A scalable method for O-antigen purification applied to various Salmonella serovars.

The surface lipopolysaccharide of gram-negative bacteria is both a virulence factor and a B cell antigen. Antibodies against O-antigen of lipopolysaccharide may confer protection against infection, and O-antigen conjugates have been designed against multiple pathogens. Here, we describe a simplified methodology for extraction and purification of the O-antigen core portion of Salmonella lipopolysaccharide, suitable for large-scale production. Lipopolysaccharide extraction and delipidation are performed by acetic acid hydrolysis of whole bacterial culture and can take place directly in a bioreactor, without previous isolation and inactivation of bacteria. Further O-antigen core purification consists of rapid filtration and precipitation steps, without using enzymes or hazardous chemicals. The process was successfully applied to various Salmonella enterica serovars (Paratyphi A, Typhimurium, and Enteritidis), obtaining good yields of high-quality material, suitable for conjugate vaccine preparations.

Determination of free polysaccharide in Vi glycoconjugate vaccine against typhoid fever.

Glycoconjugate vaccines based on the Vi capsular polysaccharide directed against Salmonella enterica serovar Typhi are licensed or in development against typhoid fever, an important cause of morbidity and mortality in developing countries. Quantification of free polysaccharide in conjugate vaccines is an important quality control for release, to monitor vaccine stability and to ensure appropriate immune response. However, we found that existing separation methods based on size are not appropriate as free Vi non-specifically binds to unconjugated and conjugated protein. We developed a method based on free Vi separation by Capto Adhere resin and quantification by HPAEC-PAD. The method has been tested for conjugates of Vi derived from Citrobacter freundii with different carrier proteins such as CRM197, Tetanus Toxoid and Diphtheria Toxoid.

Comparison between an enzyme-linked immunosorbent assay using a detergent-soluble Leishmania infantum antigen and indirect immunofluorescence for the diagnosis of canine leishmaniosis.

Serum samples collected from 290 dogs--186 Leishmania-infected and 104 control animals--were screened to detect the presence of specific antibodies to Leishmania infantum antigens in Tuscany, Italy. Two different techniques were compared: an indirect immunofluorescence assay (IFA) and an indirect enzyme-linked immunosorbent assay (ELISA). The ELISA was developed using a detergent-soluble antigen of L. infantum promastigotes. Triton X-100 and protease inhibitors were used as detergent and to increase reproducibility of the assay, respectively. A strong correlation between the anti-Leishmania antibody levels obtained by ELISA and those obtained using IFA was observed. The ELISA appeared to be somewhat more sensitive than IFA (99.5% vs. 98.4%), while its specificity was lower (97.1% vs. 100%), even if not significantly different. Immunoblotting analysis, using the detergent-soluble L. infantum antigen, demonstrated that proteins of M(r) 30 and 73 kDa were recognized by all positive sera, regardless of the serum titre. Furthermore, antigens of M(r) 16, 18, 26, 33, 50 and 117 kDa were also frequently reactive with a large proportion of sera from infected dogs. This ELISA demonstrated a high sensitivity and specificity as well as the IFA, and it seems to be a suitable assay for large scale epidemiological studies.

[Intensive care in gastroenterology. 1st experiences and verification]. / Terapia intensiva in gastroenterologia. Prime esperienze e verifiche.

The history of intensive care in gastroenterology is briefly discussed. The insertion of intensive care units in the hospital scene is also examined and illustrative examples of emergency cases treated in recent years at the Rome United Hospitals gastroenterology division are presented. It is felt that centres tailored to hospital dimensions should be formed, though their cost is likely to prove a bar to their early institution. Intensive care can, it is urged, be furnished by specialist departments, provided they manage to find a new dimension in keeping with the more difficult tasks involved.

[The topical therapy of ulcerative colitis. A multicenter study with beclomethasone dipropionate foam]. / Terapia topica della colite ulcerosa. Studio multicentrico con beclometasone dipropionato schiuma.

BACKGROUND AND AIMS: The use of steroids was recently extended to the various forms of ulcerative rectocolitis by the introduction of topical formulations, above all steroids with an hepatic "first pass" devoid of systemic interference. The aim of this study was to evaluate the efficacy and tolerability of Beclomethasone dipropionate (BDP) in a rectal foam formulation, in the treatment of patients suffering from ulcerative colitis. METHODS: The experimental protocol took the form of a 28-day open prospective trial using BDP rectal foam in patients suffering from ulcerative colitis. Endoscopic, histological, clinical and tolerability parameters were evaluated. The centres taking part in the trial collected data for 60 cases out of a total of 80 patients enrolled in the study, of both sexes and aged between 20 and 81 years old, suffering from proctosigmoiditis (46.7%) and ulcerative rectocolitis (53.3%). RESULTS: Endoscopic parameters showed an improvement after 28 days of treatment in 74.5% of patients; a clinical improvement was achieved in 65.2% of cases. In percentage terms of the mean value of all the improved parameters, histological parameters were altered in 56.9% of patients. With regard to tolerability 82% of patients judged the treatment to be good/excellent. CONCLUSIONS: In conclusion, in line with recent reports regarding other pharmaceutical forms of BDP, including the use of rectal foam, these data confirm the efficacy and tolerability of this molecule and emphasise the validity of its use in the treatment of ulcerative colitis and proctosigmoiditis.

