts from ragweed pollen grains contain novel trypsin and chymotrypsin-like serine peptidases which are described in this report. The molecular mass of the chymotrypsin-like enzyme was 82 kDa, had a pH optimum near 9.0, and its activity was unaffected by chelating or reducing agents. It was inhibited by diisopropyl fluorophosphate (DFP), a general serine class inhibitor, and more specifically N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a chymotrypsin-like proteinase inhibitor. In addition to various synthetic substrates, the neuropeptides, vasoactive intestinal peptide (VIP) and substance P, which are required for normalized lung functions, were also rapidly hydrolysed. Activity toward protein substrates was not detected with the exception of the inactivation of alpha-1-proteinase inhibitor (alpha-1-PI) which occurred through cleavage within the reactive site loop. The 'trypsin-like' enzyme has a molecular mass near 80 kDa, a blocked N-terminus, a pH optimum near 9.0, and requires Ca++ for stability and activity, but not reducing agents. It is inhibited by DFP, and more specifically the trypsin-like proteinase inhibitor, N-p-tosyl-L-lysine chloromethyl ketone (TLCK). Again, activity toward protein substrates was not detected, but various synthetic substrates and biologically active peptides were efficiently cleaved. Significantly, atrial natriuretic peptide (ANP) and angiotensin 2 (ATII), whose degradation would amplify kinin activity and influence inflammatory diseases of the respiratory tract and nasal passages, were also rapidly hydrolyzed.

Development of an algorithm for production of inactivated arbovirus antigens in cell culture.

Arboviruses are medically important pathogens that cause human disease ranging from a mild fever to encephalitis. Laboratory diagnosis is essential to differentiate arbovirus infections from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the virus-specific immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Antigen production in cell culture has largely replaced the use of suckling mice; however, the methods are not directly transferable. The development of a cell culture antigen production algorithm for nine arboviruses from the three main arbovirus families, Flaviviridae, Togaviridae, and Bunyaviridae, is described here. Virus cell culture growth and harvest conditions were optimized, inactivation methods were evaluated, and concentration procedures were compared for each virus. Antigen performance was evaluated by the MAC-ELISA at each step of the procedure. The antigen production algorithm is a framework for standardization of methodology and quality control; however, a single antigen production protocol was not applicable to all arboviruses and needed to be optimized for each virus.

Identification, assessment and treatment of women suffering from post traumatic stress after abortion.

PIP: Abortion has been reported to cause post-traumatic stress disorders in women. The manifestation of these disorders is usually delayed, and the causative effect of abortion is largely denied. Such denial is a major contributing factor to the development of the stress. This paper seeks to provide a model for assessment, treatment, and evaluation of therapeutic effectiveness that can be used when post-traumatic stress is diagnosed as a result of abortion. The possibilities of a delayed stress reaction should be examined when clients experience 1) flashbacks of the surgical procedure, people involved, or physical surroundings; 2) nightmares of being trapped in a man-made disaster and being unable to help others who are suffering from, participating in, or witnessing a terrorist act, ritual murder, or human sacrifice; 3) the sudden onset of sadomasochistic interactions or accident proneness where the client receives punishment/pain to alleviate hidden guilt; 4) sexual dysfunctions with no physiological basis; 5) uncharacteristic emotional outbursts; 6) the sudden onset of psychosomatic symptoms, and 7) impacted grief reactions. A detailed case study is presented to illustrate a number of clinical issues relevant to the treatment of a couple which had unresolved feelings about the wife's abortion before she met the husband and the abortion of the husband's girlfriend before he met his wife. Clinical experience suggests that the resolution of this conflict can be accomplished through short-term therapy, and further research is being conducted to determine how well treatment gains are being maintained.^ieng

Marital/family developmental theory as a context for understanding and treating inhibited sexual desire.

In this article, the author offers a theoretically based, heuristic model for understanding the etiology and maintenance of inhibited sexual desire (ISD) within the context of a developing relationship system. Empirical studies of premarital relationship formation, progression and maintenance form the basis of this discussion. The interplay of intrapersonal and interactional dynamics is highlighted. Emphasis is placed upon understanding ISD according to social exchange formulations of premarital and marital behavior. Particular attention is given to the interactants' nonverbalized rules and implicitly held expectations for the sexual exchange process. Guidelines for assessment are offered and implications for intervention are drawn.

A replication and validation of the Spousal Inventory of Desired Changes and Relationship Barriers (SIDCARB): elaborations on diagnostic and clinical utilization.

In this article, the authors present the findings of a study designed to validate the factor structure of the Spousal Inventory of Desired Changes and Relationship Barriers (SIDCARB). Results from this research reveal that the original factor structure remained essentially the same. Scoring and interpretations of the couple profile are discussed and treatment decisions based upon the couple profile are outlined. Although the development of SIDCARB grew out of a social learning-behavioral philosophy of marital and family interaction, this instrument yields information that can be useful to structural marital and family therapists as well.

SIDCARB: a clinical tool for rapid assessment of social exchange inequities and relationship barriers.

