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Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: AF: SELAS Pointgyn, Laboratoire Innotech International, Respiratory and Women’s Health Products, Center for observational and real-world evidence (CORE);CLR: Ferring;CRJ:Bayer, Bristol Myers Squibb, Merck Serono Healthcare, Mylan, Roche, Theramex;ES: Evalformsanté;HB: Organon, Bayer, Gédéon Richter, MSD vaccins, Exeltix, Besins;LMM: Ferring, Vichy, Ipsen, Evalformsanté, Besins, Gédéon Richter, Effik, Theramex;MB: Gédéon Richter;ML: Theramex, Gédéon Richter, Ferring, IBSA Pharma, Merck, Serono, Organon and Exeltis;SM: HAS;TB:Nordic Pharma;VB: Merck, IBSA Pharma and Besins-HealthCare, Theramex, Gedeon Richter, Ferring, IBSA Pharma, Merck Serono, Organon and Exeltis;VG: PFIZER and HAC pharma;XD: Sanofi, Astellas, Coloplast, Hologic, IBSA Pharma, Laborie, Mylan, Pierre-Fabre, Elsevier-Masson, Regimedia and HealthEvents;YA: Conférences Hermes;YC: HAS. Other authors declared no C.O.I.

Objective: To provide guidelines for the pelvic clinical exam in gynecology and obstetrics., Material and Methods: A multidisciplinary experts consensus committee of 45 experts was formed, including representatives of patients' associations and users of the health system. The entire guidelines process was conducted independently of any funding. The authors were advised to follow the rules of the Grading of Recommendations Assessment, Development and Evaluation (GRADE®) system to guide assessment of quality of evidence. The potential drawbacks of making strong recommendations in the presence of low-quality evidence were emphasized., Methods: The committee studied 40 questions within 4 fields for symptomatic or asymptomatic women (emergency conditions, gynecological consultation, gynecological diseases, obstetrics, and pregnancy). Each question was formulated in a PICO (Patients, Intervention, Comparison, Outcome) format and the evidence profiles were produced. The literature review and recommendations were made according to the GRADE® methodology., Results: The experts' synthesis work and the application of the GRADE method resulted in 27 recommendations. Among the formalized recommendations, 17 present a strong agreement, 7 a weak agreement and 3 an expert consensus agreement. Thirteen questions resulted in an absence of recommendation due to lack of evidence in the literature., Conclusions: The need to perform clinical examination in gynecological and obstetrics patients was specified in 27 pre-defined situations based on scientific evidence. More research is required to investigate the benefit in other cases., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

This paper provides an overview of ' Kia Tika, Kia Pono - Honouring Truths ' (Te Rōpū Arotahi 2022), an ethical framework to guide engagement with tamariki (children) and rangatahi (young people) who are care experienced (that is, who currently or at some stage in their lives have been in foster or residential care). Centring the voices and priorities of rangatahi with care experience, ' Kia Tika, Kia Pono' is intended for use by organisations and others working across the range of sectors and services that seek to engage tamariki and rangatahi who are care experienced in governance, policy making, service design, media or research. Its purpose is to ensure that these efforts are ethical, meaningful, and culturally safe. Grounded in Te Tiriti o Waitangi and participatory rights frameworks, 'Kia Tika, Kia Pono' is responsive to the cultural context of New Zealand. It is also distinctive in its centring of rangatahi with care experience as both knowledge-holders and knowledge-creators. In summarising the key elements of the 'Kia Tika, Kia Pono' framework, we also draw upon our insights from the research process regarding participatory practice with rangatahi with care experience. Kupu Māori/glossary of Māori words: Aroha: love, compassion, empathy; hapū: kinship group, sub-tribe, sub-nation, to be pregnant; hui: gathering, meeting, assembly, seminar, conference; iwi: extended kinship group, tribe, nation, people, bone; kai: food, meal; karakia: incantation; a set form of words to state or make effective a ritual activity; kaupapa: purpose, agenda; koha gift; especially one maintaining social relationships and has connotations of reciprocity; korowai: ornamented cloak; mana: spiritually sanctioned or endorsed influence, power, and authority; manaakitanga: showing and receiving care, respect, kindness, and hospitality; māramatanga: enlightenment, insight, understanding; mauri: life principle, life force, vital essence; pono: to be absolutely true, unfeigned, genuine; rangatahi: younger generation; rangatira: chiefly, esteemed, leader; tamariki: children; Te Tiriti o Waitangi: the Māori version of the Treaty of Waitangi; tika: what is right/good for any particular situation; tikanga Māori: customary system of values and practices that have been developed over time and are deeply embedded in the social context; tūāpapa: foundation, platform; ūkaipō: a place of nurturing and of spiritual and emotional strength; wairuatanga: spirituality; wānanga: to meet, discuss, deliberate, consider; whanau: to be born, extended family, family group; whanaungatanga: relationship, kinship, sense of family connection; a relationship through shared experiences and working together which provides people with a sense of belonging., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 The Royal Society of New Zealand.)

Background: Mycobacterium avium subspecies paratuberculosis (Map) is an infectious enteric pathogen that causes Johne's disease in livestock. Determining genetic diversity is prerequisite to understanding the epidemiology and biology of Map. We performed the first whole genome sequencing (WGS) of 141 global Map isolates that encompass the main molecular strain types currently reported. We investigated the phylogeny of the Map strains, the diversity of the genome and the limitations of commonly used genotyping methods., Results: Single nucleotide polymorphism (SNP) and phylogenetic analyses confirmed two major lineages concordant with the former Type S and Type C designations. The Type I and Type III strain groups are subtypes of Type S, and Type B strains are a subtype of Type C and not restricted to Bison species. We found that the genome-wide SNPs detected provided greater resolution between isolates than currently employed genotyping methods. Furthermore, the SNP used for IS1311 typing is not informative, as it is likely to have occurred after Type S and C strains diverged and does not assign all strains to the correct lineage. Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) differentiates Type S from Type C but provides limited resolution between isolates within these lineages and the polymorphisms detected do not necessarily accurately reflect the phylogenetic relationships between strains. WGS of passaged strains and coalescent analysis of the collection revealed a very high level of genetic stability, with the substitution rate estimated to be less than 0.5 SNPs per genome per year., Conclusions: This study clarifies the phylogenetic relationships between the previously described Map strain groups, and highlights the limitations of current genotyping techniques. Map isolates exhibit restricted genetic diversity and a substitution rate consistent with a monomorphic pathogen. WGS provides the ultimate level of resolution for differentiation between strains. However, WGS alone will not be sufficient for tracing and tracking Map infections, yet importantly it can provide a phylogenetic context for affirming epidemiological connections.

Mycobacterium kansasii (M. kansasii) is a major cause of non-tuberculous mycobacterial pulmonary disease in the South African gold-mining workforce, but the source of infection and molecular epidemiology are unknown. This study investigated the presence of M. kansasii in gold and coal mine and associated hostel water supplies and compared the genetic diversity of clinical and environmental isolates of M. kansasii. Five M. kansasii and ten other potentially pathogenic mycobacteria were cultured mainly from showerhead biofilms. Polymerase chain reaction-restriction analysis of the hsp65 gene on 196 clinical and environmental M. kansasii isolates revealed 160 subtype I, eight subtype II and six subtype IV strains. Twenty-two isolates did not show the typical M. kansasii restriction patterns, suggesting that these isolates may represent new subtypes of M. kansasii. In contrast to the clonal population structure found amongst the subtype I isolates from studies in other countries, DNA fingerprinting of 114 clinical and three environmental subtype I isolates demonstrated genetic diversity amongst the isolates. This study demonstrated that showerheads are possible sources of M. kansasii and other pathogenic non-tuberculous mycobacterial infection in a gold-mining region, that subtype I is the major clinical isolate of M. kansasii strain and that this subtype exhibits genetic diversity.

