Antiserum Treatment Research Articles

PRODUCTION OF POLYCLONAL ANTIBODIES AGAINST INDIAN PAPER WASP ROPALIDIA MARGINATA VENOM TOXINS AND THEIR EFFICACY IN THE REVERSAL OF TOXIC EFFECTS

Objective: In this study, albino mice were injected with a sub-lethal dosage of purified wasp Ropalidia Marginata venom toxins to assess the effectiveness of polyclonal anti-venom antibodies. Methods: To neutralize the toxic effects, polyclonal antibodies were generated by immunizing albino mice. The antibody underwent partial purification using ammonium sulphate treatment and octanoic acid precipitation. To detect the presence of antibodies in the antiserum, an immunodouble diffusion test was conducted using Ouchterlony's method (1962). This involved allowing both antigens and antibodies to diffuse radially towards each other from their respective wells. When they reached an equivalence zone, a precipitation complex of antigen and antibody became visible as a concentric band, indicating the development of the combination. To quantitatively determine the amount of antibodies in the antiserum, the equivalency zone approach was used. Results: Experimental mice were injected with a combination containing 400, 800, and 1200 µg of pure antibody, which had been treated serum biomolecules, including metabolic enzymes, completely reversed in the experimental with 40% of the LD50 of wasp venom the elevated serum parameters were glucose, pyruvic acid, lipid, protein and free amino acid, reached to normal (100%) in the treated with 40% of LD50 of the venom and polyclonal treated after 6 h of administration. Anti-serum treatment also successfully normalized the alteration in serum enzyme just after 4h. Similarly, anti-serum treatment also successfully normalized the alteration in serum enzyme just after 4h treated with 40% of LD50 of the venom. Serum ACP content was obtained as 125.35% after 40% of LD50 venom injection, which was get normalized up to 102.81% after 4 h of the anti-venom treatment. Serum ALP content of 114.8% elevation was reversed back to 102.40% after anti-venom treatment. The GPT level significantly reversed up to 102.5%, while it was 130% in the venom-treated mice. A complete reversal was obtained in GPT level, which was obtained as 104.54% in the venom-treated animal. Similarly, LDH which was elevated up to 112.45 % in venom-injected mice was successfully reversed up to 100.25% after anti-venom treatment. Similarly, Ache concentration was fully recovered after anti-venom treatment 6 h, all animals (group B-E) that had received 40% of the LD50 of venom treated with pure antiserum. Conclusion: The venom-injected group showed a complete restoration of serum protein, free amino acid, uric acid, cholesterol, pyruvic acid, total lipid, and glucose level in experimental mice.

Mortality, incidence and seasonality of canine and feline patients treated with tick antiserum in three far North Queensland veterinary clinics from 2000 to 2020.

Tick paralysis is a paralysis caused by bites from Ixodes holocyclus, affecting an estimated 10,000 companion animals in Australia annually. Despite tick antiserum being the cornerstone of treatment, there are no large-scale general practice studies that examine survival outcomes in tick antiserum-treated animals. In this retrospective study, clinical records from three far north Queensland general practice veterinary clinics were searched for tick antiserum-treated canine and feline patients were seen between 2000 and 2020. Patient records were assessed for survival outcomes,then logistic regression and Bayesian structural time-series model were used to assess trends in incidence and mortalityand the relationship between these and time of year, rainfall, and species. The study included 2019 dog and 953 cat records. When patients with unknown outcomes were removed, canine mortality was 11.8% (213/1799) and feline mortality was 5.3% (46/872). Dogs were found to have 2.41 odds of dying following treatment than cats. August and September had the highest mean number of monthly treatments, and rainfall in the previous 5-8months was positively correlated with the number of patients treated in each month. The odds of mortality did not vary significantly by month or season, and from 2015 onwards, there was a significant decrease in the proportion of dogs treated by the clinics. Overall, this study provides new information on tick antiserum treatment outcomes in general practice as well as new information on tick paralysis incidence in far north Queensland.

