Expression and regulation of RAB3 proteins in osteoclasts and their precursors.

The ruffled membrane, the resorptive organelle of the osteoclast, is generated by fusion of intracytoplasmic acidifying vesicles with the plasma membrane, an event analogous to regulated exocytosis. While the ruffled membrane is essential to the bone resorptive process, the mechanisms governing its generation are unknown. However, regulated exocytosis is mediated, in part, by isoforms of the Rab3 subset of Rab GTPases. Because of similarities between exocytosis and ruffled membrane formation, we asked if Rab3 proteins are expressed by osteoclasts or their precursors, and if so, are these molecules regulated by agents known to prompt the osteoclast phenotype? We find murine osteoclast precursors, in the form of bone marrow macrophages (BMMs), express at least two Rab3 isoforms, namely A and B/C, which are individually enhanced by a variety of hematopoietic cytokines. Consistent with the osteoclastogenic properties of a number of these cytokines, differentiation of BMMs into osteoclasts, in vitro, is associated with increased expression of both isoforms, particularly Rab3B/C. Finally, Rab3B/C localizes with the avian osteoclast H+ATPase (vacuolar proton pump) and pp60c-src, both intracellularly and within acidifying vesicles derived largely from the ruffled membrane. Thus, expression of specific rab3 proteins, an event which may control formation of the osteoclast ruffled membrane, is modulated by cytokines during osteoclastogenesis.

1,25 dihydroxyvitamin D3 and dexamethasone induce the cyclooxygenase 1 gene in osteoclast-supporting stromal cells.

Commitment of members of the monocyte/macrophage family to the bone resorptive phenotype, in vitro, requires contact, of these osteoclast precursors, with osteoblasts or related stromal cells. The osteoclast-inductive properties of these stromal cells are typically expressed, however, only in the presence of steroid hormones such as 1,25 dihydroxyvitamin D (1,25D3) and dexamethasone (DEX). To gain insight into the means by which steroid treated accessory cells induce osteoclast differentiation we asked, using differential RNA display (DRD), if gene expression by this stromal cell population differs from that of their untreated, non-osteoclastogenic counterpart. We identified four known genes specifically expressed by 1,25D3/DEX-treated ST2 stromal cells: 1) a family of rat organic anion transporters, 2) Na/K ATPase ss-subunit, 3) tazarotene-induced gene 2 (TIG2), and 4) prostaglandin G/H synthase I, or cyclooxygenase 1 (Cox-1). The regulation of these genes in 1,25D3/DEX-treated ST2 cells was demonstrated by Northern blot analysis of treated (osteoclast-supporting) and untreated (non-osteoclast-supporting) ST2 cells; the genes have a limited and specific tissue mRNA expression pattern. Northern blot analysis of treated and untreated ST2 cell total RNA using either a DRD-derived Cox-1 cDNA or a Cox-1 specific oligonucleotide confirmed the steroid regulation of Cox-1 mRNA. Surprisingly, there is no detectable expression by untreated or steroid exposed ST2 cells, of Cox-2, the classical regulated cyclooxygenase isoform. In contrast to 1, 25D3/DEX, serum treatment rapidly induces Cox-2 mRNA, substantiating the capacity of ST2 cells to express the gene. These data establish that steroid induction of the osteoclastogenic properties of stromal cells is attended by Cox gene expression, a phenomenon consistent with the capacity of eicosinoids to impact the resorptive process. The response of osteoclast-supporting ST2 cells to 1,25D3/DEX treatment may be one prostaglandin-mediated event which specifically involves Cox-1 regulation.

Twelve-step and mutual-help programs for addictive disorders.