In this study the suitability of different solid media was investigated for the isolation of Mycobacterium avium subsp. paratuberculosis (Map) in order to identify the optimum single or combination of media to permit the isolation of all strain types from small ruminants. A subset of these Map strains was then further characterized by molecular typing methods to assess the genetic diversity of Map strains in the study area (Northern Greece). Map strains were isolated from tissues and faeces of infected goats (n=52) and sheep (n=8) and were analysed for polymorphisms in IS1311 to classify the strain type as Type C or S. The study found that M7H11 supplemented with mycobactin j, OADC and new born calf serum (M7H11+Mj) is the best single choice of medium for the primary isolation of Map of both Type C and S from small ruminants. The combination of M7H11+Mj and Herrolds egg yolk medium supplemented with mycobactin j and sodium pyruvate allowed the detection of all Map isolates in this study. Nineteen Map isolates were characterised by pulsed-field gel electrophoresis and the isolates demonstrated significant genetic diversity. Twelve different SnaBI and 16 distinct SpeI profiles were detected of which 25 have not been described previously and are new profiles. The combination of both enzyme profiles gave 13 different multiplex profiles. Ten different multiplex profiles were detected in goats and three in sheep. One ovine isolate gave the same multiplex profile as a caprine isolate and two different profiles were found within a single goat herd., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Mycobacterium avium subspecies paratuberculosis (Map) is the aetiological agent of Johne's disease or paratuberculosis and is included within the Mycobacterium avium complex (MAC). Map strains are of two major types often referred to as 'Sheep' or 'S-type' and 'Cattle' or 'C-type'. With the advent of more discriminatory typing techniques it has been possible to further classify the S-type strains into two groups referred to as Type I and Type III. This study was undertaken to genotype a large panel of S-type small ruminant isolates from different hosts and geographical origins and to compare them with a large panel of well documented C-type isolates to assess the genetic diversity of these strain types. Methods used included Mycobacterial Interspersed Repetitive Units - Variable-Number Tandem Repeat analysis (MIRU-VNTR), analysis of Large Sequence Polymorphisms by PCR (LSP analysis), Single Nucleotide Polymorphism (SNP) analysis of gyr genes, Pulsed-Field Gel Electrophoresis (PFGE) and Restriction Fragment Length Polymorphism analysis coupled with hybridization to IS900 (IS900-RFLP) analysis., Results: The presence of LSP(A)4 and absence of LSP(A)20 was confirmed in all 24 Map S-type strains analysed. SNPs within the gyr genes divided the S-type strains into types I and III. Twenty four PFGE multiplex profiles and eleven different IS900-RFLP profiles were identified among the S-type isolates, some of them not previously published. Both PFGE and IS900-RFLP segregated the S-type strains into types I and III and the results concurred with those of the gyr SNP analysis. Nine MIRU-VNTR genotypes were identified in these isolates. MIRU-VNTR analysis differentiated Map strains from other members of Mycobacterium avium Complex, and Map S-type from C-type but not type I from III. Pigmented Map isolates were found of type I or III., Conclusion: This is the largest panel of S-type strains investigated to date. The S-type strains could be further divided into two subtypes, I and III by some of the typing techniques (IS900-RFLP, PFGE and SNP analysis of the gyr genes). MIRU-VNTR did not divide the strains into the subtypes I and III but did detect genetic differences between isolates within each of the subtypes. Pigmentation is not exclusively associated with type I strains.

Background: Mycobacterium avium subspecies paratuberculosis (Map) causes an infectious chronic enteritis (paratuberculosis or Johne's disease) principally of ruminants. The epidemiology of Map is poorly understood, particularly with respect to the role of wildlife reservoirs and the controversial issue of zoonotic potential (Crohn's disease). Genotypic discrimination of Map isolates is pivotal to descriptive epidemiology and resolving these issues. This study was undertaken to determine the genetic diversity of Map, enhance our understanding of the host range and distribution and assess the potential for interspecies transmission., Results: 164 Map isolates from seven European countries representing 19 different host species were genotyped by standardized IS900--restriction fragment length polymorphism (IS900-RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) analyses. Six PstI and 17 BstEII IS900-RFLP, 31 multiplex [SnaBI-SpeI] PFGE profiles and 23 MIRU-VNTR profiles were detected. AFLP gave insufficient discrimination of isolates for meaningful genetic analysis. Point estimates for Simpson's index of diversity calculated for the individual typing techniques were in the range of 0.636 to 0.664 but a combination of all three methods increased the discriminating power to 0.879, sufficient for investigating transmission dynamics. Two predominant strain types were detected across Europe with all three typing techniques. Evidence for interspecies transmission between wildlife and domestic ruminants on the same property was demonstrated in four cases, between wildlife species on the same property in two cases and between different species of domestic livestock on one property., Conclusion: The results of this study showed that it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of Map infections. Furthermore, the combination of genotyping techniques may depend on the geographical location of the population to be tested. Identical genotypes were obtained from Map isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of Map between wildlife species and domestic livestock on the same property provides further evidence to support a role for wildlife reservoirs of infection.

The proapoptotic B-cell lymphoma-2 family protein Bax is a key regulatory point in the intrinsic apoptotic pathway. However, the factors controlling the process of Bax activation and translocation to mitochondria have yet to be fully identified and characterized. We performed affinity chromatography using peptides corresponding to the mitochondrial-targeting region of Bax, which is normally sequestered within the inactive structure. The molecular chaperone nucleophosmin was identified as a novel Bax-binding protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Reciprocal co-immunoprecipitation and proximity assays confirmed the Bax-nucleophosmin protein-protein interaction and verified that nucleophosmin only bound to activated conformationally altered Bax. Confocal microscopy in a cell-based apoptosis model, demonstrated that nucleophosmin translocation from nucleolus to cytosol preceded Bax movement. Specific knockdown of nucleophosmin expression using RNAi attenuated apoptosis as measured by mitochondrial cytochrome c release and activation of the caspase cascade. In a mouse model of ischaemic stroke, subcellular fractionation studies verified that nucleophosmin translocation occurred within 3 h, at a time before Bax translocation but after Bax conformational changes have occurred. Thus, we have elucidated a novel molecular mechanism whereby Bax becomes activated and translocates to the mitochondria to orchestrate mitochondrial dysfunction and apoptotic cell death, which opens new avenues for therapeutic intervention.

