First episode of psychosis - an audit of service engagement and management at 1-2 year follow-up.

BACKGROUND: The study aimed to develop and implement local audit standards for management and service engagement in the follow-up of patients suffering from a 'first episode of psychosis'. MATERIAL AND METHODS: Audit standards, developed following a literature review and consultation with colleagues, were incorporated into a questionnaire for distribution to the community keyworkers of a 'first episode of psychosis' cohort at 1-2 years of follow-up. RESULTS: Most satisfied standards for engagement (91%) and maintenance medication (91%). Forty-two to sixty-three per cent had received psychological, family and educational interventions but these often lacked theoretical basis and detailed content. Admission, deliberate self-harm and forensic contacts were infrequent. Less than half had any structured daytime activity. Priorities identified for improving services for this group include adequate staff training in psychosocial interventions and more active planning and resourcing of day care and other constructive daytime activities. CONCLUSIONS: Simple locally-developed audit standards such as those described for a 'first episode of psychosis' population can offer a useful way of assessing service delivery and highlighting areas for development.

B-cell-autonomous somatic mutation deficit following bone marrow transplant.

Hematopoietic stem cell transplantation is characterized by a prolonged period of humoral immunodeficiency. We have previously shown that the deficiencies are probably not due to the failure to utilize the appropriate V regions in the pre-immune repertoire. However, a striking observation, which correlated with the absence of immunoglobulin IgD(-) cells and was consistent with a defect in antigen-driven responses, was that rearrangements in bone marrow transplant (BMT) recipients exhibited much less somatic mutation than did rearrangements obtained from healthy subjects. In this paper, we present evidence suggesting that naive B cells obtained from BMT recipients lack the capacity to accumulate somatic mutations in a T-cell-dependent manner compared with healthy subjects. This appears to be a B-cell-autonomous deficit because T cells from some patients, which were not able to support the accumulation of mutations in autologous naive B cells, were able to support accumulation of mutations in heterologous healthy-subject naive B cells.

Impact of HIV-1 infection on VH3 gene repertoire of naive human B cells.

B cells of the largest Ig variable heavy chain gene (VH) family, VH3, are reportedly decreased in patients with late stage HIV-1 disease. This deficit may contribute to their impaired responses to infections and vaccines. We confirmed that the VH3 family was underrepresented in serum IgM proteins, with a 45% decrease in patients with advanced HIV-1 disease. However, the proportion of VH3 within VH(1-6) IgM mRNA from peripheral B cells did not differ from that of control subjects (mean +/- SD, 57.1 +/- 9.7 vs 61.1 +/- 8. 7%). Similarly, within VH(1-6) IgD mRNA, which even more closely represents the unstimulated naive repertoire, the relative expression of VH3 mRNA was comparable in the two groups. Moreover, the frequency of individual genes within the VH3 family for IgD, particularly genes which encode putative HIV-1 gp120 binding sites, also was normal in HIV-1-infected patients. However, VH3 family expression for IgG mRNA was significantly decreased (17%) and VH4 IgG was increased (33%) relative to other VH families in advanced HIV-1-infected patients. Thus, the changes in VH family expression were more readily apparent in previously activated IgG "memory" B cell populations and, likely, in cells actively producing IgM rather than in resting naive cells. The presence of a relatively normal naive VH3 IgM and IgD mRNA repertoire in resting cells supports the prospect that with proper stimulation, particularly in conjunction with effective antiviral therapy, vigorous humoral immune responses to infections and vaccines may be elicited in this high-risk population.

Motif-specific probes identify individual genes and detect somatic mutations.

This report describes the correlation between motif-specific hybridization and nucleotide sequence as an approach to the identification of individual human V(H) genes using motif-specific oligonucleotide probes, complementary to specific motifs within individual V(H) genes. The sensitivity of the hybridization and post washing processes permits discrimination of single nucleotide differences between probe and target. This feature is used both to identify individual genes, as well as to detect mutations in genes by sequential hybridization with multiple probes. In addition to the general strategy, specific details are provided for the identification of 12 V(H)3 genes and 14 V(H)4 genes.

Human B cells accumulate immunoglobulin V gene somatic mutations in a cell contact-dependent manner in cultures supported by activated T cells but not in cultures supported by CD40 ligand.

The acquisition of somatic mutations in the rearranged immunoglobulin V regions in B cells occurs within the tightly regulated microenvironment of a germinal centre. The precise mechanism responsible for turning on the mutational process is unknown. To dissect the role of different components of the germinal centre in this mechanism, we have used in vitro cultures of normal human IgD+ peripheral blood B lymphocytes co-cultured with activated CD4+ T cells, or with resting CD4+ T cells, or with CD40 ligand and IL-4. We observed that if the cultures included activated CD4+ T cells, then up to 100% of VH transcripts on day 14 were somatically mutated. Transcripts were found to carry from one to 36 substitutions (median five). In contrast, in the absence of activated T cells, transcripts contained only background levels of somatic mutation irrespective of the presence of resting T cells or CD40 ligand and IL-4. Cell-cell contact was required for mutation because mutations were not detected when B cells were separated from activated T cells by a membrane.