Psychiatrists may wonder why both addiction treatment and the 12-step programs recommend abstinence. In his 50-year follow-up of two groups of alcoholics, Vaillant compared those who established secure abstinence with those who continued to drink. Secure abstinence was associated with: Living longer Better mental health Better marriages Being more responsible parents Being successful employees In considering the various routes to secure recovery, Vaillant recommended that clinicians: Offer the patient a nonchemical substitute for alcohol Remind the patient ritually that even one drink can lead to pain and relapse Repair the social and medical damage that the patient has experienced Restore the patient's self-esteem The preponderance of the research data now available indicates that the 12-step programs of AA, NA, Cocaine Anonymous, and Al-Anon are most helpful for alcohol-dependent and other drug-addicted patients as they seek to achieve secure, long-term abstinence. A growing number of clinicians is recommending that physicians become more knowledgeable and skilled in referring and supporting patients in working 12-step programs of recovery. Specific recommendations include: 1. Be familiar with 12-step activities and tools. These include meetings, home groups, sponsors, the Twelve Steps and Twelve Traditions, books, pamphlets, and slogans. To be able to discuss the meanings and applications of these tools for recovery is useful. Physicians can select those that are most suitable for the individual, recognizing that meeting attendance might not be the most important activity. 2. Support referral by facilitating a meeting between the patient and a temporary contact from the 12-step program. This means becoming familiar with local 12-step programs. Phoning the local AA or NA central office or hot line makes connecting patients to someone who will take them to a meeting that same day possible. AA and NA have committees whose members are interested in working with physicians to help get patients to meetings and to get information to physicians. These are the Cooperation with the Professional Community, Treatment Facilities, and Hospitals and Institutions committees. 3. Work with the resistance of patients. Many addicted patients are resistant to the idea of attending 12-step or mutual-help programs. Reminders of their painful personal database associated with the use of alcohol or other drugs can help break through denial. Involvement of family members and friends in the network therapy developed by Galanter can be effective in reducing resistance. Being patient and persistent in developing the therapeutic alliance helps to maintain contact during the first difficult year of recovery. Physicians should be prepared to work with patients as long as necessary to stabilize their sobriety. Zweben has suggested ways psychotherapy can help deepen work with the steps. 4. Help dual diagnosis patients understand AA's and NA's singleness of purpose. These programs work only with addiction; they do not try in any way to deal with other mental disorders. All patients have to say is, "I want to stop drinking or using drugs," and they will be welcomed and accepted at meetings (see Tradition 3). If they talk only about their psychiatric symptoms or medications, someone may suggest that they go elsewhere for help. Occasionally, well-intentioned AA or NA members tell patients to stop taking their medications. The authors always direct patients to the pamphlet The AA Member: Medications and Other Drugs. This pamphlet tells AA members not to play doctor and to take the medications their doctors prescribe. Copies of the pamphlet are widely available at many AA meetings, or they can be ordered by physicians from Alcoholics Anonymous World Services, General Service Office, Box 459, Grand Central Station, New York, NY 10163 (212-870-3400). 5. Get comfortable with the spiritual dimensions of healing. Zweber and Brown offer good suggestions for getting com

Granulocyte macrophage-colony stimulating factor reciprocally regulates alphav-associated integrins on murine osteoclast precursors.

The integrins alphavbeta3 and alphavbeta3 are expressed reciprocally during murine osteoclastogenesis in vitro. Specifically, immature osteoclast precursors, in the form of bone marrow macrophages, contain exclusively alphavbeta5, surface expression of which declines with commitment to the osteoclast phenotype, while levels of alphavbeta3 increase concomitantly. The distinct functional significance of alphavbeta5 is underscored by the integrin's capacity, unlike alphavbeta3, to mediate both attachment and spreading on ligand, of marrow macrophages, suggesting alphavbeta3 negotiates initial recognition, by osteoclast precursors, of bone matrix. Northern analysis demonstrates changes in the two beta-subunits, and not alphav, are responsible for these alterations. Treatment of early precursors with granulocyte-macrophage colony stimulating factor (GM-CSF) leads to alterations in beta3 and beta5 mRNA and alphavbeta5 and alphavbeta3, paralleling those occurring during osteoclastogenesis. Nuclear run-on and message stability studies demonstrate that while GM-CSF treatment of precursors alters beta5 transcriptionally, the changes in beta3 arise from prolonged mRNA t1/2. Similar to GM-CSF treatment, the rate of beta5 transcription falls during authentic osteoclastogenesis. In contrast to cytokine-induced alphavbeta3, however, that attending osteoclastogenesis reflects accelerated transcription of the beta3-subunit. Thus, while GM-CSF may participate in modulation of alphavbeta5 during osteoclast differentiation, signals other than those derived from the cytokine must regulate expression of alphavbeta3.

Modulation of antibody display on M13 filamentous phage.

Here we describe a phage vector for the display of single chain antibodies and polypeptides on the surface of filamentous M13 phage which permits facile manipulation of the valency of display. The gene encoding the polypeptide is fused to a synthetic copy of the major coat protein VIII gene (gpVIII) which permits incorporation into the phage during assembly of the filament. Here we examine the growth parameters of phage propagation on the subsequent selection of an anti-progesterone antibody fragment from a mixture of display phage. Our results suggest that the density of the polypeptides displayed on phage may be modulated by altering growth conditions. This ability to influence polypeptide display density on filamentous phage may provide a versatile approach for accessing complex libraries and the capture of weaker ligand receptor interactions by avidity, whilst the potential to access and discriminate between higher affinity interactions is not negated.

