The validation of potency tests: hurdles identified by EMA/CVMP/IWP.

The biological nature of IVMPs leads to some unavoidable batch to batch variation in production. The potency test is part of the quality control of the finished product intended to confirm consistency of production and that each batch is formulated equivalent to batches that have been demonstrated to be efficacious. Adequate validation of potency tests is essential to ensure that the results of the assays accurately reflect the amount, titre, or potency of the active substance measured and to indicate the limitations on the accuracy of the measurements to be expected from the test used. The CVMP/IWP published their conclusions concerning validation of potency tests in a Reflection Paper in March 2010. The test validation must demonstrate a dose response and the precision of the result should enable reliable detection of a sub-standard batch. However, the inherent variability in experimental animals often leads to unacceptably wide confidence intervals for in vivo tests which limits their ability to detect slight changes of the antigen amount. The development of in vitro methods as alternatives to in vivo potency tests is encouraged.

xid mice reveal the interplay of homeostasis and Bruton's tyrosine kinase-mediated selection at multiple stages of B cell development.

Human X-linked agammaglobulinemia (XLA) and murine X-linked immune defect (XID) are both immunodeficiencies mediated by mutations in Bruton's tyrosine kinase (Btk), yet the developmental stage(s) affected remain controversial. To further refine the placement of the XID defect(s), we used bromodeoxyuridine labeling to determine turnover, production and transition rates of developing B cell subsets in normal, xid and xid mice expressing a human Bcl-2 transgene (xid/bcl-2). We find the xid mutation manifest at two stages of B cell development. The first is early, reducing pre-B cell production by restricting pro-B to pre-B cell transit. Surprisingly, this impairment is offset by increased survival of cells progressing from the pre- to immature B cell pool, suggesting that Btk-independent homeostatic mechanisms act to maintain this compartment. The second point of action is late, substantially reducing mature B cell production. Together, these findings reconcile apparent discrepancies in the developmental stage affected by the murine versus human lesions and suggest previously unappreciated homeostatic processes that act at the pre-B to immature B cell transition. Finally, Btk likely functions differently at these two checkpoints, since ectopic Bcl-2 expression fails to directly complement the early xid lesion, yet reverses the defect impeding final B cell maturation.

Antiviral T-cell-independent type 2 antibody responses induced in vivo in the absence of T and NK cells.

Polyomavirus (PyV) infection induces protective T-cell-independent (TI) IgM and IgG responses in T-cell-deficient (TCR beta x delta-/-) mice. In this study, we show that PyV is a TI -2 antigen: B cells with a mutated Bruton's tyrosine kinase (Xid mutants) do not respond to PyV with antibody secretion in the absence of T cells. We also demonstrate that NK-cell-mediated "help" is not absolutely required for the induction of the TI-2 antibodies to PyV; thus for the first time, we provide evidence for protective IgM and IgG responses against a viral infection induced in mice lacking T and NK cells (CD3Etg). Comparison of the antibody responses observed in T- and NK-cell-deficient mice with those of mice lacking only T cells, however, suggests that NK cells may promote isotype switching to IgG2a. This effect is probably mediated by IFN gamma secretion. In support of this idea, studies on the antibody responses of PyV-infected SCID mice that had been reconstituted with IFN gamma R-/- B cells or wild-type B cells demonstrated the IFN gamma dependence of PyV-specific TI IgG2a secretion and provided evidence that IFN gamma acting directly on B cells plays an important role in TI pathways of isotype switching to IgG2a in vivo.

B cell production and turnover in CBA/Ca, CBA/N and CBA/N-bcl-2 transgenic mice: xid-mediated failure among pre B cells is unaltered by bcl-2 overexpression.