Šokový stav a další komplikace po uštknutí jedovatým hadem

The case report presents the case od 66-year-old woman with schock, severe general and grave local symptoms after a snake bite into the left limb. The species of attacking snake was not clearly specific, it should not have been clearly seen, but it was propably a common viper, a snake of viper family, which commonly occurs in our latitude. Due to the unclear origin of the bite, no specific antiserum and symptomatic treatment were given.

Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization

Rats are effective model animals and have contributed to the development of human medicine and basic research. However, the application of reproductive engineering techniques to rats is not as advanced compared with mice, and genome editing in rats has not been achieved using embryos obtained by in vitro fertilization (IVF). In this study, we conducted superovulation, IVF, and knock out and knock in using IVF rat embryos. We found that superovulation effectively occurred in the synchronized oestrus cycle and with anti-inhibin antiserum treatment in immature rats, including the Brown Norway rat, which is a very difficult rat strain to superovulate. Next, we collected superovulated oocytes under anaesthesia, and offspring derived from IVF embryos were obtained from all of the rat strains that we examined. When the tyrosinase gene was targeted by electroporation in these embryos, both alleles were disrupted with 100% efficiency. Furthermore, we conducted long DNA fragment knock in using adeno-associated virus and found that the knock-in litter was obtained with high efficiency (33.3–47.4%). Thus, in this study, we developed methods to allow the simple and efficient production of model rats.

A review on herbs against snake venom

Snakebite is major problem still depending on treatment of antisera as though they have their own limitations. Hence this review concentrates on elaborating various herbs and herbal antidotes that can be used against snake bites. Such components can help overcome the limitations of anti-venoms as they are naturally obtained. Herbs can also be easily available and quickly given to victims. Also, they are cost effective as compared to anti-venoms. Various parts of the plants such as roots, rhizomes, leaves, fruits, seeds and their extracts can be utilized to neutralize the snake venom of various snake species. There are various evident Anti-Venom limitations that can interfere with the efficiency of the anti-venom. This article covers the list of some species of the plants which counteracts selective snake’s venoms. There is detailed specification of herbs related to their origin, the venom of snake it acts on, dose and other related parameters.

Revisiting the Concept of Targeting Only Bacillus anthracis Toxins as a Treatment for Anthrax.

Protective antigen (PA)-based vaccines are effective in preventing the development of fatal anthrax disease both in humans and in relevant animal models. The Bacillus anthracis toxins lethal toxin (lethal factor [LF] plus PA) and edema toxin (edema factor [EF] plus PA) are essential for the establishment of the infection, as inactivation of these toxins results in attenuation of the pathogen. Since the toxins reach high toxemia levels at the bacteremic stages of the disease, the CDC's recommendations include combining antibiotic treatment with antitoxin (anti-PA) immunotherapy. We demonstrate here that while treatment with a highly potent neutralizing monoclonal antibody was highly efficient as postexposure prophylaxis treatment, it failed to protect rabbits with any detectable bacteremia (≥10 CFU/ml). In addition, we show that while PA vaccination was effective against a subcutaneous spore challenge, it failed to protect rabbits against systemic challenges (intravenous injection of vegetative bacteria) with the wild-type Vollum strain or a toxin-deficient mutant. To test the possibility that additional proteins, which are secreted by the bacteria under pathogenicity-stimulating conditions in vitro, may contribute to the vaccine's potency, we immunized rabbits with a secreted protein fraction from a toxin-null mutant. The antiserum raised against the secreted fraction reacts with the bacteria in an immunofluorescence assay. Immunization with the secreted protein fraction did not protect the rabbits against a systemic challenge with the fully pathogenic bacteria. Full protection was obtained only by a combined vaccination with PA and the secreted protein fraction. Therefore, these results indicate that an effective antiserum treatment in advanced stages of anthrax must include toxin-neutralizing antibodies in combination with antibodies against bacterial cell targets.