Substance use disorders. Difficulties in diagnoses.

Patients attempt, consciously or unconsciously, to minimize or disguise their substance use, in part to preserve shreds of self-respect, avoid guilt and shame, and avoid the real or imagined criticism of others. It is ironic that substance users not only use the substance to gain access to pleasant events or escape or avoid unpleasant events, but also to deny, minimize, or disguise that very use for the same reasons.

1,25-dihydroxyvitamin D3 regulates pp60c-src activity and expression of a pp60c-src activating phosphatase.

The nonreceptor tyrosine kinase, c-src, and the steroid hormone,1,25-dihydroxyvitamin D3(1,25(OH)2D3), are essential to development of the osteoclast phenotype. On the other hand, functional relationships between the activities of c-src and 1,25(OH)2D3 are as yet unknown. To determine if 1,25(OH)2D3 modulates c-src in osteoclastogenesis, we tested the steroid's effect on avian marrow-derived osteoclast precursors. We find c-src mRNA and immunoprecipitable c-src protein (pp60c-src) unaltered by 72 h exposure of these cells to 1,25(OH)2D3 (10(-11) to 10(-9) M). Despite no quantitative change in pp60c-src, in vitro kinase assay of the immune complex reveals 1,25(OH)2D3 dose-dependently accelerates the catalytic activity of pp60c-src, enhancing its autophosphorylation and phosphorylation of exogenous substrate. This observation represents the first documentation, in nontransformed cells, of humoral induction of pp60c-src kinase. Consistent with the fact pp60c-src is activated by dephosphorylation of tyrosine 527 (Y527), the phosphotyrosine content of the pp60c-src immunoprecipitate, measured by immunoblot, is decreased by 1,25(OH)2D3. Alternatively, mRNA and protein levels of c-src kinase (CSK), which inactivates pp60c-src by phosphorylating Y527, are not altered by the steroid. In contrast, 1,25(OH)2D3 enhances mRNA and especially protein levels of avian protein tyrosine phosphatase lambda (PTP lambda), an enzyme specifically activating pp60c-src by dephosphorylating Y527 [Fang et al. (1994): J Biol Chem 269:20194-20200]. Thus, treatment of avian osteoclast precursors with 1,25(OH)2D3 accelerates the catalytic activity of pp60c-src independent of protein expression. Activation of the kinase may occur via the Y527 dephosphorylating enzyme PTP, expression of which, we show for the first time, is regulated.

Serum antibodies from malaria-exposed people recognize conserved epitopes formed by the two epidermal growth factor motifs of MSP1(19), the carboxy-terminal fragment of the major merozoite surface protein of Plasmodium falciparum.

The major merozoite surface protein of Plasmodium falciparum (PfMSP1) is a candidate antigen for a malaria vaccine. A 19-kDa C-terminal processing product of PfMSP1 (PfMSP1(19)) is composed of two domains sharing a cysteine-rich motif with epidermal growth factor (EGF) and is the target of monoclonal antibodies which block erythrocyte invasion in vitro. We have evaluated human antibody responses to PfMSP1(19) by using recombinant proteins representing the EGF motifs encoded by the two main alleles of the MSP1 gene. We find that both EGF motifs are antigenic but that only 10 to 20% of malaria-exposed individuals have serum antibodies that recognized either of the motifs. When both EGF motifs were expressed together as a single protein, they were recognized by more than 40% of sera from malaria-exposed individuals. Major epitopes recognized by human antibodies are dependent upon the correct tertiary structure of the protein and are cross-reactive between the different allelic sequences of PfMSP1(19). This suggests that antibodies induced by vaccination with one or the other allelic forms of the protein could recognize all strains of P. falciparum. Immunoglobulin G (IgG) subclass-specific enzyme immunoassays indicate that PfMSP1(19) antibodies are predominantly of the IgG1 subclass.

Naturally acquired human antibodies which recognize the first epidermal growth factor-like module in the Plasmodium falciparum merozoite surface protein 1 do not inhibit parasite growth in vitro.