[The role of endomysium antibodies in the diagnosis and monitoring of celiac disease]. / Ruolo degli anticorpi anti endomisio nella diagnosi e nel monitoraggio della malattia celiaca.

Coeliac disease is a common cause of chronic diarrhea in children and adults. It is also frequently detected in children exclusively affected by iron deficiency anemia, hypocalcemia, short stature, dental enamel defects, epilepsy and intracranial calcifications, etc. The coeliac disease diagnosis may be facilitated by the use of some immunological tests like anti endomysial (AEA) or anti gliadin (AGA) antibodies detection. From December 1990 to September 1992 anti endomysial IgA and anti gliadin IgG antibodies were respectively detected in 1680 and 1598 sera from children and adults affected by chronic diarrhea, failure to thrive or other symptoms compatible with coeliac disease diagnosis. According to ESPGAN criteria at that time coeliac disease diagnosis was made in 73 cases. In our experience AEA IgA show to have a better sensitivity and specificity in the diagnosis of coeliac disease rather than AGA IgG (97.5% vs 95.1% and 99.5% vs 98.3% respectively).

Production of a conjugate vaccine for Salmonella enterica serovar Typhi from Citrobacter Vi.

A conjugate vaccine for Salmonella enterica serovar Typhi was produced by chemically linking Vi, purified from Citrobacter, to the non-toxic mutant diphtheria toxin CRM(197) via an adipic dihydrazide spacer using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide coupling chemistry. The polysaccharide purification process was developed based on Vi precipitation from culture supernatant with cetyl trimethylammonium bromide (CTAB), solubilization of the CTA-polysaccharide salt with ethanol followed by exchange of the CTA(+) counter ion with Na(+). The purified Vi polysaccharide was fully O-acetylated and with high purity. The conjugation process was optimized to obtain a scalable process that has been used for GMP production at pilot scale of vaccine currently in clinical trials.

Mycological findings in feline immunodeficiency virus-infected cats.

Thirty-five FIV-seropositive cats and 55 FIV-seronegative matched cats were examined for yeasts (oropharyngeal swabs) and dermatophytes (hair brushings). The frequency of isolation of Candida albicans and Cryptococcus neoformans was significantly higher in the former group. The only dermatophyte isolated was Microsporum canis. Its prevalence was three times higher among FIV-infected cats than among control animals.

Serum neutralization of feline immunodeficiency virus is markedly dependent on passage history of the virus and host system.

Sera from feline immunodeficiency virus (FIV)-infected cats exhibited extremely low levels of neutralizing antibodies against virus passaged a few times in vitro (low passage), when residual infectivity was assayed in the CD3+ CD4- CD8- MBM lymphoid cell line or mitogen-activated peripheral blood mononuclear cells. By sharp contrast, elevated titers of highly efficient neutralizing activity against FIV were measured, by use of high-passage virus, in assays on either the fibroblastoid CrFK or MBM cell line. However, high-passage virus behaved the same as low-passage virus after one in vivo passage in a specific-pathogen-free cat and reisolation. Subneutralizing concentrations of infected cat sera enhanced the production of low-passage virus by MBM cells, an effect not seen with high-passage virus in CrFK cells. These qualitative and quantitative discrepancies could not be attributed to differences in the amount of immunoreactive viral material, to the amount of infectious virus present in the viral stocks, or to the presence of anti-cell antibodies. The observed effects were most likely due to the different passage history of the viral preparations used. The observation that neutralizing antibodies detected with high-passage virus were broadly cross-reactive in assays with CrFK cells but isolate specific in MBM cells suggests also that the cell substrate can influence the result of FIV neutralization assays. This possibility could not be tested directly because FIV adapted to grow in CrFK cells had little infectivity for lymphoid cells and vice versa. In vitro exposure to infected cat sera had little or no effect on the ability of in vivo-passaged FIV to infect cats. These data reveal no obvious relationship between titers against high-passage virus and ability to block infectivity of FIV in cats and suggest caution in the use of such assays to measure vaccine efficacy. In conclusion, by contrast with what has been previously reported for the use of CrFK cells and high-passage virus, both natural and experimental infections of cats with FIV generate poor neutralizing antibody responses with regard to in vivo protection.

Detection of salivary antibodies in cats infected with feline immunodeficiency virus.

The saliva of cats infected with feline immunodeficiency virus was examined for total immunoglobulin content and antiviral antibodies. Seropositive cats showed an increase in salivary immunoglobulin G levels, which was only partly attributable to the enhanced prevalence of oral inflammatory lesions, compared with the levels in seronegative cats. Immunoglobulin G, but not immunoglobulin M, levels in serum were also increased. Salivary antibodies were determined by indirect immunofluorescence and Western blot (immunoblot) analysis. All but 1 of the 16 seropositive cats examined were positive, while all 16 control cats were negative. The presence of oral lesions was not a prerequisite for antibody detection in saliva. It was concluded that salivary antibody might be usefully exploited for diagnostic and epidemiologic purposes.