Viral vaccine stabilizers: status and trends.

Vaccine stability is a key factor to preserve vaccine potency and efficiency, as its potency decays over time and during temperature changes. The choice of stabilizers for viral vaccine formulation depends mainly on the vaccine type. More specifically, the choice is determined by the properties and structure of the active pharmaceutical ingredient or viral antigen(s) in the vaccine. In this review, we analyze key formulation components in different vaccine types. We discuss some of the major driving forces in the improvement of vaccine thermostability: increasing demand for cost-effective production of thermostable vaccine with lower dependency on cold chain, stricter regulatory policies for animal-origin materials, and the return of the research investment from the industry point of view. Moreover, we provide an overview of existing licensed viral vaccine types, including their production platform, presentation, delivery route, known stabilizers content and available thermostability data. In addition, we compare the data of licensed vaccines to published experimental vaccines, in order to discuss the current trends in vaccine stabilizers development.

New and cost effective cell-based assay for Dialyzed Leukocyte Extract (DLE)-induced Jurkat cells proliferation under azathioprine treatment.

The human Dialyzed Leukocyte Extract (DLE) is a heterogeneous mix of oligopeptides of <10kDa, extracted from leukocytes of healthy donors. There is significant clinical evidence of improvement using DLE during treatment of allergies, cancer,immunodeficiencies, and in mycotic and viral infections. Nevertheless, the DLE exact nature and mechanism of action have been elusive for more than 50 years. DLE biological activity testing is necessary in DLE production and quality control. Both in vitro and in vivo assays exist: E-rosette test, induction of delayed type hypersensitivity in mice, leukocyte migration and IFN-γ secretion. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic burden, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. Here we are reporting a new DLE biological activity cell-based assay. The A20 and Jurkat cell lines were treated with (+Aza) or without (-Aza) azathioprine, DLE (+DLE) or both (+Aza/+DLE). After 72h, the cell proliferation was analyzed by the MTT or BrdU incorporation assays. In +Aza/+DLE treated cells, we observed a significant higher proliferation, when compared with +Aza/-DLE. In the absence of Aza, cells did not present any proliferation difference between -DLE or +DLE treatments. Both assays, MTT and BrdU showed similar results, being the MTT test more cost effective and we select it for validation as DLE biological assay using Jurkat cells only. We tested three different lyophilized DLE batches and we found consistent results with acceptable assay reproducibility and linearity. The DLE capacity for rescuing Jurkat cell proliferation during +Aza treatment was consistent using different liquid and lyophilized DLE batches, presenting also consistent chromatographic profiles. Finally, DLE treatment in Jurkat cells did not result into significant IL-2 of IFN-γ secretion, and known lymphocyte proliferative drugs failed to rescue Jurkat cells viability in presence of +Aza, as +DLE treatment did in our MTT assay. In conclusion, our new cell-based MTT assay has excellent DLE biological activity consistency, robustness and is cost effective, presenting important advantages over previous DLE activity in vitro and in vivo assays.

Salmonella typhimurium em linfonodos mesentéricos de ovinos ao abate / Presence of Salmonella typhimurium in ovine mesenteric lymph nodes at slaughter

O rebanho de ovinos no Brasil está estimado em mais de 16 milhões de cabeças. Embora o consumo da carne desta espécie ainda seja pequeno, comparado ao de outros países, o consumo de carne, inclusive ovina, tem sido associado às doenças transmitidas por alimentos, em especial a salmonelose. No presente estudo, investigou-se a ocorrência de salmonelas em linfonodos mesentéricos e conteúdo intestinal de 175 ovinos ao abate. "Pools" constituído por cinco amostras de contéudo fecal ou 5 amostras de linfonodos de 25 g foram pre-enriquecidos em 250 mL de água peptonada tamponada e incubados a 37° C por 18-24 horas. Uma alíquota de 0,1 mL do préenriquecimento foi transferida para 9,9 mL de caldo de enriquecimento Rappaport-Vassiliadis e 1,0 mL do pré-enriquecimento foi transferido para 10 mL de caldo tetrationato Muller-Kaufmann, incubados a 42° C for 24h. 10 µL do caldo de enriquecimento foi semeado superfície de placas de ágar BPLS e ágar XLT4 incubadas a 37º C for 24-48h. Colônias suspeitas de salmonela foram testadas por provas bioquímicas e serologicas. Os testes bioquímicos utilizados para identificação de Salmonella foram TSI (triple sugar iron àgar), LIA (lysine iron àgar) e ágar ureia. Sorotipagem foi realizada no Laboratório de Enterobactérias do Instituto Osvaldo Cruz. Isolou-se Salmonella Tiphymurium de um pool de linfonodos mesentéricos, provenientes de cinco animais. O fato de se observar a ocorrência de salmonela em ovino portador sadio alerta para necessidade de monitorar este micro-organismo também nesta espécie, especialmente quando destinada ao abate, com vistas à produção de alimentos seguros.