Pepsin is a positive regulator of Ac-cathB-2 involved in the rat gut penetration of Angiostrongylus cantonensis.

BackgroundAngiostrongyliasis caused by the rat lungworm, Angiostrongylus cantonensis (A. cantonensis), has globally spread from the traditional epidemic areas. The small intestine is the site where the third-stage larvae (L3) of this worm entered the host body, and parasite proteases are involved in this process. Ac-cathB-2, a cathepsin B-like cysteine of A. cantonensis, was formerly isolated from the insoluble part of fragmentised Escherichia coli without activity. The unplanned low activity of prokaryotic expression proteins and difficulties in genetic modification hindered understanding the function of this protein.MethodsThe recombinant Ac-cathB-2 was expressed and harvested from 293 T cells and the enzymatic property and the effects of processing on the activity of the recombinant protease were investigated in vitro. The expression of Ac-cathB-2 in response to external stimulation was assessed, and the function of this protease during host gut penetration was observed by using antiserum for inhibition.ResultsOf the life-cycle stages studied, L3 expressed the highest level of Ac-cathB-2 gene and released the corresponding gene product from the body. The expression of this gene was rapidly upregulated after incubating L3 in small intestine homogenate of rat. Recombinant Ac-cathB-2 was harvested from 293 T cell culture medium. This protease was activated by pepsin-HCl and the enabled Ac-cathB-2 could subsequently digest laminin and fibronectin readily. Moreover, the small intestine isolated from rat was disrupted after incubating with the activated Ac-cathB-2, resulting in the detachment of epithelial cells. Antiserum treatment inhibited the hydrolytic ability of recombinant Ac-cathB-2 by 82.7 %, and also reduced the tissue penetration of activated L3 by 41.2 %. Additionally, pre-incubation of L3 with artificial gastric acid increased the number of penetrating larvae by 53.2 %, and this alteration could be partly blocked by antiserum treatment.ConclusionWe believe that Ac-cathB-2 from A. cantonensis might help the worm to penetrate the rat gut, because the protease was able to degrade the tissue components of host. Nevertheless, our results further indicated that host pepsin played a beneficial role in this process by cleaving Ac-cathB-2 for activation. Thus, Ac-cathB-2 may probably represent an important target for the control of A. cantonensis infection.

Anti-serum with anti-autoantibody activity decreases autoantibody-positive B lymphocytes and type 1 diabetes of female NOD mice

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by an autoimmune-mediated loss of insulin secreting β-cells. Each B lymphocyte clone that escapes immune tolerance produces a specific antibody. No specific treatment against autoantibodies is available for autoimmune diseases. We have developed a strategy to produce an antiserum against autoantibodies for the treatment of T1DM. Non-obese diabetic (NOD) but not Balb/c mouse serum contains autoantibodies. Antisera were produced by immunizing Balb/c mice with affinity-purified IgG from NOD or BALB/c mice along with the immune adjuvant (hereafter, NIgG or BIgG, respectively). A bolus administration of NIgG significantly reduced serum autoantibodies, autoantibody-positive B lymphocytes in the spleens of NOD mice, mortality and morbidity of diabetes, blood glucose and islet immune infiltration, whereas it increased islet mass in NOD mice for at least 26 weeks. NIgG antiserum treatment has no significant effect on CD3+, CD4+ or CD8+ T cells and B220+ or CD19+ B cells. BIgG also imparted a moderate therapeutic effect, although it was considerably lower than that of NIgG. NIgG did not cross-react with allogeneic serum. NIgG showed no effect on Balb/c mice. The results show the feasibility of producing antiserum against autoantibodies to prevent and treat autoimmune-induced T1DM with a single bolus administration.