The CBA/N strain carries xid, a murine btk missense mutation that reduces peripheral B cell numbers. Using in vivo BrdU labeling and cytofluorimetry, we have compared the magnitude, production rates, and turnover rates of each B lineage subset in the marrow and periphery of CBA/Ca and CBA/N mice. Our results show the pro-B compartment is largely unaffected by xid. In contrast, the pre-B cell pool is markedly reduced, reflecting a diminished production rate and unaltered turnover time. Despite diminished pre-B cell formation, the size of the immature B cell pool is relatively normal in CBA/N mice, due to increased proportional survival of pre-B cells. In addition, we have assessed the marrow and peripheral B cell subsets of CBA/N mice transgenic for bcl-2. These results indicate that while the bcl-2 transgene promotes lengthened survival in most B cell subsets, the pro/pre-B cell losses mediated by xid are not abrogated by bcl-2 overexpression. Taken together, these findings suggest that the initial [not readable: see text] from the pro- to pre-B cell pools, and that anomalies in subsequent compartments likely reflects the action of homeostatic mechanisms compensating for compromised pre-B cell production.

Expression of a human coxsackie/adenovirus receptor transgene permits adenovirus infection of primary lymphocytes.

Replication-defective adenoviruses are effective vehicles for gene transfer, both for the repair of defective genes and for studies of gene function in primary cells. Many cell types, including lymphocytes, are refractory to adenovirus infection because they lack the Coxsackie/adenovirus receptor (CAR) needed for virus attachment. To extend the advantages of adenovirus-mediated gene transfer to primary lymphoid populations and other cell types lacking endogenous CAR, we produced a mouse that expresses human (h) CAR as a transgene under control of a murine MHC class I promoter. hCAR protein is expressed on T and B lymphocytes from a variety of organs (spleen, lymph node, bone marrow, thymus, and peritoneum). These lymphocytes are susceptible to adenovirus infection, as demonstrated by reporter green fluorescent protein gene expression, with the fraction of expressing cells as high as 70%. Some lymphocyte subpopulations required stimulation subsequent to adenovirus infection for reporter expression. This activation requirement is a restriction imposed by the promoter used in the adenovirus construct. In subpopulations requiring activation, the elongation factor 1 promoter was far superior to a hCMV promoter for directing green fluorescent protein production. We also find that hCAR mRNA is produced in nonlymphoid tissues from all founder lines, including tissues that do not express endogenous murine CAR, suggesting the opportunity for effecting gene delivery to and testing gene function in a wide variety of primary cell types previously resistant to gene transfer.

Antigens of the sheep scab mite Psoroptes ovis.

Sheep scab caused by the mite Psoroptes ovis (Hering) is a highly contagious disease of sheep. As a first step in developing a mite-derived vaccine for controlling the disease, the soluble antigens in mite extracts which induce an immune response in sheep were identified by electrophoretic and immunoblotting techniques. At least 22 proteins were present in P. ovis extracts as revealed by Coomassie Blue staining. Mite-infested sheep serum recognised six antigenic bands in the extracts with approximate relative molecular weights ranging from 12 to 183 kDa. A deeply staining band at 31.2 kDa and another at 41.8 kDa are of particular diagnostic value. Immunoblotting studies showed that there was no cross reactivity between P. ovis and two other ectoparasites of sheep in the UK, the sheep louse Bovicola ovis (Schrank) and the sheep tick Ixodes ricinus L.

Regulation of B cell survival in xid mice by the proto-oncogene bcl-2.

CBA/N mice carry an X-linked immunodeficiency (xid) due to a point mutation in the Bruton's tyrosine kinase (btk) gene. xid mice have a smaller peripheral B cell pool than normal animals, lack CD5+ B cells (B1), and are hyporesponsive to mitogenic anti-Igs and thymus-independent type 2 Ags. The proto-oncogene bcl-2 affects B cell homeostasis by suppressing programmed cell death. We hypothesized that reduced bcl-2 expression could enhance programmed cell death in xid B cells, directly causing poor peripheral B cell survival and indirectly affecting Ag responsiveness. We measured and compared levels of endogenous Bcl-2 protein and spontaneous apoptosis in xid and normal B cells, and determined the effect of a human bcl-2/Ig minigene on B cell survival and Ag responsiveness in bcl-2 transgenics. The amount of endogenous Bcl-2 was reduced fivefold in freshly isolated xid B cells compared with that in normal cells, but was equal in xid and normal T cells. Attrition by spontaneous apoptosis was significantly higher in cultured xid B cells. Expression of the bcl-2 transgene suppressed apoptosis equally in normal and xid B cells, prolonged in vitro survival, and markedly expanded in vivo the follicular B cell population normally reduced in xid mice. However, most xid defects persisted; xid/bcl-2 mice remained deficient in B1 cells and hyporesponsive to anti-Igs, thymus-independent type 1 Ags, and thymus-independent type 2 Ags. The data suggest that signal transduction pathways using Btk independently regulate B cell survival and Ag responsiveness.