Merozoite surface protein 1, one of the major surface proteins of the invasive blood stage of the malaria parasite, is a prime candidate for the development of a vaccine against the human disease. Previously, monoclonal antibodies which both inhibited the growth of Plasmodium falciparum in vitro and bound to the first of two epidermal growth factor-like modules located near the carboxy terminus of the protein had been identified. In this study, we have used affinity chromatography on a recombinant fusion protein corresponding to the first epidermal growth factor-like module in P. falciparum merozoite surface protein 1 to prepare antibody induced by natural infection. The antibody was purified from the total immunoglobulin G fraction of adult West African donors, shown to passively confer immunity against falciparum malaria. Such affinity-purified antibodies were shown to recognize the native protein by a number of separate criteria and to block the binding of an inhibitory monoclonal antibody, but they failed to inhibit parasite invasion in an in vitro growth assay. These results indicate that antibody alone is not sufficient to interfere with erythrocyte invasion.

Advances in the treatment of alcoholism.

The Council on Scientific Affairs of the California Medical Association presents the following inventory of items of progress in psychiatry. Each item, in the judgment of a panel of knowledgeable physicians, has recently become reasonably firmly established, both as to scientific fact and important clinical significance. The items are presented in simple epitome, and an authoritative reference, both to the item itself and to the subject as a whole, is generally given for those who may be unfamiliar with a particular item. The purpose is to assist busy practitioners, students, researchers, and scholars to stay abreast of these items of progress in psychiatry that have recently achieved a substantial degree of authoritative acceptance, whether in their own field of special interest or another. The items of progress listed below were selected by the Advisory Panel to the Section on Psychiatry of the California Medical Association, and the summaries were prepared under its direction.

Cloning of the promoter for the avian integrin beta 3 subunit gene and its regulation by 1,25-dihydroxyvitamin D3.

We have cloned the promoter of the avian beta 3 integrin gene. Using a probe comprising the 5'-untranslated region of an avian macrophage beta 3 cDNA, characterized by 5' rapid amplification of cDNA ends, several clones were isolated from an avian genomic library. One major and one minor transcriptional start site were identified at +1 and -47 base pairs, respectively, with the latter coinciding with a consensus sequence of an initiator. DNA sequence analysis of 800 base pairs 5' of the transcriptional start site fails to reveal either a TATA or CAAT box. In addition to an initiator, the first 200 base pairs contain consensus sequences for the binding of AP-1 and SP-1. A 3.5-kilobase fragment located immediately upstream of the transcriptional start site exhibits functional promoter activity, and deletion analysis reveals both suppressor and enhancer elements. In light of our observation that 1,25-dihydroxyvitamin D3 (D3) accelerates beta 3 transcription, we determined whether the avian beta 3 promoter contains a vitamin D response element (VDRE). Transfected reporter constructs containing the first 1.5 kilobases upstream of the major beta 3 transcriptional start site respond to D3 with enhanced luciferase activity. Analysis of this region reveals a classical VDRE consensus sequence, located at -756 to -770. The following observations support the hypothesis that this sequence represents a functional VDRE: 1) a 600-base pair genomic fragment or a 29-base pair oligomer, each containing the putative VDRE, respond to D3 when transfected into HD11 cells; 2) a 67-base pair DNA fragment derived from genomic DNA and containing the candidate beta 3 VDRE specifically binds the vitamin D receptor-retinoid X receptor beta complex; and 3) avian osteoclast precursor-derived nuclear extracts bind to a synthetic oligomer containing the beta 3 VDRE-like sequence and, in turn, are specifically displaced by unlabeled beta 3 VDRE and anti-vitamin D receptor antibody.

A conserved parasite serine protease processes the Plasmodium falciparum merozoite surface protein-1.

The merozoite surface protein-1 of the human malaria parasite Plasmodium falciparum undergoes an extracellular proteolytic cleavage (secondary processing) intrinsic to successful erythrocyte invasion. In the T9/96 clone of P. falciparum the protease responsible has been characterised as a membrane-associated, calcium-dependent activity, sensitive to irreversible inhibitors of serine proteases. Here we extend these studies and show that secondary processing activity in intact merozoites of P. falciparum strains expressing the alternative dimorphic type of merozoite surface protein-1 has identical characteristics, and that the cleavage site is close to or identical to that in the protein from T9/96. The protease responsible is shown to be parasite-derived, and able to catalyse processing of native substrate only when present in the same membrane. Cleavage of the substrate follows apparent first order kinetics for at least 2 half-lives. It is concluded that secondary processing of both dimorphic forms of the P. falciparum merozoite surface protein-1 is a conserved event, mediated by a mechanistically conserved protease located on the merozoite surface. These observations provide clues to the identity of the protease and show that, irrespective of the dimorphic type, secondary processing results in the same, highly conserved region of the merozoite surface protein-1 remaining on the surface of the invading merozoite.