Objective: A Multiple-Indicator Cluster Survey (MICS) was conducted at mid-decade in more than 60 developing countries to measure progress towards the year 2000 World Summit for Children goals. These goals included the protection of at least 90% of children against neonatal tetanus through the immunization of their mothers, as measured by tetanus toxoid (TT) coverage. In the Central African Republic (CAR), serological testing was added to the MICS to understand better the relationship between survey estimates of TT coverage and the prevalence of serological protection., Methods: In the CAR MICS, mothers of children younger than one year of age gave verbal histories of the TT vaccinations they had received, using the MICS TT questionnaire. A subsample of mothers was tested for tetanus antitoxin, using a double-antigen enzyme-linked immunoadsorbent assay (ELISA). Seropositivity was defined as a titre of > or =0.01 IU/ml, and TT coverage was defined as the proportion of mothers protected at delivery, according to their history of TT vaccinations., Findings: Among the 222 mothers in the subsample, weighted TT coverage was 74.4% (95% Confidence Interval (CI); 67.0% - 81.7%) and tetanus antitoxin seroprevalence was 88.7% (95% CI; 83.2% - 94.2%). The weighted median antitoxin titre was 0.35 IU/ml., Conclusions: Tetanus toxoid coverage in the CAR was lower than the prevalence of serological protection against neonatal tetanus. If this relationship holds for other countries, TT coverage estimates from the MICS may underestimate the extent to which the year 2000 goal for protecting children against neonatal tetanus was reached. We also showed that a high level of serological protection had been achieved in a country facing major public health challenges and resource constraints.

This study compared a tetravalent DTaP-IPV vaccine (Di-Te-Ki-Pol vaccine "SSI") with the vaccination regimen used in Denmark at that time, DT-IPV plus wholecell pertussis vaccine. Two hundred and seventy children were included at their five-week routine examination. The children were allocated to one of the two vaccination regimens. No hypotonic-hyporesponsive episodes or other vaccine-related serious adverse events were seen. Local reactions, febrile and crying episodes following the investigational vaccine were similar to the reactions seen after Di-Te-Pol vaccine. All children achieved protective antibody levels to polio, diphtheria and tetanus after completing the vaccination schedule. A significantly better response to pertussis toxin was seen after the investigational vaccine. We conclude that the Di-Te-Ki-Pol vaccine is safe and immunogenic when used according to the schedule tested.

Unlabelled: We evaluated the safety and immunogenicity of a single dose of a new serogroup C O-deacetylated meningococcal polysaccharide-tetanus toxoid conjugate vaccine in 30 healthy adult volunteers. The vaccine was well tolerated with no serious adverse events and minimal local reactions and systemic symptoms. All subjects developed a fourfold or greater increase in serum bactericidal antibody (SBA) to serogroup C meningococcus. SBA geometric mean titre increased from 11 to 3649 (p<0.001). Serogroup C-specific IgG levels increased postvaccination from 0.65 to 17.02 microg/ml (p<0.001). Bactericidal titres pre- and postimmunisation showed significant correlation with serogroup C-specific IgG (r(2)=0.693). Antibody levels fell by 6 months postvaccination, however, meningococcal C IgG avidity increased indicating the successful induction of a T-cell-dependent antibody response., Conclusion: meningococcal C-tetanus toxoid conjugate vaccine is immunogenic and well tolerated in healthy adults.

To evaluate Bordetella pertussis as a cause of persistent cough in adults, we examined 201 patients who had a cough for 2-12 weeks and no pulmonary disease. We obtained the following at presentation: medical history, chest radiograph, respiratory function measurement, nasopharyngeal aspirate for polymerase chain reaction (PCR), nasopharyngeal swab specimen for culture, and a blood sample (acute serum). Four weeks later a second blood sample (convalescent serum) was obtained. Control sera were obtained from 164 age-matched healthy blood donors with no history of cough during the previous 12 weeks. Four patients were B. pertussis culture-positive; 11 (including the culture-positive patients) were B. pertussis PCR-positive; and 33, including 10 of the 11 PCR-positive patients, had serological evidence of recent B. pertussis infection. Pertussis-positive and -negative patients could not be discriminated by a history of cough. We conclude that B. pertussis infection is a common cause of persistent cough in adults. This is of concern, because these patients may be B. pertussis reservoirs from which transmission may occur to infants, in whom the disease can be devastating.

Potency and/or immunogenicity of three different Haemophilus influenzae type b-conjugated vaccines (Hib) and a DTaP-IPV vaccine alone, and their mutual interactions in DTaP-IPV-Hib combination was tested. In a mouse model, only combination of Act-Hib, in which tetanus toxoid (TT) was as active as non-conjugated TT, significantly increased the immunogenicity and potency of TT component of DTaP-IPV vaccine. Also, only combination of Hib-TITER, in which CRM197 was used as the carrier with DTaP-IPV, increased the potency of diphtheria toxoid (DT) component of DTaP-IPV vaccine significantly. It shows that the additive effect of tested Hib vaccines on immunogenicity and/or potency of TT and DT was mostly due to the existence of TT and CRM197, respectively, as the carrier in the mentioned Hib vaccines. No difference was shown in inoculation of DTaP-IPV and Hib conjugated vaccines in the same syringe or at separate sites. DTaP-IPV had dual effects on anti-Hib capsular polysaccharide (HibCP) responses to Hib vaccines in the mouse model. This duality was probably related to the carrier B-cell epitopes activity of Hib conjugated vaccines. The immunogenicity of TT component of Act-Hib and Amvax Hib-TT in the guinea pig model was shown and combination of mentioned Hib vaccines with DTaP-IPV, remarkably increased anti-TT antibody responses to the TT component of DTaP-IPV vaccine. These confirmed our results in the mouse model. Using two different protocols to evaluate the guinea pig model for induction of anti-HibCP immunity showed that a "long interval" protocol does not have any advantage over the "short interval" protocol. Also, combination of DTaP-IPV with Hib vaccines did not have any noticeable effect on anti-HibCP antibodies in the guinea pig model. Taken together, our observations in laboratory animal models may facilitate a better understanding of the mutual interactions between the different antigen components of a combined vaccine such as DTaP-IPV-Hib vaccine., (Copyright 1999 The International Association for Biologicals.)

NAVA's acellular pertussis vaccine is based on highly purified pertussis toxin (PT) inactivated with H(2)O(2). PT was analysed using advanced biochemical methodology including mass spectroscopy (LC/MS), yielding mass and peptide mapping information on the subunits. Pertactin, adenylate cyclase, and Fim 1, 2 were below detection levels and only trace amounts of filamentous haemagglutinin (FHA) have been identified as a minor impurity. The vaccine does not induce anti-FHA antibodies during the course of a 3-dose primary immunization series in infants. B and T cell epitopes are preserved to a higher extent after H(2)O(2)detoxification when compared with chemical inactivation with formaldehyde, thus providing new information explaining why vaccines employing formaldehyde detoxified PT may need additional pertussis components added to induce high levels of protection. Anti-PT antibodies generated by NAVA diphtheria, tetanus, and acellular pertussis vaccine (DTaP) showed a positive correlation with protection against WHO-defined pertussis. The safety profiles for these vaccines showed low reactogenicity with no serious adverse events due to the vaccines., (Copyright 1999 The International Association for Biologicals.)