Radiation-induced apoptosis is differentially regulated in primary B cells from normal mice and mice with the CBA/N X-linked immunodeficiency.

Normal B cells responsive to thymus independent-type 1 Ags (TI-1) are resistant to low doses of ionizing radiation in vivo (200-300 cGy), compared with TI-1 responsive B cells of mice with the CBA/N X-linked immunodeficiency (xid). This difference in radiosensitivity is an intrinsic B cell property; normal B cells adoptively transferred into xid mice remain TI-1-responsive after irradiation in situ. Because irradiation induces programmed cell death (PCD) in lymphocytes, we determined whether PCD were regulated differently in normal and xid B cells. B cells isolated immediately after irradiation from normal or xid donors when cultured without stimulators became apoptotic with the same kinetics and to the same extent, showing that apoptosis was induced equally in both populations. Apoptosis could be suppressed and mitogenesis could be induced frequently, however, if irradiated B cells were cultured with B cell activators. When activators using separate signal transduction pathways were compared, a hierarchy of efficiency at effecting apoptosis rescue was observed, and activators used singly without effect could synergize to protect. xid B cells were more resistant to rescue than normal B cells unless PMA was used as a stimulant. Although the mechanism of activator-induced rescue was not established, selective overexpression of a bcl-2 transgene rendered xid B cells radioresistant. The data suggest that a signal(s) delivered to irradiated B cells in the in vivo microenvironment suppresses apoptosis and that xid B cells and a radiosensitive subpopulation of normal B cells are refractory to this signal(s).

Progression of a vesicular stomatitis virus infection in primary lymphocytes is restricted at multiple levels during B cell activation.

Small resting B cells do not support a productive vesicular stomatitis virus (VSV) infection, but are induced by B cell activators to become fully permissive for VSV replication. Nonpermissive B cell populations restrict VSV expression at multiple points: transcript levels, translation, and maturation. Unstimulated resting G0 B cells can be infected by VSV and support the synthesis of all VSV mRNAs. Steady-state levels of viral transcripts are selectively enhanced by T cell-derived cytokines to an extent comparable with that seen for cytokine-regulated cellular mRNAs. However, viral proteins are not detected in immunoprecipitates from unstimulated or cytokine-stimulated B cells despite the fact that viral mRNAs are associated with polysomes and can be translated in vitro. This translational block is released by stimulation of infected B cells with mitogenic anti-lg or LPS, or non-mitogenic PMA. VSV virion maturation is also regulated by activation signals, because neither anti-lg nor PMA-stimulated B cells produce high levels of infectious VSV particles. Because anti-lg stimulation supports viral genome replication, maturational arrest is apparently at virus assembly or release. PMA and ionomycin induces changes beyond those seen with anti-lg, because these B cells produce PFUs at levels comparable with those seen with LPS-activated B cells and VSV-permissive cell lines. Activation-dependent regulation of virus expression provides a new paradigm for assessing activator-induced events in B cell differentiation not revealed by previous assessments of proliferation of Ab synthesis.

Bilateral leg pain following lidocaine spinal anaesthesia.