Regulation of mouse bone marrow macrophage mannose receptor expression and activation by prostaglandin E and IFN-gamma.

The macrophage mannose receptor mediates the clearance of microorganisms and glycoproteins containing terminal mannose oligosaccharides. Cell surface expression of this receptor progresses with macrophage differentiation, and thus may be critical to the scavenger function of tissue and circulating macrophages. Bone marrow macrophages, which were used in this study, differentiate in culture and express functional mannose receptors. The cytokine IFN-gamma triggered activation of these macrophages and down-regulated cell surface expression of the mannose receptor after 48 h. Macrophage activation, as assessed by the generation of superoxide radicals, was inversely correlated with mannose receptor expression. The number of surface receptors was diminished by exposure to IFN-gamma, whereas the binding affinity of the mannose receptor remained unchanged. Treatment with IFN-gamma reduced receptor biosynthesis yet did not alter receptor degradation. Mannose receptor biosynthesis is up-regulated by PG of the E series, and these anti-inflammatory agents reversed the effects of IFN-gamma on receptor expression. Down-regulation of the mannose receptor by IFN-gamma was fully reversible by PGE, indicating that receptor levels are dependent on the functional state of the cell rather than being linked to terminal cell differentiation. The regulation of the receptor by cytokines and anti-inflammatory reagents suggests that the mannose receptor plays a critical role in macrophage scavenger functions and potentially in modulating inflammatory reactions.

Training of residents and medical students in the diagnosis and treatment of dual diagnosis patients.

Treatment of dual diagnosis patients requires simultaneous treatment of the addictive and the mental disorders. Available data suggest that this does not happen often. In a survey of several psychiatric services, the unit chiefs reported that dual diagnoses were underreported, no plans were present for combined treatment, families were infrequently involved, and few referrals were made for combined treatment. There is a need for competent, experienced clinician teachers who have had positive experience with the treatment of dual disorders. The training of addiction and mental health professionals must include cooperation, understanding, and respect for each other. Cross-training is needed in chemotherapy, psychotherapy, abstinence from alcohol and other addictive drugs, 12-Step programs, spiritual issues, and milieu therapy. Negative attitudes and ignorance must be overcome for this training to take place. Faculty Fellow training programs have provided a beginning in this direction, but have so far involved few professional schools. Some examples of training with regard to referrals, prescribing, and psychotherapy are given. The importance of supervised clinical experience in treating dual diagnosis patients is emphasized. The provision of this experience provides a challenge to specialists in addiction medicine and addiction psychiatry.

Monoclonal antibodies that inhibit Plasmodium falciparum invasion in vitro recognise the first growth factor-like domain of merozoite surface protein-1.

A major protein found on the surface of the invasive stage of the malaria parasite Plasmodium falciparum, merozoite surface protein-1 (MSP1), has been proposed as a vaccine candidate. Antibodies which recognise a single fragment of this molecule (MSP1(19)), composed of 2 regions related to epidermal growth factor (EGF), also inhibit parasite growth in vitro. It is shown by direct expression of the individual EGF-like domains in Escherichia coli, that the first domain is the target of growth-inhibitory antibodies. A single amino acid difference influences the binding of some antibodies to this domain.

Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin alpha v beta 3 potentiate bone resorption.

We have investigated the mechanism by which osteoclasts adhere to and resorb bone. We show that these cells express beta 1 and beta 3 integrins which are involved in attachment to purified bone matrix proteins. Binding to osteopontin and bone sialoprotein is mediated by alpha v beta 3, while a beta 1 integrin is responsible for attachment to fibronectin. Both the rapid attachment by osteoclasts to intact bone particles and their subsequent resorption are blocked by a monoclonal antibody directed to the alpha v beta 3 complex but not by an antibody against beta 1 integrins. Attachment of osteoclasts to bone is also inhibited with soluble osteopontin, Arg-Gly-Asp-containing peptides derived from both osteopontin and bone sialoprotein, or a monospecific polyclonal antibody against osteopontin. We conclude that both osteoclast adherence to bone and subsequent resorption of its matrix are dependent on interactions between the bone matrix proteins osteopontin and/or bone sialoprotein and the integrin alpha v beta 3. Moreover, collagen, which constitutes 90% of its organic matrix, is minimally involved in binding of chicken osteoclasts to bone.