Tuberculosis in Dr. Granville’s mummy

Source: Donoghue HD, Lee OY, Minnikin DE, Besra GS, Taylor JH, Spigelman M.

Tuberculosis in Dr Granville's mummy: a molecular re-examination of the earliest

known Egyptian mummy to be scientifically examined and given a medical diagnosis.

Proc Biol Sci. 2010 Jan 7;277(1678):51-6.

 

As soon as I read the title of this interesting article, I knew that I need to write a blog on it. Luckily the full text of the paper was also available online. This work is a collaborative effort of well known researchers from Centre for Infectious Diseases, University College, London, School of Bioscience, University of Birmingham, Department of Ancient Egypt and Sudan, The British Museum, London and Kuvin Centre for Study of Infectious and Tropical Diseases, The Hebrew University, Israel.

Dr. Augustus Bozzi Granville was an eminent physician and obstetrician who examined an Egyptian mummy by scientific autopsy in 1825. The mummy was of a 50 year old woman named, lady Irtyersenu. She was from necropolis of Thebes, of the 26^th Dynasty dated to 600BC. After detailed observation of the wrappings and the exterior features of the mummy, Granville decided to perform a detailed autopsy. This mummy is commonly called as Granville's mummy. Granville observed a mass around the right ovary, which was described by him as ovarian dropsy or cancer and he proposed that this was the cause of her death. Recent studies have however, shown that this was actually a benign cystadenoma of the ovary, which is described as non-fatal. Other studies carried out recently mentioned the presence of a pulmonary exudate, which can be a potentially fatal pathological condition. Many studies were carried out to determine the exact cause of death of the mummy. During the early 1990s, several attempts were made to detect microbial or human DNA from the Granville mummy by PCR. But these studies were not successful.

Based on paleopathological changes, tuberculosis has long been recognized in Egyptian mummies. Furthermore, M. tuberculosis complex DNA has been detected and characterized from the predynastic era, the Old, Middle and New kingdoms. Thus, authors of this paper sought to determine whether lady Istyersenu had tuberculosis, which might have been the cause of her death. For this they used two strategies. These were detection of M. tuberculosis by PCR and direct detection of specific cell wall mycolic acid biomarkers of the M. tuberculosis complex, using HPLC.

For performing these experiments they used samples taken during 1990 (lung tissue, bone from the right and left femurs and hand) and some additional samples were taken. Special precautions were taken during DNA extraction and PCR procedures.

DNA from the M. tuberculosis complex was detected in lung samples, gall bladder and membranous tissue by PCR, but the results were inconsistent. The authors could not perform additional characterization of M. tb DNA, as this DNA was extremely fragile. However, they performed DNA sequencing from nested PCR products. These sequences were homologous with those of M. tb. The HPLC analysis showed a good correlation between the profiles from samples collected from different sites and presence of specific biomarkers for Tb was clear. The authors thus proposed that assuming that the pulmonary exudate was the prime source of the infection, it appears that the lady Irtyersenu had pulmonary tuberculosis that had disseminated to other sites in the body.

NOD Like Receptors (NLRs) in Human B Cells

We all know that innate immune response provides first line of defense against invading micro-organisms. Cells of the innate immune system express certain proteins that are called pattern recognition receptors (PRRs). These receptors identify microbe associated molecular patterns (MAMP) or pathogen associated molecular patterns (PAMP), which are associated with microbial pathogenesis. These PRRs can be divided into two types based on their presence on/in the cells. These two types include membrane bound PRRs (most famous example is Toll like rector (TLR), and cytoplasmic PRRs.