The objective of this study was to investigate whether a tetravalent vaccine containing diphtheria, tetanus, monocomponent acellular pertussis and inactivated poliovirus (DTaP-IPV) was immunogenic and safe compared with the vaccination regime used in Denmark at the time of the study. The study was performed as an open controlled study in which 270 Danish children were enrolled at their 5 weeks' routine examination. The children were allocated to receive either (i) DTaP-IPV (12.5 Lf, 7 Lf, 40 microg, 40, 8, 32 DU) at 3, 5 and 12 months of age (n = 186) or (ii) DT-IPV (50 Lf, 12.5 Lf, 40, 8, 32 DU) at 5, 6 and 15 months of age plus whole-cell pertussis vaccine (> or = 4 IU) at 5 and 9 weeks and at 10 months of age (n = 84). No hypotonic hyporesponsive episodes or other vaccine-related serious adverse events were seen. Local reactions, febrile and crying episodes with the investigational vaccine (DTaP-IPV) were similar to the reactions seen with the existing DT-IPV vaccine. One month after completing the vaccination schedule, all children had antibodies above the defined protective antibody concentrations to polio, tetanus and diphtheria. For pertussis toxin, there was a significantly better response in the investigational vaccine group. We therefore conclude that, when used according to the schedule tested, the tetravalent DTaP-IPV vaccine is safe and immunogenic. In addition, the number of visits and the number of injections necessary are reduced with this vaccine and vaccination schedule.

A genetically detoxified pneumolysin, pneumolysoid (PLD), was investigated as a carrier protein for pneumococcal capsular polysaccharide (CPS). Such a CPS-PLD conjugate might provide additional protection against pneumococcal infections and resultant tissue damage. A single point mutant of pneumolysin was selected, which lacked measurable haemolytic activity, but exhibited the overall structural and immunological properties of the wild type. PLD conjugates were prepared from CPS serotypes 6B, 14, 19F, and 23F by reductive amination. The structural features of free PLD, as well as the corresponding CPS-PLD, as assessed by circular dichroism spectroscopy, were virtually indistinguishable from the wild type counterpart. Each of the CPS monovalent and tetravalent conjugate formulations were examined for immunogenicity in mice at both 0.5 and 2.0 micrograms CPS per dose. Tetanus toxoid (TT) conjugates were similarly created and used for comparison. The resultant conjugate vaccines elicited high levels of CPS-specific IgG that was opsonophagocytic for all serotypes tested. Opsonophagocytic titres, expressed as reciprocal dilutions resulting in 50% killing using HL-60 cells, ranged from 100 to 30,000, depending on the serotype and formulation. In general, the lower dose and tetravalent formulations yielded the best responses for all serotypes (i.e., either equivalent or better than the higher dose and monovalent formulations). The PLD conjugates were also generally equivalent to or better in CPS-specific responses than the TT conjugates. In particular, both the PLD conjugate and the tetravalent formulations induced responses for type 23F CPS that were approximately an order of magnitude greater than that of the corresponding TT conjugate and monovalent formulations. In addition, all the PLD conjugates elicited high levels of pneumolysin-specific IgG which were shown to neutralize pneumolysin-induced haemolytic activity in vitro. As a result of these findings, PLD appears to provide an advantageous alternative to conventional carrier proteins for pneumococcal multivalent CPS conjugate vaccines.

In anticipation of future combination vaccines, a recombinant class 3 porin (rPorB) of group B meningococci was evaluated as an alternative carrier protein for a Haemophilus influenzae type b (Hib) polyribosylribotol phosphate (PRP) conjugate vaccine. The use of rPorB may avoid undesirable immunologic interactions among vaccine components, including epitopic suppression from conventional carriers (e.g. tetanus toxoid [TT]), as well as provide desirable immunomodulatory effects. Rats were found to be more reliable and consistent than mice or guinea pigs for studying antibody responses to the Hib conjugates. Different Hib conjugates, Hib-TT and Hib-rPorB, consisting of PRP conjugated by reductive amination to TT or rPorB, were compared in rats. Commercially available, licensed vaccines, HbOC (HibTITER) and PRP-T (OmniHib), were used as reference controls. Maximum geometric mean ELISA IgG titers were obtained in rats after only two doses, showing booster effects for all. However, Hib-rPorB immunization consistently resulted in responses that were 1-2 orders of magnitude greater than those for the other conjugates, including the licensed control vaccines. A maximum 4600-fold rise was observed for Hib-rPorB after two doses, and, unlike the other conjugates, a 100% response rate was always achieved without adjuvant. These results warrant further investigation of Hib-rPorB in combination with DTaP.

The purpose of this study was to analyze the effect of some pharmaceutical excipients when used for mucosal vaccine formulations and to characterize the achieved immune response. After conducting various pharmaceutical evaluations of the formulations, immunokinetic studies were performed in mice, guinea pigs, and rabbits. The kinetics and the characteristics (antibody isotypes, etc.) of the immune response were studied, as well as the induced level of toxin neutralizing IgG antibodies, which are usually used as the only measures of the potency of vaccines. Results in mice show that intranasal vaccination results in a potent and rapid immune response, similar to that seen after subcutaneous immunization. In guinea pigs and rabbits, however, the subcutaneous immunization produced significantly stronger response than did intranasal vaccination. The most promising excipients were found to be either Polysorbate 20 or Cremophor EL in an aqueous mixture together with caprylic/capric glyceride. The results indicate that nontoxic and pharmaceutically acceptable excipients can be used for mucosal vaccination, providing an interesting alternative to parenteral vaccination.

Immunogenicity and vaccine potency of carrier proteins of two different PRP-tetanus toxoid (PRP-T) conjugated vaccines, produced using different size PRP (Act-Hib & Amvax Hib-T), and one PRP-CRM197 (Hib-TITER) were studied. The immunogenicity and the vaccine potency of the carrier component of the tested PRP-conjugated vaccines, and their influences on the potency of the tetanus toxoid (T) and of the diphtheria toxoid (D) component of diphtheria toxoid-tetanus toxoid-acellular pertussis-inactivated polio vaccine (DTaP-IPV) were variable. The T component of Act-Hib (large size PRP) was as immunogenic and potent as the T component of the DTaP-IPV vaccine, and a combination of Act-Hib and DTaP-IPV resulted in a more than five-fold increase in the potency of the T However, Amvax Hib-T (small size PRP) did not show any anti-T response on its own, and a combination of Amvax Hib-T and DTaP-IPV did not affect the T potency of the DTaP-IPV vaccine. In immunogenicity studies with multiple shots, Hib-TITER (small size PRP) produced significantly less anti-D antibodies than non-conjugated D. Hib-TITER did not show any D potency on its own, while a combination of a Hib-TITER and DTaP-IPV increased the potency of the D component of DTaP-IPV vaccine significantly. Thus, in the case of a combination of T-and D-containing vaccines with a PRP-conjugated vaccine in which either T or CRMI 97 has been used as the carrier, the influence of these carriers on basic immunogenicity and vaccine potency of T and D, respectively, should be considered carefully. We propose the techniques employed in this study for the quality control of combined vaccines consisting of diphtheria, tetanus, and PRP-conjugated vaccines.

Double-antigen ELISAs for detection and quantification of anti-tetanus or anti-diphtheria antibodies in serum have been developed. The assays showed good correlations with established toxin neutralizing assays and were functionally specific for IgG antibodies. The double-antigen set-up allows specific antibodies to bind to antigen-coated microtitre wells with one arm and the free arm to bind to biotin-labelled antigen. The amount of antibodies able to bind labelled antigen was assessed by adding enzyme-conjugated streptavidin and colour substrate followed by measurement of the colour using an ELISA reader. The double-antigen principle makes it possible to compare samples of different species on the same plate, permitting the direct use of existing international references of animal or human origin. The double-antigen ELISAs showed a detection limit of 0.00002 IU/ml for both antibodies and were suitable for quantifying antibodies in blood samples collected on filter paper as well as in serum. The assays required no special equipment compared to traditional ELISA.