Therapeutic effect of post-exposure treatment with antiserum on severe fever with thrombocytopenia syndrome (SFTS) in a mouse model of SFTS virus infection

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral disease that is endemic in China, Korea and Japan. No effective vaccine or specific treatment for SFTS is currently available. Here, we used a mouse model to examine the effects of ribavirin, site-1 protease inhibitor PF-429242, steroids, and combination of minocycline and ciprofloxacin (MC) on SFTS infection. The antiserum from a patient who recovered from SFTS was also examined for its effect on mice. Administration of antiserum completely protected mice against lethal infection with SFTSV. It could also protect mice from showing clinical signs of the disease due to non-lethal infection. MC-treatment resulted in prolonged survival times during lethal infection. Although other agents had no significant protective effects, they did not provide detrimental effects that could lead to progression of the disease in mice. Our results suggest that antiserum treatment may be clinically useful for post-exposure prophylaxis against SFTSV infection.

Effects of gonadotropin on Fas and/or FasL expression and proliferation in rat ovary

Although gonadotropin is a dominant hormone involved in promoting ovarian follicle development in females, the mechanism by which gonadotropin regulates follicular development is still unknown. To systematically evaluate the effectiveness of the gonadotropin on apoptosis and proliferation of ovarian cells in vivo, rats were injected subcutaneously with eCG and/or anti-eCG antiserum. Equine chorionic gonadotropin treatment increased ovarian cell proliferation and expression of FSH receptors (FSHR) as revealed by increased immunostaining of proliferating cell nuclear antigen and FSHR in rat ovary. These effects did not occur in a follicular stage–dependent manner. Moreover, these actions were abolished by anti-eCG antiserum. However, granulosa cells exhibited more intense Fas- and FasL-positive immunostaining during all follicular stages in the anti-eCG antiserum group. We used Western blot analysis to confirm these results; Fas and FasL protein contents in rat ovaries were decreased by eCG. Meanwhile, proliferating cell nuclear antigen and FSHR expression were upregulated by eCG. However, all these eCG-induced regulations were reversed by anti-eCG antiserum treatment. Furthermore, there were no significant differences between the anti-eCG antiserum and control groups. These results indicate that eCG promotes follicular development via downregulation of death-inducer Fas/FasL expression and promotion of ovarian cell proliferation, which is partially mediated by FSHR.

CD176 antiserum treatment leads to a therapeutic response in a murine model of leukemia

CD176 (Thomsen-Friedenreich antigen) is a tumor-associated carbohydrate structure. CD176 is expressed at the surface of human leukemic cells but is almost absent in normal and benign adult human tissues. Therefore, CD176 could be a promising target for antitumor immunotherapy. In the present study, pre-immunization with asialoglycophorin A (containing the CD176 oligosaccharide chains) was able to significantly improve the survival time of mice carrying CD176+ leukemia as compared to the control mice without the immunization. Furthermore, the passive transfer of CD176 antiserum which reacted only with the tumor-associated CD176 in cancer cells, was able to effectively prolong the survival time of CD176+ leukemia mice. In particular, the CD176 antiserum treatment could inhibit the growth and spreading of CD176+ leukemic cells in bone marrow, spleen, liver, and lung as evidenced by histopathological examination. CD176 antiserum could induce the apoptosis of CD176+ leukemic cells in vivo in a manner as previously observed in vitro. The data provided strong evidence that both CD176 antigen-based active immunotherapy and CD176 antibody-based passive immunotherapy lead to a therapeutic response in CD176+ leukemia mice. Therefore, both CD176 vaccine and CD176 antibody drug may be beneficial for the treatment of CD176+ leukemia patients.