VH usage and somatic hypermutation in peripheral blood B cells of patients with rheumatoid arthritis (RA).

The human antibody repertoire has been demonstrated to have a marked V-gene-dependent bias that is conserved between individuals. In RA patients, certain heavy chain V genes (VH) have been found to be preferentially used for encoding autoantibodies. To determine if such preferential use of VH genes in autoantibodies is associated with a general distortion of the V gene repertoire in RA patients, the VH composition of peripheral blood B cells was analysed among four RA patients and four age- and sex-matched healthy controls. Usage of individual VH genes (eight VH3 and three VH4 genes tested by hybridization with a set of gene-specific oligonucleotide probes) was highly biased among RA patients, but no evidence of a distortion in the bias was observed compared with healthy controls. However, the occurrence of somatic mutations in these VH genes (estimated by differential hybridization with motif-specific oligonucleotide probes targeted to CDR and FR of the tested genes, and by DNA sequence analysis) was strikingly different between patients and healthy subjects. The number of VH3 rearrangements that had accumulated somatic mutations and the number of mutations per rearrangement were significantly elevated in three of the four RA patients. A slight but not significant elevation in mutations among rearranged VH4 genes was also observed in these patients. These data suggest that although usage of individual VH genes among peripheral blood B cells is not affected by the disease, the autoimmune process may involve a significant fraction of the B cell compartment.

The human heavy chain Ig V region gene repertoire is biased at all stages of B cell ontogeny, including early pre-B cells.

The expressed human Ig repertoire is not an equal representation of all V(H) segments present in genomic DNA. Studies have shown that a restricted set of V(H) gene segments are over-represented in Ab repertoires of fetal/neonatal and adult B cells. Additionally, this restricted set of V(H) genes is frequently expressed by autoimmune and tumor B cells. To investigate at which developmental stage a bias in the repertoire begins, we compared the V(H)3 and V(H)4 family repertoires of pre-B and immature B cells from bone marrow and mature B cells from peripheral blood of two adults. We found that the V4-34 and V4-59 gene segments of the V(H)4 family and the V3-23 gene segment of the V(H)3 family dominate the repertoires of the surface Ig-negative early pre-B as well as immature and mature B cells. Furthermore, the pattern of utilization of other V(H)3 family members suggests that certain genes that are frequently rearranged during early stages of B cell development are subsequently disfavored during later stages of B cell maturation. We conclude that the over-representation of certain V genes could arise from sequential mechanisms operating at both early and later stages of B cell development. These V(H)-mediated mechanisms might include preferential rearrangement and/or efficiency of pairing with the surrogate light chain at the surface Ig-negative, early pre-B cell stage and ligand selection at more mature, surface Ig-positive, B cell stages.

Analysis of rearranged immunoglobulin heavy chain variable region genes obtained from a bone marrow transplant (BMT) recipient.

Haematopoietic stem cell transplantation has been used for the treatment of many different malignant and non-malignant diseases. The immune system of transplant recipients must be regenerated from the transplant inoculum, and it is not surprising that many transplant recipients are deficient in generating specific antibody responses to exogenous stimuli. This B cell immunodeficiency in these patients is associated with clinically significant infections, although the underlying mechanism remains unknown. We have previously shown that the pattern of usage of V(H) genes was similar between healthy subjects and BMT recipients, indicating that the immunodeficiency was not due to a dramatic imbalance in V(H) utilization. However, motif-specific hybridization analysis indicated that the accumulation of somatic mutations was much greater among rearrangements in controls than in BMT recipients. The failure of BMT recipients to accumulate somatic mutations in rearranged V(H) genes correlates with an absence of IgD- B cells, and is consistent with a defect in antigen-driven B cell responses. In the current study, which extends those findings, we have determined the nucleotide sequences of 68 heavy chain rearrangements from one patient as well as 39 rearrangements from a healthy control. Analysis of these sequences made possible a more precise definition of variable region configuration and of the status of somatic mutation in this BMT recipient. The results validate the hybridization data and support the conclusion that, although somatic hypermutation and, by inference, antigen-driven responses are detected in BMT recipients, they are deficient compared with healthy subjects as late as 1 year after transplant.

VH repertoire in human B lymphocytes stimulated by CD40 ligand and IL-4: evidence for positive and negative selection mechanisms coupled to CD40 activation.