The NOD like receptors (NLRs) are the cytoplasmic PRRs. The NLR family comprise of 20 intracellular proteins that recognize primarily bacterial structures. They are divided into three subfamilies, NODs, NALPs and NAIPs. The best characterized members of the NOD sub-family are NOD1 and NOD2. NOD1 recognizes a molecule called meso-DAP, specific for Gram negative bacteria and NOD2 recognizes MDP, a component of all types of bacterial peptidoglycans. In the NALP subfamily, NALP1beta and NALP3 are most well-known members. Members of NALP family respond to danger signals released from injured or dying cells, leading to formation of inflammasomes. The members of NAIP family detect intracellular flagellin and also lead to the formation of inflammasome. Expression of NLRs has been detected in many cells including DCs, macrophages, monocytes, epithelial and endothelial cells and osteoblasts. However, at present it is not known whether B cells also express NLRs or not. In the present study, the authors thus investigated the expression of various NLRs and their function in human peripheral and tonsillar B cell subsets. The authors also explored the cooperative roles of NLRs and selected TLRs in B cell activation.

This paper is from the laboratory of Dr. Lars-Olaf Cardell, Division of ENT Diseases, CLINTEC, Karolinska Institute, Sweden and is published in Journal of leukocyte Biology, September 2010 issue.

Petterson T, Jendholm J, Månsson A, Bjartell A, Riesbeck K, Cardell LO. Effects of NOD-like receptors in human B lymphocytes and crosstalk between NOD1/NOD2 and Toll-like receptors. J Leukoc Biol. 2010 Sep 15.

 

A summary of major results obtained by the investigators is as follows:

• Expression of NLRs in human B lymphocytes: Freshly isolated peripheral and tonsillar and B lymphocytes (from patients) were used and mRNA expression of NOD1, NOD2, NALP1, NALP3, NAIP, and IPAF was analyzed using real-time PCR. The results of the PCR assay showed that peripheral B cells expressed all the NLRs tested in this experiment, except IPAF. In contrast, tonsillar cells only expressed NOD1, NALP1, and NAIP. Furthermore, flow cytometric and immunohistochemistry analyses were done to confirm that results of real-time PCR. For these analyses, rabbit polyclonal Ab against NOD1 and NAIP and mouse mAb against NOD2, NALP1 and NALP3 were used. It was observed that peripheral B cells expressed all the tested receptors, while tonsillar B cells showed expression of NOD1, NALP1 and NAIP.
  
• B cell receptor triggering is required for NLR-induced B cell activation: To determine whether NLRs have the capability to induce B cell activation upon stimulation with their ligands, purified B cells were isolated and cultured with or without iE-DAP (NOD1 ligand) and MDP (NOD2 ligand) in the presence or absence of BCR i.e., alpha IgM or IgD-binding protein MID. It was observed that when B cells were cultured with NOD1/2 ligands only, no proliferation was observed. However, when cells were stimulated with NLR-ligands in combination with BCR cross-linking via IgM or IgD, a profound increase in B cell proliferation was observed. To examine the role of physical T cell help in NOD induced B cell proliferation, the authors added rCD40L to highly purified B cells. They observed that CD40L in combination with the NLR ligands did not lead any additional proliferation of B cells. To find out more about NLR-induce B cell activation, the investigators stimulated B cells with NOD1/2 ligands together with BCR cross-linking (IgM/IgD) and analyzed expression of cell surface glycoproteins and viability. The number of CD27+, CD69+, CD71+, CD80+, CD86+, and CD95+ B cells was found to be increased. In peripheral B cells, the expression of all these markers were found to be elevated in comparison to unstimulated cells or to those stimulated with BCR alone. In tonsillar B cells, only stimulation with NOD1 ligand increased the percentage of CD71+, CD80+, CD86+, and CD95+ cells. It was also observed that viability of B cells cultured with NOD1 ligands along with BCR was slightly enhanced than controls. However, culture with NOD2 ligands along with BCR did not cause any increase in viability. As tonsillar B cells showed lower B cell responses, the next question was why? To answer this question, investigators determined the level of NLR proteins in naïve (CD19+IgD+CD38-), germinal center (CD19+IgD-CD38+) and memory (CD19+IgD-CD38-) B cells. However, they could not find any difference between blood derived and tonsillar cells.
  