Epidemiological and experimental studies have shown increased frequency and severity of infections after intense, long-term exercise. This study examines whether an in vivo impairment of the cell-mediated immunity and antibody production can be demonstrated after intense, long-term exercise. Twenty-two male triathletes performed one-half an ironman (group A). Vaccinations with tetanus and diphtheritis toxoid and purified pneumococcal polysaccharide were given after the exercise. Furthermore, a skin test with seven different antigens was applied on the forearm. Antibody titers were measured before and 2 wk after the exercise. The skin test was read 48 h after the application. Eleven non-exercising triathletes (group B) and 22 moderately trained men (group C) were used as control groups. Group A revealed a significantly lower skin test response to the tetanus antigen than both groups B and C. In group A, a smaller cumulative response (sum of the diameters of indurations and number of positive skin test spots) was found than in both groups B and C. No differences in antibody titers were found among the three groups. Thus, the in vivo cell-mediated immunity was impaired in the first days after prolonged, high intensity exercise, whereas there was no impairment of the in vivo antibody production measured 2 wk after vaccination.

To identify immunologically important domains on filamentous hemagglutinin (FHA), a Bordetella pertussis protein included in new acellular pertussis vaccines (ACPVs), a series of monoclonal antibodies, sera from infants vaccinated with ACPVs or whole cell pertussis vaccine (WCPV), and sera from patients with pertussis were analyzed by immunoblots containing FHA fragments and recombinant FHA proteins. Immunodominant domains located at the COOH-terminus of FHA (type I domain) and near the NH2-terminus (type II domain) were defined by the reactivity with monoclonal antibodies. The sera from patients with pertussis and sera from infants vaccinated with WCPV or with 6 different investigational ACPVs specifically recognized well-defined regions within the type I and type II domains. Identification of these prominent immunologic epitopes on FHA should be useful for the construction of more well-defined pertussis vaccines and for the interpretation of human serologic responses, which may correlate with efficacy of pertussis vaccines.

To assess the effect of the Expanded Program on Immunization (EPI) in rural Africa, blood samples were collected in two Kenyan sublocations. Serum antibodies against tetanus toxoid were measured in 155 individuals 1-70 years of age. Titers greater than the protective level of 0.01 IU/ml were found in 47% of the population. Protection was significantly higher in children born after the launching of the EPI (68%) and in women who had been at childbearing age since then (69%). Significantly lower protection was demonstrated in other age and sex-groups. The level of protection in children was equal in the two populations, whereas protection in fertile women was significantly lower in the population living a long distance from a health center. Diphtheria anti-toxin was measured in the samples from one sublocation, and 70 of 84 individuals (83%) had antibody levels greater than the protective level. No age or sex difference could be found, and there was no correlation between response levels to diphtheria and tetanus. This implicates natural infections as an important source of diphtheria antibodies. Our findings demonstrate a need for better coverage of the adult population against tetanus. Furthermore, diphtheria transmission still appears to take place, underscoring the importance of diphtheria vaccination of travelers to rural Africa.

The booster responses of three different formulations of intranasal (i.n.) diphtheria-tetanus (D-T) vaccines were determined in military recruits and compared with a conventional subcutaneous D-T vaccine. The vaccines for mucosal delivery were sprayed into one nostril and contained D and T toxoids in an enhancer mixture of polysorbate and caprylic/capric glycerides. All of the vaccines gave rise mainly to a systemic IgG response. Among 51 persons with anti-D antibody concentrations in serum below a protective level of 0.01 international units (IU ml-1) before vaccination, all except two attained protective antibody concentrations 4 weeks after vaccination. The median increase in anti-D antibody concentration was 113-fold with the most efficient i.n. formulation. The median increase in anti-T antibody level was 2.4-fold, however, the pre-vaccination levels for this antigen were very high. Within the examined levels, the booster response depended mainly on the dose of the antigen in the vaccine rather than on the concentration of the vehicle mixture. Compared with the parenteral D-T vaccine containing aluminium hydroxide as an adjuvant, all of the tested i.n. formulations showed somewhat lower immunogenicity in man as well as in pre-clinical guinea-pig studies. Among 215 persons immunized i.n., 61% preferred this route of administration rather than a parenteral injection, although the formulations were all associated with varying local symptoms, frequently stinging and pronounced, nasal secretion.

Diphtheria may occur even among previously vaccinated persons and knowledge of the duration of immunity is of crucial importance when designing effective vaccination programmes. In a follow-up study of 42 representative probands revaccinated 8 years previously, a continuous fall-off in antitoxic immunity was demonstrated. 98% were still protected (antitoxin concentration > 0.01 IU/ml). From the distribution of titres in the group the individual risk of susceptibility 8 years after revaccination was calculated to be 0.8/1000 (0.2-2.9/1000, 95% confidence limits). Thus, repeated revaccinations are required to secure continuous protection. The fall-off pattern for diphtheria antitoxin was approximately the same as for tetanus antitoxin. Peak values following revaccination are decisive for the duration of immunity. As peak values following vaccination depend on naturally acquired immunity and consequently decrease as indigenous diphtheria in a population disappears, highly potent vaccines are required to secure long-term immunity following diphtheria revaccination. The effects of dose and adjuvant are discussed.

Adverse reactions and antibody levels were compared following a booster vaccination of 177 Danish military recruits with a plain, an aluminium hydroxide (0.5 mg Al per human dose, HD) and a calcium phosphate (0.25 mg Ca per HD) adsorbed diphtheria-tetanus (D-T) vaccine. The calcium phosphate adsorbed vaccine was given in a HD of 3 Lf of D and T toxoids and proved to be of equal efficacy as the aluminium hydroxide adsorbed vaccine which was injected in a dose containing twice the antigen amount. The calcium phosphate vaccine caused fewer adverse reactions than the one adsorbed to aluminium hydroxide. The plain vaccine (6 Lf per HD of D and T toxoid) had the highest efficacy with a similar low occurrence of adverse reactions as the calcium phosphate adsorbed vaccine. Potency assays in mice were in accordance with these immunogenicity results in man if a two dose immunization schedule was followed, but not if the vaccines were compared after a single immunization as requested by the procedure for potency testing according to current WHO and European Pharmacopoeia requirements. Both of the adsorbed vaccines primed mice for specific IgE antibody formation. This could be detected after a second immunization with either of the adsorbed vaccines or with the plain D-T vaccine. Also in humans, immunization with the plain vaccine boosted specific IgE formation to a detectable level. This may be ascribed to adjuvant priming during the primary vaccination series some 20 years previously.