Spinal anaesthesia is considered to be a safe and effective method of providing anaesthesia for a variety of surgical procedures. Recently, observations have been made that associate the use of hyperbaric lidocaine with bilateral leg pain. We report nine patients who developed strikingly similar neurological symptoms following routine spinal anaesthesia using hyperbaric lidocaine 5% solutions. All patients had their anaesthesia and surgery in the ambulatory or "short stay" care setting. In each patient, moderate to severe, bilateral, posterior, leg pain developed within 24 hr of the anaesthetic administration. The pain was described as either sharp or cramping with or without associated back pain. None of the patients demonstrated objective neurological deficits. In all cases the symptoms resolved fully within one week. The dose of lidocaine administered in these nine patients ranged from 40 to 100 mg. Although the aetiology of the symptoms is not clear the local anaesthetic or its formulation may have been responsible.

Rapid restoration of B-cell function in XID mice by intravenous transfer of peritoneal cavity B cells.

The primary method employed to correct immune deficiency is bone marrow transfer. Depending upon the exact nature of the immune deficiency, however, alternative cell sources may be used to provide a more rapid reconstitution of immune function. In this report, peritoneal cavity (PerC) B cells are shown to be effective in the rapid emendation of the B-cell defect exhibited by XID mice. Restoration of normal numbers of splenic IgM antibody-secreting cells (ASC) and serum IgM levels were observed 4 and 7 days, respectively, after the i.v. transfer of 3 x 10(6) PerC. This regimen also restored responsiveness to thymus-independent type 2 (TI-2) antigens in XID recipients. Transfer of 30 x 10(6) spleen (SP) cells restored these functions in XID recipients but at a considerably slower rate. The data indicate that introducing a small number of PerC B cells into systemic circulation results in the rapid restoration of serum IgM levels in unirradiated XID mice.

Antibodies to Chlamydia species in patients attending a genitourinary clinic and the impact of antibodies to C. pneumoniae and C. psittaci on the sensitivity and the specificity of C. trachomatis serology tests.

In a retrospective study, the prevalence of antibodies to Chlamydia trachomatis serovars D to K, C. pneumoniae, and C. psittaci in cases attending a genitourinary clinic was examined. Blood samples were collected from 7,002 cases attending the clinic in Doncaster, England between May 1983 and May 1990. Sera from these samples were tested by a modified microimmunofluorescence test using panels of microdots of egg-grown, purified elementary bodies representing a pool of C. trachomatis D to K, a single C. pneumoniae agent, a single C. psittaci agent, and a negative control. Serum specimens were tested for the presence of IgG and IgM at starting dilutions of 1/16 and 1/8, respectively. Chlamydial IgG at a level of 1/16 or higher and IgM at a level of 1/8 or higher was present in 66.6% and 2.6% of samples, respectively. Species-specific or cross-reactive IgG against C. trachomatis D to K, C. pneumoniae, and C. psittaci was present in 32.6%, 25.1%, and 0.1% of the samples, respectively. In 8.7% of samples, the level of IgG was similar against two or all three species (group-specific). IgM against C. trachomatis D to K, C. pneumoniae, or C. psittaci was present in 2.5%, 0.03%, and 0.04% of the samples, respectively. The results of the study show that antibodies to C. pneumoniae and C. psittaci account for up to half of all chlamydia IgG positive cases attending genitourinary clinics.(ABSTRACT TRUNCATED AT 250 WORDS)

Causes of conjunctivitis and keratoconjunctivitis in Karachi, Pakistan.

The causes of conjunctivitis and keratoconjunctivitis in 388 patients who attended eye casualty departments in Karachi, Pakistan, during a 5 month period were investigated. Most of these infections were diagnosed as adenovirus (291, 75%) or bacterial (71, 18.3%). Of the remainder, 9 cases (2.3%) were caused by herpes simplex virus and 7 (1.8%) by Chalmydia trachomatis. There was no evidence of typical active trachoma in this urban population. Bacteria or Candida albicans were also grown from 44 of the adenovirus cases (15%). Many of the bacteria grown from eyes in this study were resistant to antibiotics, probably because of inadequate and/or inappropriate self-medication with antibiotics in this community.

Experimental conjunctival infection of lambs with a strain of Chlamydia psittaci isolated from the eyes of a sheep naturally affected with keratoconjunctivitis.