• NLR stimulation augments TLR-induced B cell proliferation: To study the interplay between NLRs and TLRs on B cells, the investigators incubated B cells with suboptimal concentration of TLR agonists in combination with NOD1/2 ligands and BCR cross-linking. It was observed that proliferation of peripheral B cells was enhanced, when they were incubated with TLR agonists. In contrast, only co-stimulation with NOD1 ligand increased the proliferation in tonsillar B cells.
  

Conclusions:

1. This is the first study to demonstrate the presence of a range of NLRs in purified human B cells.
   
2. The study shows that peripheral B cells express NOD1, NOD2, NALP1, NALP3 and NAIP, while tonsillar B cells express NOD1, NALP1 and NAIP.
   
3. Activation of B cells by NLR ligands required BCR cross linking and did not require T cell help.
   
4. NLRs enhanced TLR-induced activation of B cells.

CTLA-4 controls regulatory T cell peripheral homeostasis and is required for suppression of pancreatic islet autoimmunity

CTLA-4 is a member of the immunoglobulin super-family. It is expressed on the surface of helper T cells and transmits an inhibitory signal to T cells. Earlier studies have linked polymorphisms in CTLA-4 gene with several autoimmune diseases including type1 diabetes, thyroiditis and SLE. Severe disease has been observed in CTLA-4 deficient mouse. Several reasons have been proposed for this, although the exact is not known. These reasons include the ability of CTLA-4 to raise the threshold of T cell activation, to modulate T cell motility, alter T cell thymic selection or control the function of regulatory T cells (Tregs). Much work has been done to identify the role of CTLA-4 in Tregs function.

This paper is from the labs of Dr. Lucy. S. Walker, University of Birmingham Medical School, Birmingham, UK. The authors have tried to analyze the role of CTLA-4 in Tregs and the results are as follows:

• Treg are over-expressed in CTLA4-/- mice: To quantify Tregs in CTLA4 knock out mice (CTLA4-/-), authors used intracellular Foxp3 staining. Foxp3 (Forkhead box P3) is a specific marker for Tregs. The authors observed that Foxp3+ Tregs were over-expressed in CTLA4-/- mice in both peripheral LN and spleen in comparison to controls.
  
• 
  CTLA4-/-Treg show unaltered thymic selection but increased peripheral proliferation: The increased expression of Tregs in CTLA4-/- mice can possibly indicate thymic selection or enhanced proliferation in the periphery. To answer these questions, the authors stained thymus cells for CD4, CD8 and Foxp3. They found that the proportion of CD4+Foxp3+ cells was not much different in two groups of mice. To determine whether there was an increased proliferation of Tregs in CTLA4-/- mice, spleen sections from CTLA4-/- animals and controls were stained for CD4, Foxp3, and Ki67 (nuclear protein expressed by proliferating cells). The results showed increased proliferation of Foxp3+ cells in CTLA4-/- mice compared to controls.
  
• CTLA4 regulates proliferation of OVA-specific Treg: Interpretation of data obtained from CTLA4-/- animals is complicated by the lethal lymphoproliferative syndrome that they develop. To overcome this, CTLA4-/- mice can be bred onto a TCR transgenic background (DO11.10), directly their specificity to a non self antigen (OVA). These animals can further be maintained in a rag deficient background. Such DO11/CTLA4-/-/rag-/- mice do not develop lymphoproliferative syndrome and their T cells bear a naïve phenotype. But the DO11/rag-/- animals do not develop Treg. The authors therefore crossed these mice with those that expressed OVA under the control of RIP. This strategy allowed the authors to study Treg in mice that lack CTLA4 but have an intact CD28. In DO11+ mice that did not express the RIP-mOVA ag, DO11 T cells did not develop into Tregs. In antigen positive mice, a fraction of DO11+ T cells developed into Tregs. Thus, CTLA4 signaling is not an obligate step in Treg differentiation. The authors next examined whether CTLA4 expression limited Treg proliferation in the periphery. They observed that Treg in peripheral lymphoid organs of DO11xRIP-mOVA/rag-/- mice remained largely undivided even in the absence of CTLA4. In contrast, Tregs isolated from Pancreas exhibited greater proliferation in mice that lacked CTLA4. All these findings support the idea that CTLA4 signaling controls Treg proliferation in response to encounter with self antigens.
  