The occurrence of dermatophilosis on cattle in the tropics is closely associated with infestation by Amblyomma variegatum ticks. Animals that are more susceptible to the disease also tend to carry higher tick burdens. Longitudinal studies carried out on the Accra Plains suggest that N'Dama cattle acquire an enhanced degree of resistance to these ticks following initial exposure. Lesions on zebu-type Ghana Sanga cattle tend to be confined to the predilection feeding sites of adult A. variegatum even when relatively large numbers of ticks are present suggesting that these cattle are able to contain the effects of the tick. Further studies are required to investigate the nature of the apparent resistance of indigenous cattle to the tick and/or its effects and the potential for controlling the disease by immunoprophylaxis using tick derived immunogens. Current methods of controlling the disease depend on the control of ticks using chemical acaricides. Susceptibility to A. variegatum associated dermatophilosis varies between breeds of cattle therefore the level of tick control required is breed dependant. Rigorous tick control is necessary for the prevention of dermatophilosis on highly susceptible exotic breeds of cattle. The limited use of acaricides applied either at the predilection feeding sites of A. variegatum and/or at selected times when the level of challenge increases is sufficient to control the occurrence of the disease on indigenous cattle.

A close association was found between infestation with Amblyomma variegatum and the occurrence of dermatophilosis on cattle on the coastal Plains of Ghana. Animals belonging to breeds traditionally regarded as more susceptible to the disease also tended to carry higher tick burdens. The basis of the association between infestation with this tick and the occurrence of dermatophilosis would appear to involve tick-induced immunosuppression.

A dual, double antigen, time-resolved fluorescence immunoassay (DELFIA) for the simultaneous detection and quantitation of diphtheria (D) and tetanus (T) antibodies in sera has been developed. In the double antigen format one arm of the antibody binds to antigen coated microtitre wells and the other arm binds to labelled antigen to provide a fluorescent signal. This assay was found to be functionally specific for IgG antibodies and showed a good correlation with established toxin neutralization assays. Furthermore, the double antigen set-up was species independent, permitting the direct use of existing international references of animal origin to measure protective antibody levels in humans in international units (IU/ml). The detection limit corresponded to 0.0003 IU/ml with Eu(3+)-labelled toxoids and to 0.0035 IU/ml using Sm(3+)-labelled toxoids. The assay was fast with a high capacity making it a suitable method for serological surveillance studies.

In intranasal vaccination it is important that the adjuvant does not have any toxic effect on the sensitive nasal mucosa. In this study a histological and clinical evaluation of the effects of two different adjuvants in a vaccine containing detoxified diphtheria (DT) and tetanus toxoid (TT) in guinea pigs was done. The guinea pigs were divided in four groups and treated daily for 14 days with different formulations. Group I with saline, Groups 2 and 3 with the vaccines in a non-ionic surfactant formulation containing glycerides and Group 4 with tetraethyleneglycol formulation containing glycofurol. The guinea pigs in Groups 1, 2 and 4 were sacrificed on day 15 and Group 3, 1 week later and the tissues processed for histological examination. The animals remained healthy during the treatment and minor clinical signs, such as nose-blowing, decreased with time. The histological appearance, including the development of lymphoid tissue, was comparable in all groups. A specific toxic effect on the nasal mucosa by the different vaccine and adjuvant formulations was not observed.

Two diphtheria-tetanus vaccines (DT), adsorbed to either aluminium hydroxide or calcium phosphate but identical with respect to toxoid origin and amounts, were compared in full potency assays in mice according to the European Pharmacopoeia (EP) and in a reduced potency assay in guinea-pigs using a double dose immunization schedule. The efficacy of the vaccines was compared in a clinical trial with revaccination of 313 military recruits. The reduced potency assay gave a better reflection of the efficacy of the two vaccines in humans than the required assays of the EP. For release of combined, final vaccine formulations the reduced potency assay suggested will reduce the number of animals in quality control.

Patients with B cell chronic lymphocytic leukemia (B-CLL) have decreased capacity to mount relevant antibody responses upon immunization, and development of hypogammaglobulinemia is part of the natural history of the disease. We investigated the influence of histamine type-2 (H2) receptor blockade by ranitidine on the in vivo antibody production in B-CLL patients following vaccination. Anti-polysaccharide antibodies in B-CLL patients, vaccinated with a tetanus-toxoid conjugated vaccine against Haemophilus influenzae type-B (Hib), reached long-term protective levels in more than 90% of B-CLL patients randomized to ranitidine treatment, as compared to 43% of the untreated patients (P = 0.024). No difference in the response to vaccination against influenza virus types A and B protein could be detected between the two groups. Plasma histamine levels were 2-fold to 20-fold higher in 23 out of 31 B-CLL patients, compared to normal controls, and these levels showed a significant positive correlation to disease duration. These findings indicate the possibility of improving in vivo antibody production against a highly relevant pathogen in B-CLL patients by histamine type-2 receptor blockade, and the combined finding of an immune-stimulatory effect of ranitidine and increased plasma histamine levels, strongly suggests the involvement of histamine in the pathogenesis of B-CLL immunodeficiency.

Isolates of Bordetella parapertussis, recovered from sheep or man, were characterised by reaction with specific anti-Bordetella lipopolysaccharide monoclonal antibodies, production of filamentous haemagglutinin, fatty acid patterns, and antibiotic sensitivity. Generally, the isolates lay within one of four groups, with separation of the ovine isolates into two groups. Reactions with specific monoclonal antibodies against lipopolysaccharide separated the ovine isolates into these two groupings. Analysis of the cellular fatty acid compositions by cluster analysis differentiated between the human and the ovine strains and also showed variation within the ovine isolates. When the production of filamentous haemagglutinin was analysed in an ELISA system, a similar pattern emerged. Varying concentrations of filamentous haemagglutinin (11-429 ng (mg total protein)-1) were extracted from the human isolates and the one group of ovine isolates with no significant protein detected in the other ovine group. These studies demonstrate variation between and within B. parapertussis isolates recovered from two mammalian sources.

The potencies of two diphtheria-tetanus vaccines (DT) adsorbed to either aluminium hydroxide or calcium phosphate were compared in mice and guinea pigs. The vaccines were made from the same batches of purified toxoids and contained the same amounts of antigens. Immunizations were done once or twice with different doses of vaccine injected undiluted, diluted in saline or diluted in the corresponding adjuvant. The various potency assays showed that the adjuvanticity of calcium phosphate was lower than or equal to aluminium hydroxide. Despite the range of potency assays done, none of the methods reflected the efficacy of these vaccines in revaccination of humans. A simplified potency assay is suggested for release of final vaccine formulations to reduce the number of animals in quality control.

Diphtheria and tetanus antibody levels were measured before and four weeks after booster vaccination of 313 Danish military recruits participating in a clinical trial to compare aluminium hydroxide and calcium phosphate as adjuvants in diphtheria-tetanus vaccines (DT). Twenty-eight percent of the men had a diphtheria pre-vaccination content below a protective level of 0.01 IU ml-1. The calcium phosphate adsorbed vaccine showed the highest efficacy for both antigens. Adverse reactions were rare but more frequent in the calcium group than in the aluminium group. No correlation was found between pre- or post-vaccination levels and adverse reactions and both vaccines gave rise to specific IgE formation. The results show that calcium phosphate is more effective but not a safer alternative to aluminium hydroxide when compared in vaccines containing 1.0 mg ml-1 of Ca or of Al.