The ovine flock in Brazil is estimated at over 16 million head. Despite that meat consumption of this species is still small when compared to other countries, general meat consumption, including mutton, has been associated to food borne diseases, especially salmonellosis. In the present study, the occurrence of salmonella in mesenteric lymph nodes and intestinal content of 175 ovines during slaughter was investigated. A pool of 5 feces samples or 5 lymph node samples of 25 grams was pre-enriched in 250 mL of buffered peptone water at 37° C for 18-24h. Following this, 0.1 mL of pre-enriched broth was transferred to 9.9 mL of Rappaport-Vassiliadis enrichment broth and 1.0 mL of pre-enriched broth was transferred to 10 mL of Muller-Kaufmann tetrationate broth, incubated at 42° C for 24h. Then, a 10 µL of the enrichment broth was spread on the surface of a BPLS and an XLT4 plate, both incubated at 37º C for 24-48h. Suspected Salmonella colonies were picked from the agar and tested with biochemical and serological methods. Biochemical testing was carried out for the identification of Salmonella, using the TSI (triple sugar iron agar), LIA (lysine iron agar) and urea agar tests. Serotyping was done at the Laboratory of Enterobactérias of the Instituto Osvaldo Cruz. Salmonella Tiphymurium was isolated from a pool of mesenteric lymph nodes from 5 animals. That Salmonella was observed in healthy carrier ovines points out the necessity of monitoring this microorganism in this species as well, especially when animals are destined to slaughter, so to assure safe food production.

An early function of the adenoviral E1B 55 kDa protein is required for the nuclear relocalization of the cellular p53 protein in adenovirus-infected normal human cells.

It is well established that the human subgroup C adenovirus type 5 (Ad5) E1B 55 kDa protein can regulate the activity and concentration of the cellular tumor suppressor, p53. However, the contribution(s) of these functions of the E1B protein to viral reproduction remains unclear. To investigate this issue, we examined properties of p53 in normal human cells infected by E1B mutant viruses that display defective entry into the late phase or viral late mRNA export. The steady-state concentrations of p53 were significantly higher in cells infected by the E1B 55 kDa null mutant Hr6 or three mutants carrying small insertions in the E1B 55 kDa protein coding sequence than in Ad5-infected cells. Nevertheless, none of the mutants induced apoptosis in infected cells. Rather, the localization of p53 to E1B containing nuclear sites observed during infection by Ad5 was prevented by mutations that impair interaction of the E1B protein with p53 and/or with the E4 Orf6 protein. These results indicate that the E1B protein fulfills an early function that correlates efficient entry into the late phase with the localization of E1B and p53 in the nucleus of Ad5-infected normal human cells.

Plasma and testicular testosterone levels, volume density and number of Leydig cells and spermatogenic efficiency of rabbits.

Plasma and tissue testosterone concentrations were determined by radioimmunoassay in 12 eight-month-old sexually mature New Zealand White rabbits and evaluated for possible associations with spermatogenic efficiency as well as with volume density and number of Leydig cells. Testicular tissue was processed histologically and histometry was performed in order to quantify germ cells, Sertoli cells and Leydig cells. Spermatogenic efficiency, reported as the ratios among germ cells (spermatogonia, primary spermatocytes and round spermatids) and by the ratio of germ cells to Sertoli cells, was not associated with testosterone levels. However, Leydig cell parameters such as number of Leydig cells per gram of testis, total number of Leydig cells per testis and percent cell volume of Leydig cell nuclei were correlated significantly with testosterone levels. The statistically significant correlation (r = 0.82, P<0.05) observed between testosterone levels and the number of Leydig cells per gram of testis suggests that, in the rabbit, the latter parameter can serve as a criterion for monitoring testosterone levels in this species under normal conditions.