• Ab-mediated CTLA4 blockade augments Treg proliferation in BALB/c mice: The authors next tested whether Ab-mediated CTLA4 blockade in normal animals enhanced Treg proliferation, as shown in CTLA4 knockout mice. Mice, which were treated with anti-CTLA4 mAb showed increased ki67 within CD4+Foxp3+ cells after 8 days. Three days following blocking, Foxp3+ cells (Tregs) showed significantly increased proliferation than controls (treated with control mAb). The proliferation of Foxp3- T cells was not much different from that seen in control mAB treated mice. By day 8, Foxp3- cells showed an increased proliferation in anti-CTLA4 Ab treated mice, but this increase became significant only at day 14.
  
• Ag-specific Treg deficient in CTLA4 fail to suppress autoimmune diabetes: The results of investigators raised the paradox that a fatal lymphoproliferative syndrome develops in the CTLA4 knockout mice despite an increase in Treg. To resolve this paradox the authors hypothesized two reasons. Firstly, CTLA4 deficiency might render Treg pathogenic and secondly, CTLA4 might be required for Treg suppression. To test the first hypothesis, authors utilized the fact that injection of CTLA4-/- lymphocytes into rag deficient recipient mice can transfer lymphoproliferative syndrome. Thus, they assessed the ability of purified CD4+CD25- or CD4+CD25high cells from CTLA4-/- to transfer this disease. It was observed that introduction of CD25- cells from CTLA4-/- mice induced the lymphoproliferative syndrome while recipients of CD4+CD25+high cells from CTLA4-/- mice remained healthy. To test the second possibility that CTLA4 is necessary for Treg suppression in vivo, the authors took advantage of the fact that Ag-specific Treg lacking CTLA4 can be purified from TCR transgenic mouse. CD4+CD25+ cells were isolated from DO11xRIP-mOVA/rag-/- mice that were either CTLA4 sufficient or deficient. Treg proportion was equivalent between CTLA4 deficient or sufficient groups. To test the ability of these Tregs to control diabetes in an adoptive transfer model, disease was induced by transfer of OVA-specific CD25- cells into mice expressing OVA in the pancreas and then Tregs from both groups were introduced. It was observed that mice that received CTLA4 sufficient Treg were completely protected from diabetes, while mice that received CTLA4 deficient Treg were not protected from diabetes. To further explore the basis for the failure of CTLA4-/- Treg to control diabetes, the investigators analyzed the expression of IL-10 and surface TGF-beta. No difference in IL-10 expression was observed between wild type and CTLA4-/- Tregs. The expression of TGF-beta was higher in CTLA4-/- Tregs. The investigators further investigated the hypothesis that Treg-expressed CTLA4 can deplete antigen presenting cells of co-stimulatory ligands. The authors found that the presence of Treg resulted in marked down-regulation of CD86 expression on APC and this down-regulation could be blocked by using anti-CTLA4 Ab. Expression level of MHC class II on APC was unaltered by Treg. Taken together, these data suggest that Treg-expressed CTLA4 plays important role in the regulation of an anti-islet immune response and this reflects a capacity for Treg to influence APC in a CTLA4 dependent manner.
  