In this article, the development, standardization and clinical uses of the SIDCARB: Spousal Inventory of Desired Changes and Relationship Barriers are presented. The SIDCARB was developed to help the therapist gain some understanding of each spouse's unique perceptions of the conjugal exchange process. This instrument measures three dimensions of conjugal exchange: 1) desired changes, dissatisfaction with the equity of exchanges and commitment; 2) willingness to terminate the marriage and Internal-Psychological Barriers; and 3) External-Circumstantial Barriers. The uses of the SIDCARB as an aid to clinical assessment, treatment and evaluation are outlined, and several examples are offered. The limitations of the SIDCARB at its current state of development are discussed.

Purification and characterization of a novel endopeptidase in ragweed (Ambrosia artemisiifolia) pollen.

Ragweed (Ambrosia artemisiifolia), the major cause of late summer hay fever (allergic rhinitis) in the United States and Canada, is clinically the most important source of the seasonal aeroallergens. A novel endopeptidase was extracted from the pollen of this plant and purified by a series of column chromatographic steps. It has a molecular mass of 82 kDa according to gel filtration and SDS-polyacrylamide gel electrophoresis and a pH optimum near 9.0, and its activity is unaffected by chelating or reducing agents. A 17-amino acid amino-terminal sequence of this protein showed no similarity with any other proteases. The enzyme was inhibited by diisopropyl fluorophosphate, a general serine class inhibitor, and more specifically N-p-tosyl-L-phenylalanine chloromethyl ketone, a chymotrypsin-like proteinase inhibitor. Various synthetic substrates were efficiently cleaved with a strong preference for Phe in the P1 and P3 position and Pro in the P2 position. This specificity was confirmed through inhibition studies with both peptidyl chloromethyl ketone and organophosphate inhibitors. In addition to synthetic substrates, the neuropeptides, vasoactive intestinal peptide and substance P, which are required for normalized lung functions, were also rapidly hydrolyzed. Activity toward protein substrates was not detected with the exception of the inactivation of alpha-1-proteinase inhibitor, which occurred through cleavage within the reactive site loop. These results indicate that the purified enzyme is a novel endopeptidase, which may be involved in both the degradation of neuropeptides and the inactivation of protective proteinase inhibitors during pollen-initiated allergic reactions.

Purification and characterization of an arginine-specific peptidase from ragweed (Ambrosia artemisiifolia) pollen.

Ragweed (Ambrosia artemisiifolia) is clinically the most important source of seasonal aeroallergens, as it is responsible for the majority and most severe cases of hay fever (allergic rhinitis). Extracts from pollen grains have been shown to contain numerous proteins with various functions, including a novel serine proteolytic enzyme with chymotrypsin-like specificity that has been previously described (J. Biol. Chem. 1996; 271:26227-26232). We now report the isolation and properties of a second, trypsin-like enzyme with a molecular mass near 80 kD, from ragweed pollen extracts. This enzyme has a blocked N-terminus, a pH optimum near 9.0, and requires Ca2+ for stability and activity, but not reducing agents. The enzyme is inhibited by diisopropyl fluorophosphate, a general serine class proteinase inhibitor, and more specifically by N-p-tosyl-L-lysine chloromethyl ketone. Activity toward protein substrates was not detected, but various synthetic substrates and small biologically active peptides were efficiently cleaved, with a strong preference for Arg in the P1 position and either Arg or Gly in the P2 position. This specificity was confirmed through inhibition studies with both peptidyl chloromethyl ketone and organophosphate inhibitors. Significantly, atrial natriuretic peptide and angiotensin 2, whose degradation would amplify kinin activity and influence inflammatory diseases of the respiratory tract and nasal passages, were also rapidly hydrolyzed. Thus, the ragweed pollen endopeptidase may be involved in the inactivation of regulatory neuropeptides during pollen-initiated allergic reactions.

Case management in mental health.

Despite the growing popularity of case management in caring for clients' multiple needs, the concept of case management and its relationship to social work are yet to be defined clearly. To help shed light on the subject, this article reports on a survey of 403 case managers in community mental health centers in Georgia that examined how workers' educational level, professional identification, and demographic characteristics influenced their performance of case management tasks.

Induction of histamine release from non-immunized guinea pigs: a possible involvement of lectin-like factor(s) in pollinosis.

To investigate nonimmune pathogenic functions of pollens, vascular permeability enhancement (VPE) activity of pollen extracts was examined using guinea pigs nonimmunized against pollens. Ryegrass, ragweed, mesquite and almond, but not common cattail and sumac, induced VPE which was inhibited primarily by an anti-histamine drug. Ryegrass pollen VPE activity was extracted more at pH 7.3 than at pH 6.5 or 8.0 and the maximal activity was extracted in 30 min. Interestingly, more than 60% of the maximal activity was extracted in 5 min. The maximal VPE activity had a dose-dependency similar to histamine (3 x 10(-5) M) but lasted longer than the histamine activity. The VPE activity was inhibited by oligomannose-glycosylated ovalbumin or avidin, as well as the oligosaccharides but not by the deglycosylated proteins. These results indicate that some pollens contain lectin-like, histamine-releasing factor(s), which may be involved in part in pollinosis, by inducing mast cell degranulation through a nonimmune mechanism and resulting in allergy-like symptoms.