Non-ionic excipients, having different lipophilicity, were compared for their selection of immunological response in different organs and biological fluids. In order to express the lipophilicity of the formulation, the balance between the size and strength of the hydrophilic and lipophilic groups was used, called the hydrophile-lipophile balance (HLB) value. Mice were immunized and boosted intranasally with diphtheria toxoid, and samples were taken from the blood, spleen, nasal wash, lungs, saliva, stomach, duodenum, jejunum and the skin. In general, formulations which were highly hydrophilic and highly lipophilic were not able to augment the immunological response markedly (except for IgA). Formulations having intermediate HLB values, e.g. around 9.0, stimulated both IgG1 and IgG2a production, where the HLB = 5.5 formulation seemed to stimulate mainly IgG2b and IgG3 antibody production. On the other hand, comparing the IgA concentration in various samples with respective IgG level, increasing HLB value seems to augment the production of mainly IgA antibodies. The results indicate that the antibody isotypes may be controlled, using variations in the hydrophile-lipophile balance value.

Primary and secondary immunizations with live, attenuated yellow fever virus vaccine (17D strain) were performed in order to study the course of appearance of virus-neutralizing antibodies and immunoglobulin M (IgM) and IgG antibodies directed against the virus and the interferon-dependent enzyme 2',5'-oligoadenylate synthetase (2',5'AS) activity, determined in homogenates of peripheral B and T lymphocytes. From cellular ATP, this enzyme generates 2',5'-oligoadenylates which mediate degradation of viral mRNA by stimulation of a latent RNase. By day 4 after the first immunization, the earliest and highest 2',5'AS activity was present in the T-lymphocyte fraction. By day 7, the enzyme activity was highest in the B-lymphocyte fraction. Virus-neutralizing antibodies appeared on day 7, and IgM antibodies were present on day 12. After the second immunization, performed 2 years +/- 2 months later, the only significant increase in 2',5'AS activity was observed in the T-lymphocyte fraction. Virus-neutralizing antibodies were present from day 1, whereas no IgM antibodies were detected. By day 12, 80% of the vaccines were IgG positive. In the primary and secondary (memory) immune responses, 2',5'AS activity is expressed in the T-lymphocyte fraction prior to the appearance of antibodies directed against the virus and may serve as an early and sensitive marker of an ongoing virus infection which is otherwise difficult to detect. No change in conventional laboratory analysis parameters, such as in differential blood cell counts or total IgA, IgG, and IgM, disclosed the immune activity in either the primary or the secondary immunization.

The proliferative response of lymphocytes from tick-infested Zebu type, N'Dama and Friesian cattle and acaricide-treated Zebu types and Friesians in concanavalin A (Con A) stimulated cultures was monitored regularly for periods ranging from 11 to 27 months. The numbers of ticks on the animals and the presence of dermatophilosis were also noted. The Friesian cattle carried most and the N'Dama fewest Amblyomma variegatum ticks. The tick-infested Friesians all developed severe clinical dermatophilosis within 5 months of becoming tick-infested. Dermatophilosis lesions on the tick-infested Zebu type and N'Dama cattle were less common and less severe especially in the N'Damas. The proliferative response of lymphocytes from tick-infested Friesians in Con A stimulated cultures fell to almost half that of the acaricide-treated Friesians soon after the former became tick-infested. The tick-infested Zebu types also developed a depressed response compared with the tick-free Zebu group over a 27 month study period. However, the responses of the N'Damas was similar to that of the tick-free Zebu types. The addition of autologous serum to Con A stimulated cultures of lymphocytes derived from the tick-infested Zebu types and N'Damas suppressed their proliferative response compared with that of similar cultures for the tick-free Zebu types.

Immunogold electron microscopy and monoclonal antibodies (Mabs) were used to localize surface-related antigens of Bordetella pertussis. Unfixed organisms of B. pertussis strains which are included in the Danish whole-cell pertussis vaccine and fixed cells from a vial of vaccine were examined. Mabs to agglutinogens 2 and 3 labelled fimbria-like structures on both live and fixed cells in a serotype-specific manner. Mab against pertactin, a 69 kDa outer membrane protein, produced intense labelling of the surface of unfixed cells, whereas staining was reduced when fixed cells were examined. Mabs against filamentous haemagglutinin (FHA) stained aggregates of material between or adherent to both live and fixed cells. Negligible labelling of FHA on cell surfaces was observed. Mabs to pertussis toxin and adenylate cyclase toxin labelled loose-structured material which was adherent to or between cells, but neither of these toxin antigens was expressed on the surface of B. pertussis in Mab recognizable form. It is therefore suggested that these antigens are readily dispersed after exit from the outer membrane of B. pertussis.

The object of this work was to test the polymerase chain reaction (PCR) for demonstration of Bordetella pertussis (BP) in nasopharyngeal secretions. The method was applied to patients with recently diagnosed pertussis, as verified by BP culture. In order to test the sensitivity and specificity of PCR for the diagnosis of BP, we used known concentrations of BP, Bordetella parapertussis and Bordetella bronchiseptica in aqueous solutions. PCR was furthermore carried out on species of bacteria that might be isolated from the nasopharynx. The applicability of PCR to patient specimens was tested in 25 patients in whose nasopharyngeal secretions BP had been demonstrated after 4-7 days of culture. The detection limit of PCR in aqueous solution was 1-2 BP bacteria per reaction tube. PCR was 100% specific for BP, showing no response with other Bordetella species or other bacteria known to colonize the nasopharynx. Of 25 patient specimens, 16 were PCR-positive 4-7 days after the positive primary culture had been established; only 5 out of 13 patient specimens were positive by repeated conventional nasopharyngeal culture at that time. We conclude that PCR is a possible alternative to culture for the demonstration of BP, as PCR is considerably faster than culture and might be more sensitive.

The seasonal abundance of adult ticks on cattle and their association with dermatophilosis were investigated in five herds on the coastal plains of Ghana over a 26-month period. Four genera, Amblyomma, Boophilus, Rhipicephalus and Hyalomma were identified, A. variegatum being the predominant species occurring throughout the year with two peaks of infestation, one in April-May and the other in November. A significant positive correlation was revealed between A. variegatum and dermatophilosis in four of the five herds. Significant positive correlations were found between H. m. rufipes and dermatophilosis in two of the herds and between Rh. senegalensis and dermatophilosis in one herd. Negative correlations of statistical significance were observed between Boophilus species and dermatophilosis in three of the herds. Nevertheless, it was considered that A. variegatum was the most important tick factor involved in the pathogenesis of the disease.

The leukocyte adhesion molecule CR3 (CD11b/CD18, Mac-1) promotes leukocyte transmigration into tissues by engaging an unknown cognate ligand on the surface of vascular endothelial cells. Filamentous hemagglutinin (FHA), an adhesin of the bacterium Bordetella pertussis, binds to CR3. We hypothesized that FHA mimics the native ligand for the CR3 integrin on endothelial cells and predicted that anti-FHA antibodies should bind to endothelial cells, interfere with leukocyte recruitment, and induce endothelial permeability. Anti-FHA monoclonal antibodies bound to cerebral microvessels in sections from human brain and upon intravenous injection into rabbits. Antibody binding correlated with the ability to recognize two polypeptides in extracts of human cerebral vessels that were also bound by CD18. In vivo, antibody binding not only interfered with transmigration of leukocytes into cerebrospinal fluid but also induced a dose-dependent reversible increase in blood-brain barrier permeability sufficient to improve delivery of intravenously administered therapeutic agents to brain parenchyma.