 

Is human papilloma virus viral load a clinically useful predictive marker: a longitudinal study

Human papilloma viruses (HPV) are a group of more than 100 related viruses and they are one of the primary causes of cervical cancer in women. These are divided into low risk and high risk types based on their ability to cause cancer. There is need to distinguish between those women who test positive for high risk (HR) human papilloma virus (HPV) types and are likely to acquire cervical neoplasia and those women who test positive for HR-HPV but who are not at increase risk. This distinction would require one to study biomarkers. One such biomarker is HPV viral load. It has been observed that in women who are positive for HR HPV types, cytological abnormalities were found to be positively associated with HPV viral load. However, the relationship between HPV viral load and cervical neoplasia is not clear and seems to be complex. In this study, the authors have tried to determine this relationship by measuring viral load of HPV16 and HPV18, HR-HPV types in serial samples taken during the follow up of a group of young women who were recruited soon after they first had sexual intercourse.

Results:

Viral load waxes and wanes during follow up: In the study, 118 women who had an incident of HPV infection (HPV16 or HPV18) as detected by GP5+/GP6+ system and who provided at least three samples for qPCR during follow-up, at least one of which was positive, were studied. Sixty women were tested positive for HPV16 using qPCR. Of these 60, 41 tested positive in all three samples; 10 in two; and 9 in one. Fifty eight women were tested positive for HPV18. Of these 58, 39 were positive in all three samples, 5 in two and 14 in one. In the 60 women with HPV16 infection, median copy number was 7.7 in their first qPCR positive sample and 7.8 in their last positive sample. In the 58 women with HPV18 infection, median copy number was 2.3 in their first qPCR positive sample and 2.4 in their last positive sample. Viral load appeared to increase and decrease during follow up. Among 43 women with HPV16 infection and among 35 women with HPV18 infection, maximum viral load observed during follow up was found to be increased than that detected in the first qPCR positive sample and it was greater than that detected in the last qPCR positive sample in 37 of these women with HPV-16 infection and 32 with HPV18 infection.

Increase viral load is associated with an increase risk of acquiring an incident cervical cytological abnormality: There was no significant difference in the maximum HPV16 viral load or the maximum HPV18 viral load between women who subsequently acquired an incident cytological abnormality and those who did not. However, when viral load was modelled as a log10-transformed continuous covariate, controlling for whether or not a woman had ever tested positive for the relevant type using qPCR, it was observed that a 10 fold increase in HPV viral load was associated with a significantly increased risk of acquiring an incident cervical cytopathological abnormality in women with HPV16 or HPv18 infection.

 

Conclusions: Thus, the study concludes that while large relative increase in the viral load was associated with an increase risk of cytological abnormality, a single measurement of viral load done at an intermediate point during the natural history of HPV infection, does not reliably predict the risk of acquiring cervical neoplasia.

 

Source: Constandinou-Williams C, Collins SI, Roberts S, Young LS, Woodman CB, Murray

PG. Is human papillomavirus viral load a clinically useful predictive marker? A

longitudinal study. Cancer Epidemiol Biomarkers Prev. 2010 Mar;19(3):832-7.

Adenovirus vector vaccination induces expansion of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to HIV-1

A recently conducted phase 2B HIV vaccine trial in high risk HIV-negative volunteers (the STEP study) using an adenovirus vaccine (Ad5 vector expressing Gag, Pol and Nef) was ineffective. In that study, individuals were divided into two groups on the basis of their Ad5 antibody titers. Besides being ineffective, individuals who received this vaccine were more susceptible to HIV-1 infection in comparison to the placebo. This susceptibility was more significant in the group of vaccinees with high Ad5 antibody titers. The reason for this is not clear.

Natural Ad5 infection occurs via gut or nasopharynx and the virus replicates in the epithelial cells of mucosal tissues, inducing mucosal immunity. Thus, the authors of this study hypothesized that vaccination of individuals immune to Ad5 with adenovirus vector would activate and expand T cells expressing a mucosal homing phenotype, and these cells would migrate to the gut mucosa, increasing the number of permissive HIV-1 target cells and the risk of infection.

Citation: Benlahrech A, Harris J, Meiser A, Papagatsias T, Hornig J, Hayes P,

Lieber A, Athanasopoulos T, Bachy V, Csomor E, Daniels R, Fisher K, Gotch F, 

Seymour L, Logan K, Barbagallo R, Klavinskis L, Dickson G, Patterson S. 