In the normal immune system, B cells are thought to be negatively or positively selected at various checkpoints during their maturation; a process that maintains a broad immunoglobulin repertoire while eliminating non-functional or potentially harmful autoreactive antibodies. This study tested the hypothesis that utilization of certain immunoglobulin heavy chain variable region (VH) genes, possibly as a consequence of intrinsic affinity for various ligands, directs positive or negative B cell selection coupled to B cell activation in the periphery during the immune response. The specific prediction that the VH repertoire of CD40-activated B cells would differ from the repertoire of unstimulated cells from the same donor, was tested by assessing VH utilization among human B cell clones grown in vitro, following stimulation with CD40 ligand (CD40L) and IL-4. The results showed that, although utilization of the known VH families and of individual VH3 genes was similar to that found in unstimulated B lymphocytes of the same donor, utilization of individual VH4 genes in CD40-activated B cells displayed a pattern that was markedly different from that of the unstimulated B cells. An allele of V4-61, V4-61b, was over-represented among the activated cells and, in contrast, the V4-34 gene (known to encode cold agglutinins with strong autoreactive properties) was modestly represented among the VH4 activated B cells, although V4-34 was overwhelmingly predominant in the repertoire of resting B cells. These results point to the existence of selection mechanisms that operate during B cell activation in the periphery. These mechanisms may favor B cells utilizing certain VH genes and disfavor the cells that utilize other genes, possibly because utilization of the latter confers autoreactivity.

Non-stochastic utilization of Ig V region genes in unselected human peripheral B cells.

Limited evidence based on a few subjects suggests that human peripheral blood B cells may express a non-stochastic assortment of V region genes. To determine if non-stochastic utilization was a generally applicable rule, the identities of rearranged V region gene segments were determined in unselected peripheral blood B cells from 12 subjects (five male, seven female), ranging in age from 35 to 72 years. The analysis was limited to V region genes belonging to the VH3 gene family. More than 4500 independent VH3-containing rearrangements were analysed. The frequency of occurrence of eight individual VH3 gene segments contained in rearrangements was assessed using gene specific oligonucleotide probes. Usage of elements was not uniform. Three elements, which have been known to encode autoantibodies as well as to be frequently rearranged during fetal development, were represented among rearrangements more frequently than were other members of the VH3 family, and in aggregate, accounted for the majority of rearrangements. These three predominant loci are clustered in an 80 kb region suggesting an influence of chromosomal location on efficiency of rearrangement. The results document a clear, statistically significant, preference for the occurrence of specific V region genes among rearrangements. The modest amount of variation observed between subjects was not associated with either age or gender. Duplications which increased gene dose may have contributed to increased gene usage. These data indicate that, in caucasians, the immunoglobulin rearrangements in adult human B cells are dominated by a few heavy chain V region genes to the exclusion of other putatively equally functional genes. Thus, the conventional notion that the adult repertoire is normalized with respect to family complexity is not confirmed by analysis of individual VH genes.

Immunoglobulin heavy chain variable region gene usage in bone marrow transplant recipients: lack of somatic mutation indicates a maturational arrest.

Many recipients of bone marrow transplant (BMT) make normal amounts of serum immunoglobulin but are deficient in generating specific antibody responses to exogenous stimuli. To determine if abnormal usage of VH genes contributes to this immunodeficiency, the usage of VH genes was determined in peripheral blood B cells of four BMT recipients, two of whom had developed chronic graft versus host disease. The pattern of usage of VH3 or VH4 genes assessed at either 90 days or approximately 1 year after transplant was similar to that observed in healthy subjects and was marked by the over utilization of two elements, one VH3 and one VH4. However, the repertoires of each of the four BMT recipients appeared to be less complex than the repertoires of healthy subjects. The differences were a consequence of the accumulation of somatic mutations among rearrangements in the controls but not in the BMT recipients. The failure to accumulate somatic mutations in rearranged VH genes is consistent with a defect in antigen driven B-cell responses. These results indicate the although the VH gene content of the repertoire has normalized by 90 days posttransplant, a maturational arrest in B-cell differentiation associated with antigen activation persists for at least 1 year after BMT.

Analysis of the VH3 repertoire among genetically disparate individuals.

It has been shown that a conserved but highly biased subset of the germline VH complement is utilized in the peripheral VH repertoire of Caucasians. To distinguish between VH-autonomous effects and background genetic effects, we have now assessed the incidence of rearrangement of eight VH3 gene segments in preimmune CD19+IgD+B cells from genetically disparate subjects of diverse ethnic backgrounds. Results indicate that the set of gene segments utilized for rearrangements by these subjects is similar, and predominantly reflects the bias previously observed in Caucasians. The preimmune repertoire of each subject is dominated by two or three gene segments, with a predominant expression of V3-23 in all the subjects. Variation in the use of individual gene segments, including V3-23, was observed among the subjects. These results indicate that the processes that favor the preferential use of particular genes is largely independent of genes outside the VH locus. The minor variation in utilization of VH segments, however, is probably influenced by the genetic background of the individual.