Plasma and testicular testosterone levels, volume density and number of Leydig cells and spermatogenic efficiency of rabbits

Plasma and tissue testosterone concentrations were determined by radioimmunoassay in 12 eight-month-old sexually mature New Zealand White rabbits and evaluated for possible associations with spermatogenic efficiency as well as with volume density and number of Leydig cells. Testicular tissue was processed histologically and histometry was performed in order to quantify germ cells, Sertoli cells and Leydig cells. Spermatogenic efficiency, reported as the ratios among germ cells (spermatogonia, primary spermatocytes and round spermatids) and by the ratio of germ cells to Sertoli cells, was not associated with testosterone levels. However, Leydig cell parameters such as number of Leydig cells per gram of testis, total number of Leydig cells per testis and percent cell volume of Leydig cell nuclei were correlated significantly with testosterone levels. The statistically significant correlation (r = 0.82, P<0.05) observed between testosterone levels and the number of Leydig cells per gram of testis suggests that, in the rabbit, the latter parameter can serve as a criterion for monitoring testosterone levels in this species under normal conditions

Duration of spermatogenesis and sperm transit time through the epididymis in the Piau boar.

The Piau boar is a rustic breed of economical importance in Brazil. The duration of spermatogenesis and sperm transit through the epididymis in Piau boars was estimated using intratesticular injections of tritiated thymidine. Animals were sacrificed 1 h, 7 days, 14 days, 21 days, 34 days, and 36 days after injections. Each cycle of spermatogenesis in Piau boars lasts 9 +/- 0.2 days. At least 9 days are necessary for spermatozoa to traverse the entire epididymis. Considering that the total duration of spermatogenesis takes about 4.5 seminiferous epithelium cycles, spermatogenesis was estimated to take 40.6 days. The primary spermatocytes life span is 13.5 days, while spermiogenesis in Piau boars lasts 14.5 days. Staging in Piau boars was based on tubular morphology system. The relative stage frequencies in these boars, based on approximately 1200 seminiferous tubule cross-sections for each animal, were as follows: stage 1, 11.7 +/- 0.7%; stage 2, 14.3 +/- 0.3%; stage 3, 5.4 +/- 0.1%; stage 4, 12.1 +/- 0.6%; stage 5, 9.6 +/- 0.4%; stage 6, 17.2 +/- 0.4%; stage 7, 15.4 +/- 0.8%; and stage 8, 14.3 +/- 0.9%. The duration of spermatogenic events and the relative stage frequencies in Piau boars differ slightly from those observed in improved swine breeds.

Alteraçöes etárias na espermatogênese do cäo. II. Populaçäo celular dos túbulos seminíferos e rendimento espermatogênico / Age changes in dog spermatogenesis. II. Cell population of seminiferous tubules and spermatogenic yield

Analisou-se quantitativamente a espermatogênese de 40 cäes adultos, sadios, sem raça definida (SRD), distribuídos nos seguintes grupos etários: 1 a 2,5 anos; 3 a 4 anos; 5 a 6 anos; 7 a 9 anos e acima de 10 anos. O estudo envolveu frequência relativa dos estágios do ciclo de epitélio seminífero, populaçäo celular dos túbulos seminíferos, rendimento intrínseco da espermatogênese e índice de células de Sertoli. O rendimento espermatogênico atingiu níveis máximos em animais de 3 a 6 anos de idade, configurando-se, nesta faixa etária, a plena maturidade sexual

Alteraçöes etárias na espermatogênese do cäo. I. Análise histométrica / Age changes in dog spermatogenesis. I. Histometrical analysis

Analisaram-se histometricamente os testículos de 40 cäes adultos, sadios, sem raça definida (SRD), distribuído nos seguintes grupos etários: 1,0 a 2,5 anos; 3,0 a 4,0 anos; 5,0 a 6,0 anos; 7,0 a 9,0 anos e acima de 10 anos. Os seguintes parâmetros foram abordados: peso e dimensöes testiculares, diâmetro dos túbulos seminíferos, espessura do epitélio seminífero e proporçäo volumétrica dos componentes do parênquima testicular. Os túbulos seminíferos atingiram capacidade máxima de produçäo espermática nos animais de 3,0 a 6,0 anos de idade. As principais alteraçöes observadas nos testículos dos animais velhos foram volumes percentuais aumentados de tecido conjuntivo intertubular e de células espermatogênicas degeneradas

Gonadal and extragonadal sperm reserves of the Brazilian Nelore zebu (Bos indicus).