Five ram-lambs were inoculated into the left conjunctival sac with the 15R isolate of Chlamydia psittaci, recovered from a sheep with keratoconjunctivitis. A sixth ram-lamb was kept in contact with them. The five lambs developed varying degrees of acute conjunctivitis and 14 days later C psittaci could be recovered from the inoculated eyes, from which Branhamella ovis was also isolated. The eyes were examined regularly for four months; C psittaci could not be re-isolated but the eyes developed varying degrees of follicular conjunctivitis. After four months the sheep were treated with corticosteroids in an attempt to reactivate a latent chlamydial infection but no chlamydiae could be isolated. Five months after the start of the experiment the six lambs were inoculated with 15R into the left conjunctival sacs. Acute conjunctivitis developed which was not as severe as after the first inoculation, but C psittaci could only be recovered from the left eyes of three sheep three days after inoculation. The eyes remained chronically affected by follicular conjunctivitis. Six months after the start of the experiment the left eyes were again inoculated with 15R; on this occasion acute conjunctivitis did not develop and chlamydiae could not be isolated. Chronic follicular conjunctivitis persisted until the experiment was terminated three months later.

Clinical and immunological responses of ewes following vaccination with an experimental formalin-inactivated Chlamydia psittaci (ovis) vaccine and subsequent challenge with the live organism during pregnancy.

The protection afforded by an experimental, killed, adjuvanted vaccine derived from the A22 strain of Chlamydia psittaci (ovis) against ovine enzootic abortion was studied. The vaccine was used undiluted (group A), at a dilution of 10(-3) (group B) and at a dilution of 10(-6) (group C). A fourth control group (group D) was inoculated with all components of the vaccine except the chlamydial antigen. A group of rams (group R) was also vaccinated with the chlamydial antigen diluted to 10(-3). Animals were challenged 70 days after mating with the A22 strain of C. psittaci (ovis) and were studied throughout pregnancy and the subsequent lambing period. Their cell-mediated immune responses were examined using a skin test and their humoral immune responses were studied using an ELISA. Tests for excretion of chlamydiae in their faeces and genital tract during pregnancy and after parturition and in the faeces of their lambs were made. The reproductive performance of the ewes was assessed by calculating the average weight of lambs produced per ewe in each group. The experimental vaccine protected the ewes in groups A and B against challenge with C. psittaci (ovis) as none showed clinical signs of OEA or excreted chlamydiae. The average weight of lambs produced per ewe in both groups was greater than 4 kg. Both groups seroconverted after vaccination but not all of them were positive to the skin test. The experimental vaccine at 10(-6) dilution of antigen did not protect the ewes as three of 10 ewes displayed clinical OEA and excreted chlamydiae in the products of parturition.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection of ewes vaccinated with A22 strain Chlamydia psittaci (ovis) against challenge in pregnancy with homologous and heterologous strains of the organism.

Fifty ewes were randomly divided into four groups. Groups A and B were vaccinated with an experimental vaccine derived from the A22 isolate of Chlamydia psittaci (ovis), an isolate known to cause ovine enzootic abortion (OEA). Groups C and D were unvaccinated controls. In mid-pregnancy, animals in group A and C were challenged with live A22 C. psittaci (ovis) and those in B and D were challenged with a field isolate of the organism (BS) against which the commercially available A22 vaccine appeared to offer poor protection. In group A, three animals showed clinical signs of OEA and six excreted chlamydiae. In group B, five ewes showed clinical signs of OEA and excreted chlamydiae. In group C, three ewes had clinical signs of OEA but seven excreted chlamydiae. In group D, all 11 ewes showed clinical signs of OEA and excreted chlamydiae in the products of parturition. This group produced only four live lambs with an average weight of viable lamb per ewe of 1.4 kg, whereas the other groups each produced 12 or 13 lambs with an average weight of viable lamb per ewe of more than 4 kg. The BS isolate was much more virulent than the A22 isolate for unvaccinated, pregnant ewes. However, the A22 vaccine offered significant protection against the heterologous BS isolate although on this occasion it did not appear to alter the course of disease produced by the less pathogenic A22 isolate.