The main purpose of the present study was to identify B-cell epitopes on the S4 subunit of pertussis toxin (PT) by the synthetic peptide approach. Two strategies were followed: (i) screening of two series of overlapping peptides (12- and 25-residue peptides) covering the entire S4 sequence by a panel of murine monoclonal anti-PT antibodies and various polyclonal anti-PT antisera in an enzyme-linked immunosorbent assay (ELISA), and (ii) analysis of the S4 amino acid sequence by a predictive algorithm followed by synthesis and immunization of mice with the predicted peptides coupled to diphtheria toxoid. The anti-peptide conjugate antisera were tested in an ELISA for cross-reactivity with native PT, B oligomer, and S4. Screening of the free peptides in an ELISA by the PT antisera indicated the presence of six B-cell epitope-containing domains covered by residues 18 to 32, 33 to 46, 39 to 52, 51 to 65, 71 to 84, and 91 to 106. None of the peptides, however, were recognized by the monoclonal anti-PT antibodies in an ELISA. Immunization with six computer-predicted peptides (B1 to B6) and three potential T-cell epitopes (T1 to T3) gave rise to very high antibody responses towards the homologous conjugates. With the exception of the anti-T1/diphtheria toxoid antisera, all anti-peptide conjugate antisera cross-reacted with PT in an ELISA at different levels. None of these anti-peptide conjugate antisera, however, showed any PT-neutralizing effect as measured by the Chinese hamster ovary cell assay and the leukocytosis-promoting activity test. The results of the present study suggest that discontinuous epitopes are predominant in the S4 subunit of native PT.

A novel procedure which allow the rapid screening of complex protein mixtures in cellular assays is described. A device has been developed which allows a convenient, simultaneous electroelution of separated proteins from whole SDS polyacrylamide gels into narrow chambers each containing single or a few protein bands. We have optimized the conditions of the procedure and have obtained an efficient removal of SDS, leading to non-toxic protein fractions in a physiological buffer suited for direct testing in cell cultures. The responses generated by stimulating lymphocytes with the purified products have been compared to the native protein and a corresponding preparation of protein transferred to nitrocellulose particles. The method was used to investigate murine T cell responses to secreted mycobacterial antigens during infection with M. tuberculosis. A immunodominant secreted protein fraction was purified in a semipreparative scale by the procedure and used to immunize mice. The specificity of and lymphokine production by T cells generated in these animals were investigated. The device developed has various applications and provides a tool for the possible identification of new T cell antigens of importance for protective immunity.

The recovery of acromegaly is not obtained in about 50 percent of cases treated with radiotherapy and/or transsphenoidal surgery. Somatostatin analogs prescribed in such cases are effective but need either several subcutaneous injections a day or continuous infusions with pumps. Long-acting formulations of the new somatostatin analog lanreotide should avoid such drawbacks. Nine acromegalics, not cured by pituitary surgery (associated with radiotherapy in 7) received on IM injection of a long acting formulation of lanreotide twice a month for one year. Basal evaluation included: clinical examination, routine analyses, gall bladder ultrasonography, hormonal investigation of pituitary function including GH and IgF-1 measurements, visual field evaluation and pituitary scanning. A similar evaluation was performed on months 6 and 12 of treatment. The clinical symptoms of acromegaly progressively improved during therapy. Plasma GH levels decreased significantly (P < 0.01) from 24.2 +/- 2.1 to 9.3 +/- 1.2, 6.4 +/- 1.4 and 7.9 +/- 1.1 micrograms/l on months 3, 6 and 12, respectively. Plasma IgF-1 levels were normalized, decreasing from 676 +/- 40 to 331 +/- 30, 350 +/- 36, and 317 +/- 29 ng/ml on months 3, 6 and 12, respectively. Plasma lanreotide levels remained stable throughout the treatment. Side-effects included slight and transient diarrhoea and abdominal cramps which disappeared after 6 months of treatment. No gallstones appeared during treatment. These results show that one injection, twice a month, of a long-acting formulation containing 30 mg lanreotide is able to control the evolutivity of acromegalies not cured by pituitary radiotherapy and/or transsphenoidal surgery. Such formulations are well tolerated and avoid the drawbacks of either several subcutaneous injections a day or continuous infusions of somatostatin analogs.

We have investigated the memory T-cell immune response to Mycobacterium tuberculosis infection. C57BL/6J mice infected with M. tuberculosis were found to generate long-lived memory immunity which provided a heightened state of acquired resistance to a secondary infection. The T-cell response of memory immune mice was directed to all parts of the bacilli, i.e., both secreted and somatic proteins. Major parts of the memory T-cell repertoire were maintained in a highly responsive state by cross-reactive restimulation with antigens present in the normal microbiological environment of the animals. A resting non-cross-reactive part of the memory repertoire was restimulated early during a secondary infection to expand and produce large amounts of gamma interferon. The molecular target of these T cells was identified as a secreted mycobacterial protein with a molecular mass of 3 to 9 kDa.

The treatment of acromegalics with somatostatin analogs requires continuous sc infusion using pumps or several sc injections daily. Long-acting formulations (BIM-LA) of BIM 23014 (BIM) using delayed microcapsules may provide a more convenient form of therapy. Fourteen acromegalics whose GH secretion had not been normalized by transphenoidal surgery followed, in 10 cases, by pituitary radiotherapy (performed at least 2 yr before the study) were studied. Eight of these patients participated in an initial study of the pharmacokinetics of BIM-LA, after which a 6-month efficacy study was undertaken. The 8 patients in the pharmacokinetic study had an initial blood sample collected for measurements of plasma GH and insulin-like growth factor-I (IGF-I) levels before the im injection of 30 mg BIM-LA, and blood samples were subsequently taken 2, 4, 6, and 8 h after injection and then twice a week for a month. Plasma IGF-I levels were measured on days 4, 14, 20, and 30 after the injection. Assays of plasma GH, IGF-I, and BIM levels were performed by RIAs. The results showed that plasma GH levels were markedly reduced from 26.0 +/- 2.0 to 2.5 +/- 0.2 micrograms/L 2 h after BIM-LA injection and remained lower than 5 micrograms/mL for the 11 following days. Plasma GH levels increased to 5.5 +/- 1.2 micrograms/L on day 14 and returned to basal values 23 days after injection. Similarly, plasma IGF-I decreased from an initial level of 656 +/- 43 to 324 +/- 23 ng/mL on day 4 and remained close to the normal range for the following 10 days. Plasma BIM levels reached a peak 2 h after the injection (7.2 +/- 2.3 ng/mL) and remained higher than or close to 1 ng/mL until the 14th day after injection. This initial study showed that a single injection of 30 mg BIM-LA effectively suppressed GH and IGF-I secretion for at least 14 days, in accordance with the kinetics of the drug in plasma. Based on the results of this initial study, 30 mg BIM-LA were injected twice monthly for 6 months in all 14 patients. All of the subjects had a basal evaluation before treatment with BIM-LA and were then subjected to assessment of clinical, pituitary, and hormonal parameters. Patients were evaluated after 3 and 6 months of treatment on the same basis as that previously used when starting the BIM-LA therapy. Plasma BIM levels were measured monthly. Clinical signs of acromegaly improved during the treatment.(ABSTRACT TRUNCATED AT 400 WORDS)