Gonadal and extragonadal sperm reserves were estimated through hemocytometric method in six Nelore zebu bulls, aging 4-6 years, with normal spermatogenesis, and kept at sexual rest. Gonadal sperm reserve was estimated to be 47.8 +/- 5.8 X 10(6) sperm cells/g testis parenchyma and 9.8 +/- 1.7 X 10(9) sperm cells/testis. Using a time divisor of 4.94 days the daily sperm production was estimated to be 10.0 +/- 0.9 X 10(6) sperm cells/g testis parencyma/day and 2.0 +/- 0.3 X 10(9) sperm cells/testis/day. Epididymal sperm reserve amounted 11.9 +/- 1.6 X 10(9) spermatozoa/organ, distributed as follows: 35.3 +/- 3.6% in the head, 16.9 +/- 1.7% in the body and 47.7 +/- 3.7% in the tail.

Morphological events occuring in the seminiferous tubules of the Brazilian Nelore zebu associated with puberty.

The morphological events occurring in the Brazilian Nelore zebu testis from 3 to 18 months of age are described. Spermatozoa were seen only at 16--18 months of age indicating a considerable delay in the sexual development of the Nelore zebu when compared with taurine breeds.

Age-related changes in the seminal vesicles of a Brazilian (Nelore) zebu.

Age changes in the structure of the seminal vesicles and in the rate of production of fructose and citric acid have been studied in a Brazilian (Nelore) zebu, from the fetal period to 36 months of age. At 3 and 6 months, the microscopic anatomy of the gland resembled that of the fetus; the tubules of the seminal vesicles had a reduced diameter and a low epithelial layer; only a few presented traces of secretion, and tissue contents of fructose and citric acid were accordingly low. At 12 months, the tubules were more ramified and had a larger diameter. In the 18-month-old animals the seminal vesicles presented substantial modifications; the tubules were large, with irregular lumina and surrounded by narrow stroma, the epithelial layer was higher than that of previous stages and its columnar cells had nuclei located basally. Tissue levels of fructose increased rapidly between 12 and 18 months. At 24 months, the seminal vesicles had reached the adult condition characterized by intense proliferation of tubules with irregular lumina and abundant secretory material. Numerous dark columnar cells were found in the epithelium. Seminal vesicles of Nelore zebus contain less fructose and citric acid than those of taurine bulls of comparable age.

Blood supply to the testis of a Brazilian marsupial (Didelphis azarae) and its abdominotesticular temperature gradient.

The testicular arteries of Didelphis azarae originate from the abdominal aorta either independently from each other or by way of a common trunk. Accessory testicular arteries may be found. At the spermatic cord they form a rete mirabile having 26.8 +/- 5.0 and 29.3 +/- 4.9 slender branches on the right and left sides, respectively. The arterial branches are intermingled with veins of similar caliber and number. Near the testis the branches of the rete reunite in a single vessel which then penetrates the parenchyma of the testis. Inside the testis the artery divides usually into two main branches that course toward the caudal pole. The rectal, scrotal and testicular temperatures were 32, 28.5 and 30.4 degrees C, respectively, appearing than an abdominotesticular gradient temperature exists in this animal. Whether this mechanism is thermoregulatory for the normal spermatogenesis cannot be inferred from the present work.

Microscopic anatomy of the scrotum, testis with its excurrent duct system and spermatic cord of Didelphis azarae.

The authors give a description of the microscopic anatomy of the scrotum, testis and its excurrent duct system and the structures of the spermatic cord. The scrotal skin of Didelphis azarae is hairy with the surface marked by shallow grooves and spotted with black pigmented areas. Clear cells and mitotic figures are frequently seen in the basal region. The sweat glands are tubular, apocrine, with alcian-blue- and PAS-positive secretion, presenting large myoepithelial cells. The tunica dartos is poorly developed. The tunica vaginalis is constituted by three layers, presenting patches of melanocytes in variable extensions. The tunica albuginea is composed preponderantly of collage and thin elastic fibers without muscle fibers. The interstitial tissue presents connective cells and a large number of Leydig cells. Mast cells were not observed. The tunica propria of the seminiferous tubuli is fibroelastic with two or three layers of elongated cells. The straight tubuli are divided into three different portions lined by epithelium with variable height. These tubuli at the mediastinum join each other to form a single duct near the cranial pole of the testis. The extratesticular segment of the efferent duct divides initially into two and then into three or four smaller flexuous ductuli to constitute the head of the epididymis. The spermatic cord shows a well-developed cremaster muscle. A collagenous fibrous band separates the muscle from the deferent duct and vessels. Mast cells are observed among the muscle fibers of the cremaster and the tunica adventitia of the blood vessels.