Adenovirus vector vaccination induces expansion of memory CD4 T cells with 

a mucosal homing phenotype that are readily susceptible to HIV-1. 

Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):19940-5.

 

RESULTS:

1. Adenovirus specific cytokine responses do not correlate with Ad5 antibody titers: Ad5 and Ad11 antibody titers were measured in 20 healthy donors. IFN-gamma ELSIPOT was performed on PBMCs stimulated with Ad5 or Ad11 from 15 of these donors. 73% and 66.7% of individuals showed responses against Ad5 and Ad11, respectively. When Ad5 and Ad11 IFN-gamma responses were plotted against Ad5 antibody titers, no significant correlation was observed in between the Ad5 or Ad11 IFN-gamma ELISPOT responses and Ad5 antibody titer. Furthermore, the investigators pulsed dendritic cells from the donor with Ad5, Ad11, tetanus toxoid, heat inactivated influenza or Staphylococcus enterotoxin B (SEB) and measured cytokines in lymphocytes. It was observed that majority of IFN-gamma responses against Ad5 and Ad11 were mediated by CD8 T cells. CD8 cells produced more TNF-alpha in response to stimulation with Ad5 or Ad11 in comparison to CD4 T cells. There was no or very little IL-12 production in response to either Ad5 or Ad11 by any cell. The authors then assessed the functional quality of the responding Ad5 and Ad11-specific CD4 T cells. There was no difference between Ad5 and Ad11 responding CD4 cells in terms of cytokine profile as both groups of cells predominantly produced TNF-alpha alone or in combination with IFN-gamma or IL-2.
   
2. Adenovirus induced T cell proliferation correlates with Ad5 antibody titers: The investigators next sought to determine the ability of Ad vectors to induce T cell expansion ex vivo. For this, DCs were pulsed with Ad5, Ad11, tetanus toxoid, heat inactivated influenza or Staphylococcus enterotoxin B (SEB) and co-cultured with autologous CFSE-stained T cells. It was observed that majority of proliferationg T cells (CSFR low) in response to Ad5 or Ad11 stimulation were CD4 T cells. No significant difference in CD4 or CD8 cells was observed in response to tetanus toxoid or influenza. The authors also observed a positive correlation between Ad5- or Ad11- induced CD4 T cell proliferation and Ad5 serostatus.
   
3. Alpha4beta7 expression by expanded adenovirus specific memory CD4 T cells: Since, migration of lymphocytes to gastrointestinal tissues is dependent on the expression of a heterodimer composed of alpha4 and beta7 integrins, the authors measured expression of this molecule on expanded CSFE-low (dividing) CD4 T cells co-cultured with Ad5-, Ad11-, tetanus toxoid-, heat inactivated influenza- or Staphylococcus enterotoxin B (SEB)-pulsed DCs for five days. Majority of the expanded Ad5-specific CD4 T cells expressed high levels of alpha4beta7 in comparison to CSFE high (undivided) CD4 cells.
   
4. Adenovirus induced alpha4beta7 increases correlate with Ad5 titers: The authors next determined the fold increase in the alpha4beta7 expression by stimulation of T cells with Ad5, Ad11, tetanus toxoid, influenza or SEB-pulsed DCs in relation to the background expression levels of CD4 T cells cultured with unpulsed DCs. A positive correlation between the fold increase in total CD4 T cells expressing alpha4beta7 in response to Ad5 or Ad11 stimulation was observed. The authors also found a positive correlation between the fold increase in total CD4 T cells expressing alpha4beta7 and Ad5 antibody titers.
   