The Effect of Gonadotropin on Glucose Transport and Apoptosis in Rat Ovary

Although the effects of Gonadotropin on ovarian physiology have been known for many decades, its action on glucose uptake in the rat ovary remained poorly understood. Evidence also suggests that glucose uptake is mediated by a number of glucose transporter proteins (Glut). Therefore, we examined the rat ovary for the presence of Glut1–4 and blood glucose level after eCG (equine chorionic gonadotropin) and anti-eCG antiserum treatment. All of the glucose transports were present in the ovarian oocyte, granulosa cells and theca cells in different stage follicles. The expression of Glut in ovary was up-regulated by eCG, however, anti-eCG antiserum reversed eCG action. Western blot analysis also demonstrated the content of Glut1 was higher in eCG treatment group compared with anti-eCG antiserum and control group. The same tendency was shown in other glut isoforms. Moreover, there were no significant difference between the anti-eCG antiserum and control group. In additional, the level of serum glucose in eCG treatment group was significantly higher than others, which is similar with glut expression pattern. High glucose level in blood is correlated with increased expression of glucose transporter proteins in rat ovary. Meanwhile, anti-eCG antiserum increased granulosa cell apoptosis in antral follicle compared with those in eCG group. Our observations provide potential explanation for the effects of Glut on follicular development in rat ovary and a role for eCG in the regulation of ovarian glucose uptake.

Blockade of Osteopontin Inhibits Glomerular Fibrosis in a Model of Anti-Glomerular Basement Membrane Glomerulonephritis

Background: In our rat model for anti-GBM GN, severe fibrosis follows glomerular inflammation. A potential role of extracellular matrix protein osteopontin (OPN) in glomerular fibrosis was investigated. Methods: Neutralizing OPN antiserum or control normal serum was injected into the experimental rats at late inflammatory/early fibrotic stage. Glomerular inflammation and fibrosis were determined. Results: OPN antiserum treatment had little effect on glomerular inflammation. However, the antiserum treatment resulted in a significant reduction in number of fibrotic glomeruli (50% of the controls). Histology observation showed that fibrotic tissue in glomeruli of the antiserum treated rats was mild and poorly developed. OPN antiserum treatment resulted in downregulated glomerular expression of collagen 1α1; collagen deposition in the antiserum treated rats reduced to <30% of that for normal serum controls. Conclusion: Neutralization of OPN inhibited progression of fibrosis in vivo when given at early fibrotic stage. Thus, OPN may be a therapeutic target for glomerular fibrosis.

Expression of Bone Morphogenetic Protein Receptor (BMPR) during Perinatal Ovary Development and Primordial Follicle Formation in the Hamster: Possible Regulation by FSH

To understand whether bone morphogenetic protein plays any role in the formation of primordial follicles in the hamster, we examined the temporal and spatial expression of bone morphogenetic protein receptor (BMPR) mRNA and protein in embryonic (E) 13 through postnatal day (P) 15 ovarian cells and a possible regulation by FSH during the formation of primordial follicles on P8. BMPRIA and BMPRII mRNA levels were significantly higher than that of BMPR1B throughout ovary development. BMPRIA and BMPRII mRNA levels increased significantly on E14 and declined by P5 through P6. Whereas BMPRII mRNA increased again by P7, BMPRIA mRNA levels increased through P8 concurrent with primordial follicle formation. In contrast, BMPRIB mRNA levels increased greater than 10-fold on P7-9, with a further 3-fold increase by P10. BMPR proteins were low in the somatic cells and oocytes on E13 but increased progressively during postnatal development. BMPR expression in somatic cells increased markedly on P8. Whereas BMPRII expression declined by P10 and remained steady thereafter, BMPRIA protein expression fluctuated until P15 when it became low and steady. Overall, BMPRIB immunoreactivity also declined by P10 and then remained low in the interstitial cells through P15. FSH antiserum treatment on E12 significantly attenuated receptor mRNA and protein levels by P8, but equine chorionic gonadotropin replacement on P1 reversed the inhibition. Furthermore, FSH in vitro up-regulated BMPR levels in P4 ovaries. This unique pattern of BMPR expression in the oocytes and somatic cells during perinatal ovary development suggests that BMP may play a regulatory role in primordial follicle formation. Furthermore, FSH may regulate BMP action by modulating the expression of its receptors.