Polymorphism and utilization of human VH Genes.

The human VH germline repertoire comprises approximately 100 elements, which can be grouped into seven families based on nucleotide sequence similarity. Members of different families are interspersed throughout the complex, with limited sets of alleles identified for most loci. Linkage disequilibrium between most elements is weak. Variation within the population can be attributed to differences in nucleotide sequence between allelic genes as well as to differences in the number of genes present. Gene number per haplotype varies as a result of the common occurrence of insertion/deletion polymorphisms, which may be small, involving a single element, or may be extensive, involving four or five elements. In some cases, such polymorphisms may involve duplication of a functional VH gene segment on some haplotypes and deletion of the gene on others. The resulting variation in germline composition of the VH locus may have profound effects on VH gene utilization.

Representation of rearranged VH gene segments in the human adult antibody repertoire.

The heavy chain variable region composition of the human adult Ab repertoire is poorly defined, but recent evidence suggests that peripheral blood B cells may express a nonstochastic assortment of VH genes. In this study, the contribution of individual VH gene segments to the human Ab repertoire has been assessed. As a measure of VH gene utilization, the frequency of occurrence of eight individual VH3 gene segments contained in rearrangements was assessed in the peripheral blood B cells of two adult subjects. In addition, the frequency of occurrence of rearrangements containing nine individual VH4 gene segments was analyzed in one of the subjects. More than 2500 independent rearrangements were analyzed. For controls, amplifications and subsequent identification of nonrearranged VH3 and VH4 genes from the same individuals were also performed. The results of this germ-line analysis indicated that approximately 25 VH3 gene segments and nine VH4 gene segments could be amplified quantitatively. However, usage of elements was not uniform; one VH3 element, V3-23, and one VH4 element, V4-34, were represented among rearrangements more frequently than were other members of their respective families. This pattern of VH utilization was apparent in B cells isolated from the same subject after an 8-mo interval, indicating the relative stability of the repertoire over time. These results indicate that the adult human Ab repertoire is dominated by a few VH genes demonstrating a pattern of nonrandom utilization that could involve preferential rearrangement and/or receptor-dependent selection.

Predominant V-region gene configurations in the human antibody response to Haemophilus influenzae capsule polysaccharide.

The antibody response to Haemophilus influenzae type b polysaccharide (Hib PS) is known to be encoded by a few V-region genes. We have obtained four human monoclonal Hib PS antibodies from four healthy adult subjects immunized with diphtheria toxin-conjugated Hib PS vaccine. The VH gene segments that encode for these antibodies belong to the VH3 gene family, of which two are related to the V3-23 gene and two to the VH3b subfamily. Both hybridomas that express a V3-23-related gene use short D-segments (3 bp), the JH6 gene segment and a V kappa gene derived from the A2 germline gene. The two hybridomas that express VH3b genes use D-segments of conventional length (24-33 bp), the JH4 gene segment and a non-A2 V kappa gene. Comparison of our sequences with those reported by others suggests that the above patterns of V-region gene segment association exemplify two V-region gene configurations that are predominant in the Hib PS antibody response. The first configuration is reminiscent of antibodies produced by B-1 B cells while the second is more characteristic of antibodies produced by conventional B cells. The possibility that these two configurations, in fact, represent the products of two different B cell lineages remains to be elucidated.

A fetally expressed immunoglobulin VH1 gene belongs to a complex set of alleles.

The immunoglobulin VH gene 51p1, a member of the large VH1 gene family, is preferentially expressed by B cells in the fetus and in chronic lymphocytic leukemia (CLL) and appears to be the source for many cryoglobulin rheumatoid factors. Polymorphism of 51p1 may therefore be functionally important. We have studied the germline representation of 51p1 and closely related VH elements to establish their prevalence and allelic relationship. A panel of oligonucleotide probes directed to the complementarity determining regions (CDR1 and CDR2) of 51p1 and a similar gene, hv1263, was used in restriction fragment polymorphism analysis of 48 unrelated individuals and six families. 13 VH alleles to the 51p1 locus were identified, each distinguished by its restriction fragment size, hybridization profile, or both. On some haplotypes the locus was duplicated. Null alleles were not seen. The 13 alleles were cloned, yielding nine distinct nucleotide sequences that were > 98.2% identical and included 51p1 and hv1263. These germline variations could influence specificity for antigen, because the corresponding protein sequences differed by up to five amino acids, including three nonconservative changes in the CDR. Two of the most prevalent variants contained 51p1. These findings expand the spectrum of polymorphism seen among human VH genes and elucidate the germline origin of VH1 sequences frequently expressed in autoantibodies and CLL. We conclude that the 51p1 locus is polymorphic, and that the 51p1 element is the predominant member of a complex set of alleles.