5. CCR9 and CCR5 up-regulation in response to Ad5 and Ad11 challenge: As chemokine receptor CCR9 also helps in the migration of lymphocytes to mucosal tissues, the authors next studied the level of CCR9 expression by activated adenoviral-specific memory CD4 T cells in four donors. They observed significantly increased levels of surface CCR9 in cells expanded by stimulation with first and second generation Ad5 and Ad11 vectors in comparison to CSFE high cells. Similar to CCR9, CCR5, which functions as a co-receptor for HIV-1 entry is up-regulated by T cells with effector or memory phenotype and Th1 cells. The authors thus, investigated the expression of CCR5 on expanded adenovirus specific memory T cells. CCR5 was found to be up-regulated on CSFE low CD4 T cells when co-cultured with Ad5 or Ad11 pulsed DCs in comparison to CSFE high undivided T cells.
   
6. Re-stimulated adenovirus specific CD4 T cells are more permissive to HIV-1 infection: To determine the significance of increased expression of CCR5 on expanded adenovirus specific memory CD4 T cells, the authors stimulated DCs from these donors with first or second generation Ad5 or Ad11 and co-cultured them with lymphocytes. They were then infected with the R5 virus, HIV-1. The authors observed that intracellular staining for HIV-1 p24 was significantly higher in the proliferating CFSE-low CD4 T cells in comparison to un-dividing CFSE-high cells.
   

Oral Immunization with Attenuated Salmonella enterica serovar Typhimurium Encoding Cryptosporidium parvum Cp23 and Cp40 Antigens Induces a Specific Immune Response in Mice

Source of this paper is: Benitez AJ, McNair N, Mead JR. Clinical and vaccine immunology. 2009. 16(9):1272-1278.

The live oral Salmonella vaccines have been successfully used in delivering heterologous antigens and in generating a mucosal immune response against a number of organisms including bacteria, viruses and parasites. Among the parasitic species Toxoplasma gondii and Eimeria tenella are two good eaxmples. The advantages of attenuated Salmonella vaccines include:

• They induce both cell-mediated and humoral responses
  
• They elicit both systemic and local response
  
• They are easy to administer
  
• They are affordable
  

In this paper from the laboratory of Dr. Jan R Mead, Emory University School of Medicine, Atlanta, Georgia, the authors assessed the use of an attenuated strain of Salmonella carrying specific C. parvum antigens as a vaccine receptor and the protection that it offers against Cryptosporidium parvum infection.

Cryptosporidium parvum is an obligate intracellular protozoan parasite that causes gastrointestinal symptoms when ingested by humans. Infection with C. parvum can be severe and even life threatening in immuno-compromised individuals. Treatment is available but it could it could be less efficient in immuno-deficient individuals, thus, development of a vaccine that is capable of at least inducing partial immunity in such individuals would be beneficial.

The experiments performed and results obtained are:

1. Expression of Cp23 and Cp40 S. Typhimurium constructs: Cp23 and Cp40 genes from C. parvum were PCR amplified and sub-cloned into suitable expression vectors. The resulting constructs were transformed into Salmonella vaccine strain SL3261 (this strain contains a mutation in the aromatic amino acid biosynthetic pathway and has been previously reported to be the safest vaccine strain) and the expression of proteins was tested by SDS-PAGE and Western Blotting using mouse C. parvum serum. Both proteins were stably expressed in both aerobic and anaerobic conditions and in vaccine strain SL3261 and remained soluble.
   
2. Plasmid stability and Salmonella persistence in vivo: SL3261 containing the Cp23 protein was passaged in antibiotic free medium for a period of nearly five days. The plasmid was stably maintained when plated on to medium with or without antibiotics. Furthermore, three groups of mice were inoculated orally with SL3261 and euthanized at days 3, 10 and 25 post-immunization and bacterial counts per time point were performed on homogenates of spleen, liver and intestines. These counts showed that the recombinant construct grew and persisted in the reticulo-endothelial system.
   