Physiological roles of inhibin in regulation of FSH secretion and follicular development during early pregnancy in goats

The aim of the current study was to clarify the physiological role of inhibin in controlling FSH secretion and follicular development during the early pregnancy in goats. Eight goats investigated sonographically on Days 19–21 (Day 0 = day of mating) for pregnancy were assigned into control ( n = 3) and treated ( n = 5) groups. The ovaries of all animals were daily scanned with ultrasound for follicles 2 mm or more in diameter from 1 day before to 5 days after treatment. On Day 25 postbreeding; animals received either 10 ml, of normal goat serum or antiserum against [Tyr (30)]-inhibin alpha (1–30). Jugular blood samples were collected every 6 h starting 24 h before and until 120 h after treatment. The plasma concentration of FSH increased at 6 h and remained at significantly high levels until 120 h in treated vs. control group. The plasma concentrations of estradiol showed a marked increased at 66 h, with peak levels at 120 h after treatment of antiserum. The basal concentrations of LH and the pattern of plasma concentrations of progesterone were not significantly different between the two groups. The number of medium size (3.5–5.0 mm) follicles increased considerably from Day 2, whereas small (3.5 mm or less) and large (5 mm or more) follicles increased noticeably from Day 3, as compared with pre-treatment and controls. These results clearly indicated that inhibin is a key hormone in regulation of follicular development through regulation of endogenous FSH secretion during early pregnancy in goats.

Obestatin inhibits vasopressin secretion: evidence for a physiological action in the control of fluid homeostasis

Obestatin, a product of post-translational processing of the ghrelin prohormone, has been reported to act in the brain to inhibit thirst. We extended our initial studies on water drinking by examining the effects of obestatin on hypovolemia-induced water and saline drinking and vasopressin release in male rats. Intracerebroventricular administration of obestatin significantly inhibited water, but not saline (0.3 M NaCl) drinking in response to a hypovolemic challenge. Obestatin also inhibited, in a dose-related fashion, dehydration-induced vasopressin secretion without affecting plasma oxytocin levels. Vasopressin release induced by central angiotensin II administration was attenuated significantly by prior administration of obestatin. Finally, central administration of an antiserum specific to obestatin resulted in an exaggerated basal vasopressin release and an increased vasopressin response to overnight water deprivation. Antiserum treatment also resulted in significantly increased ad libitum water drinking and drinking in response to dehydration. We conclude that this product of post-translational processing of the ghrelin prohormone may be an important contributor to the physiologic regulation of fluid and electrolyte homeostasis.

Passive Immunization Approach to Reduce Body Fat in Pigs Using Fat-specific Polyclonal Antiserum

Plasma membrane proteins from pig adipocytes, brain, heart, kidney, liver and spleen were isolated using a 32% sucrose gradient. An adult male sheep was immunized three times at 3-wk intervals with the purified pig adipocyte plasma membrane (APM) proteins. Blood samples were taken from the immunized sheep 12 d after the third immunization. Antiserum showed strong reactivity with APM proteins determined by ELISA, and the reactivity could be detected at dilutions in excess of 1:128,000. Antiserum showed very low binding affinity with proteins isolated from brain, heart, kidney, liver or spleen. Ninety weanling pigs were allocated randomly to three treatment groups and were injected i.p. with 40 ml of antiserum (n = 30) or 20 ml of lyophilized antiserum (21.5 mg/ml; n = 30). A control group (n = 30) received 40 ml of saline, and all pigs were slaughtered at 24 wk of age. The polyclonal antiserum did not change BW or ADG. Carcass percentage of pigs was numerically increased by the antiserum treatment compared with control. Both antiserum treatments did not significantly (p>0.05) affect body composition, including body fat content, relative to the control group.