3. Immunogenicity of Salmonella derived Cp23 and Cp40 antigens: Mice were immunized with SL3261 without Cp23 or Cp40 and with SL3261 with Cp23 and SL3261 with Cp40. Cp23 and Cp40 antibody production was determined in vaccinated mice at seven weeks after immunization. Specific serum Ig G levels were enhanced in mice immunized with Sl3261/Cp23 or SL3261/Cp40 in comparison to mice immunized with the control. Furthermore, the oral immunization with SL3261/Cp23 or SL3261/Cp40 induced only an IgG1 antibody response and no IgG2a was detected. These findings suggested that in response to immunization with S. Typhimurium encoding C. parvum Cp23 and Cp40 antigens, a Th2 type response is generated. It was also observed that a prior immunization with a recombinant DNA vaccine resulted in significant increase in titres of IgG specific anti-Cp23 and anti-Cp40 antibody response.
   
4. In vitro detection of neutralizing antibodies: To check whether antibodies from orally immunized mice can inhibit parasite from infecting host cells, sera from immunized mice were incubated with sporozoites and they were then allowed to infect HCT-8 cells. It was observed that sera from mice immunized with either Cp23 or Cp40 were able to reduce infection by sporozoites by 30%.
   

Conclusions:

The study demonstrates the immunogenicity of a single oral immunization with Salmonella vaccine strain SL3261 that expresses recombinant C. parvum antigens (Cp23 and Cp40) in mice. The study also shows the stability and safety of the recombinant Salmonella vector and a partial inhibition of infection by sporozoites incubated with immune sera. Although with some optimizations are still required, the use of Salmonella vaccine vector to deliver C. parvum antigens appears to be a feasible way to elicit humoral and mucosal immune response in the immunized host,

A review on Microfluidic systems for detection of pathogens

Being associated with molecular diagnostics of infectious agents for past so many years, I can easily identify with the need to develop faster, portable and more accurate methods for diagnosis of pathogenic microorganisms. For the last few years, much advancement has been made in this regard. This blog is on a very interesting review article from the laboratory of Dr. Peter Ertl, Viena, Austria on microfluidic systems for pathogen sensing.

Microfuidics is explained in Wikipedia as one that deals with the behavior, precise control, and manipulation of fluids that are geometrically constrained to a small typically sub-millimeter scale. It is a multidisciplinary field intersecting engineering, physics, chemistry, biotechnology, and microtechnology. It involves design of systems in which small volumes of fluid are used. It is used in the development of inkjet printheads, lab-on-a-chip technology, micro-propulsion and miceo-thermal technology.

The standard methods which are used for pathogen detection include pathogen culture, enzyme immuno assays and polymerase chain reaction. All these methods take 2-4 days and generally require to be performed in centralized labs. As most centralized labs are limited to larger cities, there is a need to develop near patient testing which is commonly referred to as point of care (POC) testing. Therefore robust and portable diagnostic devices capable of rapidly providing information on pathogens will also help reduce mortality rates and hospitalization in case of infectious pathogens. In the last two decades a variety of different biosensors have been developed. Still, miniaturized, low cost or disposable biosensors capable of rapid detection and accurate identification of a wide range of pathogens are required. Thus, efforts have been made to minimize the time span between sampling and results. The results of these efforts are miniature devices that do not depend upon special infrastructure and sample preparation procedures. The area of miniaturized or microfluidic analysis systems, which are also called "micro total analysis system" or "lab-on-a-chip (LOC) system".

The major advantages of the microfluidic systems are:

• To conduct measurements from small volumes of complex fluids with efficiency and speed.
  
• No need for a skilled operator
  
• Reduced consumption of reagents due to small size
  
• Ability to integrate separation and monitoring techniques within a single device.
  
• Portable LOC devices are able to provide information in even in most remote settings.
  

In this review the recent progress within microfluidic based pathogen detection is presented. A schematic overview of microfluidic based pathogen sensing is presented in this flow chart.

Source: Mairhofer J, Roppert K, Ertl P. Microfluidic systems for pathogen sensing: A Review. Sensors 2009, 9, 4804-4823.