The administration of naked plasmid DNA into the liver induces antitumor innate immunity in a murine liver metastasis model

Gene therapy is a promising strategy against advanced cancer; however, the safety of viral vectors and the effectiveness of non-viral vectors have not yet been established. Recently, a hydrodynamics-based procedure was reported to be an effective and safe method to deliver and transduce DNA into the liver. Herein, we propose a strategy for liver metastasis by a hydrodynamics-based procedure to deliver naked non-coding plasmid DNA (pDNA) into the liver as an immunocompetent organ. Mice received a rapid intravenous (i.v.) injection of naked pDNA in a large volume of saline (0.1 ml/g body weight). The single administration of a naked non-coding pDNA by the hydrodynamics-based procedure before tumor cell inoculation strongly suppressed liver metastasis formation. However, the usual i.v. injection (200 microl/body) of the same dose of naked pDNA could not suppress liver metastasis formation. Following the methylation of CpG sequences within the pDNA using CpG methylase, injection of the methylated pDNA by the hydrodynamics-based procedure could not suppress liver metastasis formation. Gadolinium chloride pretreatment did not interfere with this antitumor effect, but anti-asialo GM1 antiserum treatment did. These findings indicated that natural killer (NK) cells, not Kupffer cells, were involved in this antitumor effect. The NK cytotoxic activities of liver mononuclear cells were strongly enhanced after receiving a naked pDNA by the hydrodynamics-based procedure. These observations suggest that unmethylated CpG motifs in pDNA stimulated immune cells, resulting in the activation of NK cells in the liver to suppress liver metastases in a murine model.

Reduction in nerve growth factor availability leads to a conditioning lesion‐like effect in sympathetic neurons

Axotomized peripheral neurons are capable of regeneration, and the rate of regeneration can be enhanced by a conditioning lesion (i.e., a lesion prior to the lesion after which neurite outgrowth is measured). A possible signal that could trigger the conditioning lesion effect is the reduction in availability of a target-derived factor resulting from the disconnection of a neuron from its target tissue. We tested this hypothesis with respect to nerve growth factor (NGF) and sympathetic neurons by administering an antiserum to NGF to adult mice for 7 days prior to explantation or dissociation of the superior cervical ganglion (SCG) and subsequently measuring neurite outgrowth. The antiserum treatment dramatically lowered the concentration of NGF in the SCG and increased the rate of neurite outgrowth in both explants and cell cultures. The increase in neurite outgrowth was similar in magnitude to that seen after a conditioning lesion. To determine if exogenous NGF could block the effect of a conditioning lesion, mice were injected with NGF or cytochrome C immediately prior to unilateral axotomy of the SCG, and for 7 days thereafter. A conditioning lesion effect of similar magnitude was seen in NGF-treated and control animals. While NGF treatment increased NGF levels in the contralateral control ganglion, it did not significantly elevate levels in the axotomized ganglion. The results suggest that the decreased availability of NGF after axotomy is a sufficient stimulus to induce the conditioning lesion effect in sympathetic neurons. While NGF administration did not prevent the conditioning lesion effect, this may be due to the markedly decreased ability of sympathetic neurons to accumulate the growth factor after axotomy.

Expression of apoptotic genes in testicular germ cells and their modulation by luteinizing hormone

Gonadotropins regulate spermatogenesis by promoting survival and differentiation of germ cells. The molecular markers that are modulated by these hormones to ensure survival however have not been described in great detail. Immunoneutralization of LH in particular leads to apoptotic cell death of the spermatocytes and the round spermatids. In the present study, the expression pattern and regulation of apoptotic markers after specific immunoneutralization of LH in germ cells purified from rats has been investigated at the RNA and protein level. Of the several markers tested, Bax, caspases 1 and 2 and Fadd exhibit differential expression, with the round spermatids expressing higher levels of caspases 1 and 2, and the spermatocytes expressing higher levels of Bax and Fadd. The two cell types therefore exhibit differential expression of apoptotic markers. The cell types also differ with respect to their response to LH antiserum treatment. Fas and Bax both are up-regulated in the round spermatids after 24 h of antiserum treatment. In the spermatocytes, Fas was up-regulated as early as 12 h after antiserum treatment while Bax was up-regulated after 2 days. These results demonstrate that LH regulates survival of germ cells by modulating the levels of pro and anti-apoptotic proteins.